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University Microfilms International 300 N. Zeeb Road Ann Arbor, Ml 48106 8410426
Shank, Richard L.
EFFECTS OF SEASON AND TEMPERATURE ON THE SUSCEPTIBILITY OF STREAM INSECTS TO A COMMON ORGANOPHOSPHATE INSECTICIDE
The Ohio Stale University Ph.D. 1984
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University Microfilms International EFFECTS OF SEASON AND TEMPERATURE ON THE
SUSCEPTIBILITY OF STREAM INSECTS TO A
COMMON ORGANOPHOSPHATE INSECTICIDE
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Richard L. Shank, B.S., M.S.
The Ohio State University
1984
Reading Committee: Approved By
William Collins
David Denlinger
Sheldon Lustick
Roger Yeary Department of Entomology VITA
March 31 , 1948 ...... Born - Cleveland, Ohio
1973-1974 ...... Undergraduate Research Assistant, Water Resources Center, The Ohio State University, Columbus, Ohio
1974 ...... B.S., The Ohio State University, Columbus, Ohio
1974-1975 ...... Graduate Research Associate, Water Resources Center, The Ohio State University, Columbus, Ohio
1975-1976...... Graduate Teaching Associate, Department of Zoology, The Ohio State University, Columbus, Ohio
1976 ...... M.S., The Ohio State University, Columbus, Ohio
1976-1979 ...... Environmental Scientist, The Gnio Environmental Protection Agency, Columbus, Ohio
1979-1981 ...... Graduate Research Associate, Department of Entomology, The Ohio State University, Columbus, Ohio
1981-1984...... Environmental Scientist, The Ohio Environmental Protection Agency, Columbus, Ohio
PUBLICATIONS
"Loss of Toxicity to Receiving Water Biota Following Secon dary Treatment of a Non-ionic Surfactant." Journal of the Water Pollution Control Federation, September^ 1979. TABLE OF CONTENTS
Page VITA ...... ii
LIST OF T A B L E S ...... v
LIST OF F I G U R E S ...... vii
INTRODUCTION AND LITERATURE REVIEW ...... 1
Biochemical Adaptation to Temperature Changes ...... 4 Parathion and Organophosphate Insecticides . . 8 Inhibition of Acetylcholinesterase ...... 9 The Role of Mixed-Function Oxidases in Organo phosphate Poisoning...... 12 Toxicity Studies with Aquatic Insects ...... 14 Research Objectives ...... 16
METHODS AND MATERIALS ...... 20
Collection Sizes, Collection Methods and Holding Conditions in the Laboratory .... 20 Experimental Organisms ...... 25 Physical Characteristics of Experimental I ns e c t s ...... 28 Metabolic R a t e ...... 29 Toxicity Tests ...... 31 In vitro Acetylcholinesterase Activity .... 33 In vitro Inhibition of Acetylcholinesterase . . 35 Tn vivo Inhibition of Acetylcholinesterase and Regeneration of Inhibited Enzyme ...... 36 Statistical Analysis ...... 37
iii Page RESULTS ...... 38
Emergence Patterns and Seasonal Effects on S i z e ...... 38 Water Content ...... 40 Lipid C o n t e n t ...... 41 Respiration Rates ...... 41 Toxicity Experiments ...... 48 Acetylcholinesterase Activity ...... 57 Inhibition of Acetylcholinesterase ...... 64 In vivo Inhibition of Acetylcholinesterase in Chaoborus sp...... 89
DISCUSSION ...... 97
Respiration ...... 97 for Respiration ...... 99 To x i c i t y ...... 104 Acetylcholinesterase Activity ...... Ill Acetylcholinesterase Inhibition ...... 114 In vivo Inhibition of Acetylcholinesterase in Chaoborus sp...... 116 Regeneration of Acetylcholinesterase ...... 117
CONCLUSIONS...... 118
REFERENCES ...... 122
iv LIST OF TABLES
Table Page
1. Characteristics of Experimental Insects .... 18
2. Average Wet Weight, Water Content and Lipid Content of Experimental Animals ...... 39
3. Respiration Rates (O2 Consumption) of Experi
mental Insects at Two Temperatures, ul 0 2 /mg/hr (+S.D.)...... 42
4. Respiratory Quotients (Qin) for the Experimental A n i m a l s ...... 45
5. Comparison of Acclimatization Respiration Rates to Acclimation Respiration Rates in the Experi mental Animals ...... 47
6. Toxicity Data of Experimental Insects (48 hour ECj-q ) for Parathion (PT) , Paraoxon (PO) , With and Without PBO (ug/L) ...... 49
7. Toxicity Comparison Between Winter and Summer Forms of the Experimental Insect s...... 51
8. Toxicity Comparison of Parathion and Paraoxon for Winter and Summer Forms of the Experimental I n s e c t s ...... 52
9. Interactive Toxicity Ratios of the Experimental Insects for Parathion and P B O ...... 54
10. Interactive Toxicity Ratios of the Experimental Insects for Paraoxon and P B O ...... 56
11. Acetylcholinesterase Activity Rates (u moles substrate/mg organism/minute) at 4°C and 22°C for the Experimental I n s e c t s ...... 63
v Table Page
12. Values for Acetylcholinesterase Activity Rates of the Experimental Ins ects...... 65
13. In Vitro Inhibition of Acetylcholinesterase by Paraoxon in Experimental Insects: L n, Molar (X10‘5) ...... 66
14. Inhibition of Acetylcholinesterase in Chaoborus sp. (Summer Form) Exposed to Parathion at Z 2 ° C ...... 92
15. Acetylcholinesterase Inhibition in Chaoborus sp. (Summer Form) Held in Clean Water at 22°C After Exposure to Parathion ...... 95
vi LIST OF FIGURES
Figure Page
1. Effect of Substrate Concentration on in vitro Acetylcholinesterase Activity of Chaoborus sp. 59 (Summer Form) at 4°C and 22°C ......
2. Effect of Temperature on in vitro Acetyl cholinesterase Activity of~Cheumatopsyche sp. . 61
3. Winter Form of Allocapnia sp.: In vitro Inhibition of Acetylcholinesterase by Paraoxon at 4°C and 22°C and by Parathion at 22°C . . . 68
4. Summer Form of Stenonema femoratum: In vitro Inhibition of Acetylcholinesterase byTaraoxon at 4°C and 22°C and Parathion at 2 2 ° C ...... 70
5. Winter Form of Stenonema femoratum: In vitro Inhibition of Acetylcholinesterase byTaraoxon at 4°C and 22°C and Parathion at 2 2 ° C ...... 72
6. Summer Form of Stenonema vicarium: In vitro Inhibition of Acetylcholinesterase by Paraoxon at 4°C and 22 ° C ...... 74
7. Winter Form of Stenonema vicarium: In vitro Inhibition of Acetylcholinesterase by Paraoxon at 4°C and 22 ° C ...... 76
8. Summer Form of Cheumatopsyche sp.: In vitro Inhibition of Acetylcholinesterase by Paraoxon at 4°C and 22°C and Parathion at 2 2 ° C ...... 78
9. Winter Form of Cheumatopsyche sp. : I_n vitro Inhibition of Acetylcholinesterase by Paraoxon at 4°C and 22°C and Parathion at 2 2 ° C ...... 80
10. Summer Form of Hydropsyche sp.: In vitro Inhibition of Acetylcholinesterase by Paraoxon at 4°C and 22 ° C ...... 82
vii Figure Page
11. Winter Form of Hydropsyche sp.: In vitro Inhibition of Acetylcholinesterase by Paraoxon at 4°C and 22°C and by Parathion at 22°C .... 84
12. Summer Form of Chaoborus sp.: In vitro Inhi bition of Acetylcholinesterase Fy Paraoxon at 4°C and 22°C and Parathion at 2 2 ° C ...... 86
13. Winter Form of Chaoborus sp.: In vitro Inhi bition of Acetylcholinesterase Fy Paraoxon at 4°C and 22°C and Parathion at 2 2 ° C ...... • . 88
14. In vivo inhibition of Acetylcholinesterase in CFaoborus sp. (Summer Form) Exposed to 100 ug/L Parathion at 22°C. A and B are Duplicate Ex periments Conducted at Different Times ...... 91
viii INTRODUCTION AND LITERATURE REVIEW
One problem posed by chemical contamination of surface water is establishing a rational basis for regulating the use of chemicals so that benefits are attained without
deterioration of water as a habitat for aquatic organisms
and for human consumption and enjoyment. Generally, water
that is a safe habitat for a diverse aquatic community is
also safe for human utilization. A rational basis for regu
lating each chemical is derived from research data that are generated with aquatic organisms under natural conditions or
those that simulate natural exposure.
Pesticides are one of the largest groups of chemicals
for which water quality standards or criteria have been es tablished (National Technical Advisory Committee to the
Secretary of the Interior, 1968; NAS/NAE, 1972, U.S. EPA,
1977 and the Ohio EPA, 1978). Pesticides enter surface waters in the United States from manufacturing operations
(industrial effluents), municipal sewage, agricultural use, forest treatments, other miscellaneous uses and by transloca tion from other environmental compartments. Water pollution by pesticides is a major concern of federal and state
1 2
regulatory agencies and agencies responsible for the manage ment and protection of our natural resources. Pesticide- free water systems are not compatible with our industrial- agricultural system of production, but it is possible to establish safe upper limits of concentration of each pesti cide that is a potential contaminant of surface water. In secticides are the group of pesticides that has been given the most attention in regard to allowable stream burdens.
The setting of tolerances or limits (i.e., water quality standards and criteria) is based on biological data acquired with aquatic organisms, primarily fish and to a lesser ex tent, aquatic insects.
Acute toxicity data (24 or 96 LC^q ) for aquatic organ isms are a useful starting point for establishing standards or criteria. But pesticides approaching acutely toxic con centrations in streams would undoubtedly alter the ecology of the stream and may not be safe for human utilization.
Hence, factors other than acute toxicity become important elements in establishing criteria or standards for certain groups of pesticides. For instance, organophosphate insec ticides comprise nearly 50% of all insecticides used in the
United States today and their mode of action as poisons is the inhibition of acetylcholinesterase, an enzyme common and essential to metazoans. The inhibition is long-lasting
(regeneration times are often 10 to 15 days) to such an 3
extent that organophosphates are classified as irreversible
inhibitors of acetylcholinesterase. Consequently, stream organisms exposed intermittently to sublethal concentrations
of organophosphates may exhibit cumulative acetylcholines
terase inhibition, an effect that would not be revealed by bioconcentration studies with organophosphates since the
latter do not typically accumulate in body tissues to any extent. Parathion, the toxicant used in this study, is
representative of the organophosphate group. It is used
extensively in agriculture (about 25% of all insecticide use
in the U.S.) and has been detected in Ohio and U.S. rivers
in U.S. EPA monitoring studies.
Another important aspect of toxicity for which very little research has been conducted is in the area of seasonal temperature fluctuations. Active populations of poikilo- thermic stream vertebrates and invertebrates exist through out the year in Ohio streams. Seasonal changes of tempera ture from 0°C to 30°C are not uncommon. These changes would obviously affect uptake, mode of action, metabolism and ex cretion of xenobiotics. While the poikilothermic stream organisms may adjust biochemically to these great changes in temperature by utilizing several different strategies, it is unclear as to how this may affect their susceptibility to xenobiotics. Municipal and industrial discharges of toxic substances and run-off from agricultural areas occur 4
year-round. It is the purpose of this thesis to present
some basic knowledge on the effects of season and tempera
ture on the susceptibility of stream organisms to a common
organophosphate pesticide.
Biochemical Adaptation to Tenperature Changes
Generally a rise in temperature of 10°C causes a
doubling of reaction rates in chemical systems. This expo nential increase in reaction rate is referred to as van't
Hoff's rule (Gordon, 1972). The increase in a rate caused by a 10°C rise in temperature is called the Q-^q . It can
also be applied to respiration and other biochemical reac
tions. In ectothermic organisms, where body temperature
follows environmental temperature, Q^q values generally vary between 2 and 3 (Schmidt-Nielsen, 1979).
Environmental temperature changes pose severe problems for ectotherms in temperate climates. Survival mechanisms that have evolved to cope with seasonal variation include extreme changes in life cycles or spending dormant periods in areas protected from freezing temperature (i.e., below ground). However, aquatic habitats are never completely frozen (particularly streams) and ectotherms existing in these areas (mainly fish, invertebrates and some amphibians) are known to remain active during winter months. Thus, or ganisms that must live at temperatures as high as 30°C in 5
the summer and as low as 0°C in the winter would undoubtedly
exhibit altered biochemical activity and/or the organisms may have mechanisms to compensate for seasonal temperature changes. Bullock (1955), in a review article, suggested
that many poikilothermic organisms exhibit metabolic and physiological activity independent of temperature to varying degrees. He felt that this represents a compensation rather
than an insensitivity of metabolic activities or the rate of various biochemical functions to temperature. In a more
recent review article, Hochachka and Somero (1973) explained the various possible methods by which ectotherms possibly compensate for environmental temperature changes. They theorized that three major forms of compensation are utilized.
1. Evolutionary rate compensation - This method is
accomplished over long geologic periods of time. Or
ganisms using this method have usually adapted to one
narrow range of temperature. The best example of this
method is found in the arctic fish, Trematomus borch-
grevinki, which lives at water temperatures of -2°C
throughout the year, yet has metabolic activities and
growth rates comparable to tropical fishes. Organ
isms adapted to such narrow temperature ranges usually
cannot survive fluctuating temperatures and studies
indicate that their enzymes have a very narrow tem
perature range of activity (i.e., extremely large Q^q 6
values). Hazel and Prosser (1974), in a more recent review article, surmised that such organisms have lost their capacity for thermal acclimation.
2. Seasonal rate compensation - In studies comparing an aquatic ectotherm species that was acclimated at different temperatures, organisms acclimated to the cold demonstrated much higher metabolic rates than would have been predicted by extrapolating warm- acclimated metabolic rates of the same species, assum ing normal Q^q values (2 to 3). This was also true for metabolic rates of isolated tissues. Baldwin and
Hochachka (1970), working with rainbow trout, dis covered different isozymes of acetylcholinesterase in warm- and cold-acclimated trout, suggesting that sea sonal isozymes exist in this species. Moon and
Hochachka (1971, 1972) made a similar discovery in experiments with rainbow trout and isocitrate dehy drogenases. Hazel and Prosser (1974) cited evidence from other authors that biochemical acclimation to temperature may exist in Atlantic salmon, green sun- fish, coho salmon, brown bullhead, earthworms, Rana pipiens (leopard frog) and P. brevicornis (an Alaskan beetle). Hochachka and Somero (1973) suggested that seasonal rate compensatory responses are due to funda mental changes in cellular chemistry. 7
3. Immediate rate compensation - Some aquatic ecto therms, mainly intertidal organisms, experience sig nificant temperature changes on a diurnal basis and yet exhibit no unusual changes in metabolism (i.e., approximately a Q^q 0f unity). For example, Somero
(1969) worked with pyruvate kinase in the Alaska king crab, an organism that experiences fluctuations within a range of 4°-12°C in its environment. He found two variants of the enzyme and concluded that both variants are formed by a temperature-dependent conversion of one protein species. Newell (1966), working with an intertidal species of cockle and citing work with other intertidal organisms such as anemones, poly- chaetes, winkles and barnacles, concluded that the metabolism of these organisms is largely independent of rapid fluctuations in temperature (Q-^q of approxi mately 1.2 - 1.3). The range for this temperature independence depends on the range of temperature to which the particular organism was subjected. Hazel and
Prosser (1974), citing the same articles as Newell
(1966) plus one that utilized a species of mussel, pointed out that although active metabolism was tem perature dependent, the standard or resting metabolism was temperature independent. The range of temperature independence was again associated with the range of 8
environmental temperatures. Hazel and Prosser (1974)
also noted other authors who found similar evidence of
immediate rate compensation in such organisms as
amphipods, copepods, crayfish, oysters, garter snakes,
salamanders, frogs, desert spiders, shore crabs, desert
locusts and blowflies. It should also be noted that
this phenomenon applies to tissue preparations as well.
Although much work in this area has been done with fish
and intertidal organisms, little if any has been attempted
with aquatic insects. In addition, no evidence was found
that anyone has attempted to relate these basic metabolic
responses to temperature with the toxicity of pesticides in
aquatic insects.
Parathion and Organophosphate Insecticides
After World War II, there was a large expansion in
the number of insecticides available, due mainly to war-time
research. These new compounds were mainly organic in nature.
Many were chlorinated organic compounds such as DDT and
chlordane but in recent years, many of these compounds have
been banned due to findings that they were environmentally persistent, biologically accumulative, highly toxic and car
cinogenic .
As a result, another group of insecticides, organo phosphates (also developed during the war), has been used 9
more extensively. This is the largest and most diverse
group of insecticides in use today (McEwen and Stephenson,
1979) . These insecticides are esters of alcohols and phos
phoric acid or derivatives of phosphoric acid (O'Brien,
1967).
Parathion was discovered by Schrader in 1944. Chemi
cally, this compound is defined as o, o-diethyl o-paranitro- phenyl phosphorothioate. It is one of the most toxic and widely used insecticides in North America, is broad spectrum
in application to pest problems, is not bioaccumulative and
is metabolized rapidly by most organisms. It is not highly
soluble in water (1-24 ppm) and degrades rapidly in aquatic
systems. Therefore, it is rarely found in water or sediment
samples.
Inhibition of Acetylcholinesterase
Early in these developments, it was recognized that organophosphates were inhibitors of acetylcholinesterase, resulting in a build-up of the neurotransmitter acetycholine at nerve synapses (Colhoun, 1959; McEwen and Stephenson,
1979) . Although it was noted that phosphorothioates were not such potent inhibitors of acetylcholinesterase as their phosphate analogs, it was noted by Metcalf and March (1949) that there was little difference in toxicity between thiono forms and their oxygen analogs. Chamberlain and Hoskins 10
(1951) also noted that thiophosphates were slower in toxic action but ultimately just as toxic as the phosphate analogs.
They also observed that parathion was a poor acetylcholines terase inhibitor in vitro and that toxic effects did not appear in cockroaches until 851 inhibition of acetylcholines terase had occurred. They postulated that parathion was metabolically changed to paraoxon. Diggle and Gage (1951) found that parathion was converted to paraoxon in the liver and other tissues of rats, noting also that previous studies claiming inhibition of the enzyme by parathion was the result of impurities and that parathion is a poor inhibitor of cholinesterases. Metcalf and March (1953) made the first observation of parathion conversion to paraoxon in insects by chromatographic methods. O'Brien (1960) stated that the activation of parathion to paraoxon may result in a 10,000- fold increase in inhibitory capacity. Metcalf and March
(1949) found that paraoxon worked more rapidly and was more toxic to roaches than parathion. They also noted that the degree of cholinesterase inhibition correlated with the pro gression of toxic symptoms and that inhibition levels of greater than 901 were necessary for knockdown and death.
Bigley (1966) confirmed these same observations with house flies. In addition, he also found paraoxon to be approxi mately 100 times more soluble in water than parathion. 11
Dortland (1978) and Goodman (1979) also found decreased
enzyme activity with increased concentration of parathion.
Paraoxon has a high affinity for acetylcholinesterase
and rapidly phosphorylates the enzyme. After a few hours,
the enzyme-inhibitor complex becomes permanent and therefore
paraoxon and most organophosphates are considered to be ir
reversible inhibitors. Regeneration of the enzyme is the
only means to regain full enzyme activity. Mengle and O ’Brien
(1960) found that cholinesterase inhibition was rapid and
that houseflies recovered 90% of their enzyme activity within
one day, however, there was no recovery of inhibition in
in vitro studies. Brady and Sternburg (1966) found slow in
vivo recovery of cholinesterase activity and attributed it
to regeneration of the enzyme. Verma et al (1979) found
that certain fish required up to seven days to recover full
cholinesterase activity. They felt that, although some
organophosphate poisonings may not result in their death,
fish may be subject to predation before the enzyme activity
could be regenerated. Symons and Metcalf (1978) found that
the aquatic insect, Brachycentrus numerosus, exposed to feni-
trothion for 24 hours, required up to 19 days to recover to normal activity. More importantly, affected organisms left
the protection of their cases which also may permit predation. 12
The Role of Mixed-Function Oxidases in Organophosphate Poisoning
It was established early in the development of organo phosphate insecticides that phosphorothioates were converted enzymatically by various tissues in mammals and insects to the more toxic phosphate form by oxidative (desulfurization) reactions (Diggle et al, 1951; Metcalf and March, 1953 and
O'Brien, 1967). This reaction as well as hydroxylation of aromatic and alicyclic rings, hydroxylation of aliphatic side chains, dealkylation of aromatic and cyclic ethers, dealky- lation of substituted amines, epoxidation of double bonds and oxidation of thioethers were associated with microsomal cell preparations in insects, similar to preparations from mammalian liver (Wilkinson and Brattsten, 1973). This wide ranging, non-specific enzyme system came to be known as mixed-function oxidase (MFO). Most of these reactions act to detoxify xenobiotics, but in the case of some insecti cides, particularly thionophosphates, these enzymes actually activate the compound.
Kok and Walop (1954) found most of the oxidative ac tivity in American cockroaches to be in the fat body.
Hodgson and Plapp (1970) found oxidation of xenobiotics in houseflies to be dependent on a microsomal electron trans port system which included NADPH and a carbon monoxide bind ing pigment similar to cytochrome P-450 in mammalian liver. 13
They also found that the microsomal preparation from flies was similar in appearance to mammalian liver preparations under the electron microscope. Brattsten and Metcalf (1973) working with several species of flies found the MFO system to be responsible for the biochemical transformation of several insecticides.
Specific inhibitors of the MFO system have been dis covered that advanced our knowledge on the toxicological implications of oxidative metabolism. MFO inhibitors may be used to increase the toxicity (synergize) of some insec ticides (i.e., pyrethrins) or, in the case of activation reactions, cause a decrease (antagonize) in the toxicity of phosphorothioates. Nakatsugawa and Dahm (1965) reported that several synergists, particularly piperonyl butoxide
(PBO), could inhibit MFO activity and, therefore, prevent the activation of parathion. Philleo et al (1965) working with methylenedioxyphenyl compounds as inhibitors of the hydroxylation of naphthalene in houseflies, found that 14 of these compounds (including PBO) would inhibit this reac tion. Terriere (1968) also noted that the conversion of parathion to paraoxon was inhibited by PBO. Casida (1970) found that methylenedioxyphenyl compounds enhanced the in secticidal properties of certain chemicals by inhibiting the
MFO system in microsomes. Brattsten and Metcalf (1973) also 14
found that MFO activity could be inhibited by PBO. Estenik and Collins (1979) concluded that the MFO system in
Chironomus riparius was highly active and could be completely inhibited by PBO.
The activity of the MFO system can therefore be ap proximated in many insects by the ability of PBO to inhibit the conversion of parathion to paraoxon. This conversion would be reflected in the interactive toxicity ratio (ECj-q parathion/EC5Q parathion + PBO). A low ratio would indicate higher MFO activity. This method was also used by Estenik and Collins (1979).
Toxicity Studies with Aquatic Insects
Although some toxicity work has been conducted with aquatic insects and insecticides, very little has been done relating toxicity to temperature.
Jensen and Gaufin (1964) worked with two species of stonefly naiads and several organic pesticides. All experi ments were conducted at approximately 13°C and they estab lished lethal levels as well as effects on molting. Sanders and Cope (1968), also working with stonefly naiads (3 species) and pesticides, measured only the acute toxicity at 15.5°C.
They found the smallest species and the smallest members of each species to be the most susceptible. Mulla and
Khasawinah (1969) studied the problem of midge populations 15
as pests in sewage oxidation ponds. They evaluated the ef fectiveness of several insecticides, including parathion, at
78°F in the field and laboratory. This research was followed by field testing of seven organophosphorus insecticides against midge larvae in flood control channels (Ali and
Mulla, 1976). Federle and Collins (1976) conducted studies with three species of pond insects. They used representa tives of four classes of insecticides (organochlorines, or- ganophosphates, carbamates and pyrethrins). All tests were conducted at 25°C and only acute toxicity was measured.
Symons and Metcalf (1978) studied the effects of fenitrothion to the caddis fly larvae Brachycentrus numerosus. They ran all tests at 5°C. They measured acute toxicity as well as behavior in cases. They found that all the organisms that left their cases after a 24-hour exposure were dead within
30 days. They also discovered that survivors recovered completely in 19 days.
Several other studies have been conducted with aquatic insects and insecticides, but J. have been unable to locate any reports where temperature was varied to determine its effects on toxicity from either a seasonal or diurnal basis.
However, one study conducted on the terrestrial cabbage looper found that a reduction in temperature caused a re duction in toxicity. This study was conducted from an agri cultural control standpoint and acclimation of the organisms 16 was not done. A laboratory colony was used and tests were run at 32°C and 10°C. Decreases in toxicity with temperature varied between insecticides used (Chalfont, 1973).
Research Objectives
Six species of immature aquatic insects representing four orders (Plecoptera, Ephemeroptera, Trichoptera and
Diptera) were collected in their natural habit in summer and winter seasons for experimentation. Laboratory experi ments were conducted with these insects at temperatures of
4°C and 22°C which approximated the seasonal temperatures of the streams in which they were collected. A laboratory culture of the seventh species (Chironomus) was acclimated to comparable temperatures before tests were conducted.
The conceptual objectives of this research were:
1. To determine if winter and summer forms of aquatic
insects differ significantly in their suscepti
bility to parathion and paraoxon at temperatures
equivalent to their natural habitat.
2. To determine the relative toxicity of parathion
and paraoxon to aquatic insects at high (22°C)
and low (4°C) temperatures.
3. To determine if, in aquatic insects, oxidative
metabolism affects the toxicity of a phosphoro-
thioate insecticide (parathion) and its oxygen
analogue (paraoxon). 17
4. To determine i„f acetylcholinesterase transforma
tions occur in aquatic insects as a response to
seasonal changes.
5. To determine the threshold of in vivo acetyl
cholinesterase inhibition associated with obser
vable symptoms of toxicity of parathion in
Chaoborus.
6. To determine the stability of inhibited acetyl
cholinesterase in Chaoborus exposed to parathion
as a means of estimating the potential for cumu
lative acetylcholinesterase inhibition in insects
exposed intermittently in streams.
The experimental objectives of this research were:
1. To collect six species of aquatic insects from
their natural habitat in the summer and winter
for laboratory experimentation, adding a seventh
species which was reared in the laboratory (Table
1 provides the genera, orders and characteristics
of the aquatic insects utilized in the project).
2. To measure the average wet weight, dry weight,
percent water content and lipid content of the
insects.
3. To measure the respiratory rate (oxygen consump
tion) of winter and summer forms of aquatic in
sects at 4°C and 22°C, respectively. Table 1
Characteristics of Experimental Insects
Species Order Common Name Habitat Habits
Allocapnia sp. Plecoptera Stonefly Stream - Leaf Packs (riffles) - Detritus Feede
Stenonema femoratum Ephemeroptera Mayfly Stream - Rock Clinger (riffles) - Scraper
Stenonema vicarium Ephemeroptera Mayfly Stream - Rock Clinger (riffles) - Scraper
Cheumatopsyche sp. Trichoptera Caddisfly Stream - Case Builder (riffles) - Net Spinner
Hydropsyche sp. Trichoptera Caddisfly Stream - Case Builder (riffles) - Net Spinner
Chaoborus sp. Diptera Phantom Midge Lakes and - Pelagic ponds - Predator
Chironomus riparius Diptera True Midge Streams - Burrower - Detritus Feede
aLaboratory Colony 19
4. To measure the acute toxicity (48-hour LC^-g) of
parathion and paraoxon (with and without piperonyl
butoxide, a mixed-function oxidase inhibitor) in
winter forms of aquatic insects at 4°C and sum
mer forms at 2 2 ° C.
5. To measure the acute toxicity (48-hour LCj-g) of
parathion to the winter form of Chaoborus and the
laboratory culture of Chironomus at 22°C and 4°C,
with and without piperonyl butoxide.
6. To establish the optimum temperature and sub
strate concentration conditions for iii vitro
acetylcholinesterase activity in the aquatic in
sects, and to measure the in vitro acetylcholines
terase activity in winter and summer forms at
4°C and 22°C, respectively.
7. To measure the in vitro paraoxon inhibition (I^q ,
molar) of acetylcholinesterase in summer and win
ter forms of aquatic insects at both temperatures,
4°C and 22°C.
8. To measure the rate of inhibition of acetylcholi
nesterase in Chaoborus exposed to parathion and
to monitor the regeneration of free enzyme from
inhibited enzyme in Chaoborus held in clean water. METHODS AND MATERIALS
Collection Sizes, Collection Methods and Holding Conditions in the Laboratory
Seven species of immature aquatic insects were used
in these experiments. They were selected because of their
availability in large numbers in both winter and summer
seasons and their ease of maintenance in the laboratory.
Three species of immature stream insects (Allocapnia
sp., Stenonema femoratum and Stenonema vicarium) were col
lected from Hayden Run, a small stream fiften kilometers
north of the OSU campus. All collections were made between
its confluence with the western side of the Scioto River
and a ten meter waterfall approximately 300 meters upstream.
Groundwater contributes a portion of the stream flow in
this section and, therefore, it remained open during the winter. The stream is approximately two to three meters wide with a mean depth of 20 centimeters. The substrate is
rocky and contains very little silt. The upper reaches of
the stream pass through pasture land and remote suburban areas while the stretch below the falls flowed through a heavily shaded ravine. Stream temperatures measured randomly
20 21
during collections never exceeded 22°C in the summer and
were not less than 1°C in the winter.
Two additional species (Cheumatopsyche sp. and Hydro-
psyche sp.) were collected from the Scioto River 200 meters
below the dam at Griggs Reservoir five kilometers west of
OSU. The stream, approximately 20 meters wide at this point,
has a mean depth of 50 centimeters but this factor varied,
depending on water releases from the reservoir. The water was more turbid and contained more silt than Hayden Run.
The substrate was rocky. Winter stream collection tempera
tures varied from 0°C to 8°C but the river remained open at
this point due to rapid water movement. Summer collection
temperatures varied from 18°C to 27°C.
Collections from both streams in the summer were ob
tained by handpicking desirable specimens from rocks. Buc kets containing the specimens were kept cool by immersing them in the stream. Summer collections at both sites were made from mid-June through September. The insects were im mediately transported to the laboratory where they were segregated according to species into shallow trays of water and maintained in incubators at 22°C with aeration and 15 hours of light. Insects were utilized for experiments within two days of collection.
Winter collections were made from mid-December through
March. Due to the large accumulations of tree leaves among 22
the rocks and also the rigors of collecting at cold tempera
tures, collections were made by placing a square frame dip
net downstream of the collection point and dislodging the
insects from the rocks with small brushes. Insects and
debris, swept into the net by the stream flow, were placed
into collection buckets containing stream water and trans
ported to the laboratory. There, the insects were sorted
from the debris by hand and kept at 1°C during this process
by placing the sorting trays on ice. Sorted collections
were maintained at 4°C in incubators with aeration and a ten-
hour photophase. Insects were utilized for experiments
within two days of collection.
Laboratory tap water used for maintenance of the in
sects in the lab as well as for the experiments was collec
ted 24 hours in advance of its use. The source of this water (Columbus City System) is the Scioto River and Big
Walnut Creek. This water is softened and chlorinated by the
city treatment plants. All tap water used in these experi ments was filtered through activated charcoal using Tygon
tubing as connectors. The filter, constructed of a poly- vinylchloride tube 1 meter long and 10 centimeters in dia meter contained high grade, fine granule carbon (Barnebey-
Cheney coconut shell base AC, 6 x 10 Tyler screen). The
column was mounted vertically and the flow was against gra vity. This treatment effectively removed chlorine (Shank, 23
thesis 1976), trace organics (Shank and Collins, unpublished) and some metals (Shank and Collins, unpublished). The water was then aerated for 24 hours in Nalgene carbouys placed in incubators at the appropriate temperature (4°C or 22°C).
Chaoborus sp. was collected from three cement-lined ponds at the U.S. E.P.A. Toxicology Station in Newtown,
Ohio. Numerous other aquatic insects were present in these ponds. Chaoborus, a pelagic species, was collected with fine mesh dip nets, placed in buckets containing the pond water and transported to Columbus. During the two hour drive, temperature was maintained in the collection buckets by adding ice. Pond temperatures varied from 18°C to 30°C in the summer and 0°C to 8°C during the winter collection period. These insects were maintained in deep plastic trays placed in incubators at 22°C (15 hours of light) in the sum mer and 4°C (10 hours of light) in the winter. Light in each incubator was supplied by one 8 watt fluorescent tube.
With minimal aeration Chaoborus could be maintained under these conditions for more than two weeks in the laboratory with no observable stress. Quantities of Daphnia sp. were collected from the same ponds to provide food for Chaoborus during the holding period in the laboratory. The collected
Daphnia were maintained in the same container with the
Chaoborus, 24
Chironomus riparius was the only species that was cul
tured in the laboratory. This particular colony had been
reared in the laboratory for nine years prior to these ex
periments. A starter colony was originally collected below
a city sewage treatment facility on the Scioto River down
stream from Columbus (Estenik, 1978). They were reared in
80 liter all-glass aquaria with screen flight cages (60 cm
long, 30 cm wide and 45 cm high) mounted over the tanks to
provide a swarming and mating space for the adults. The
larvae were fed a fine grained commercial trout chow (Purina)
three times a week. The substrate in which they constructed
burrows was formed by larval excrement, organic matter de
rived from unconsumed food, etc. The midges were maintained
continuously at room temperature (20°C - 25°C) with a 14
hour photophase. Larvae used for "summer condition" experi
ments were removed directly from the aquaria. Acclimation
of larvae to simulated winter conditions was accomplished by
placing several thousand organisms with substrate from the
cultures into deep aerated trays in the incubator at 22°C
and 14 hours of light per day. Over a 30 day period the tem perature was gradually decreased (approximately 5°C per week) to 4°C and the light period shortened to 10 hours. The
insects were then used for "winter condition" experiments. 25
Experimental Organisms
General ecological data on the insects used in these
experiments are found in Table 1. Allocapnia sp.* was the
only representative of the Stoneflies (Plecoptera) and was only collectable in the winter season. These organisms are detritivores inhabiting coarse sediments, debris jams and
leaf packs in streams (Merritt and Cummins, 1978). They were found in leaf packs throughout the stream from December
through March but were most abundant in January and February.
Emergence and mating occurred during the latter months and adults were observed crawling on the snow in great numbers.
The numbers of nymphs and adults declined drastically in
March, and were not found from April through November.
Therefore, only winter nymphs were used in these experiments.
A search of the literature revealed that finding only winter forms of Allocapnia would not be unusual. Khoo (1968) reported that Capnia bifrons, a winter species closely re lated to Allocapnia, diapaused in the substratum during the warm months. Moreover, Harper and Hynes (1970), working with seven species of winter stoneflies including three species of Allocapnia, found that they diapaused as early instar nymphs deep in the gravel of the stream bottom during
*Probably Allocapnia vivapara (Claassen) due to the absence of wings in the adult males ("Usinger, 1956; Mike Glorioso, personal communication). 26
the warmer months. This pattern was verified by Coleman and
Hynes (1970a, 1970b). Our observations in the laboratory
indicated that this species could be kept at warmer tempera
tures (10°C) for no more than a few hours.
Last nymphal instars of two species of mayflies used
in the experiments were both of the genus Stenonema (S.
femoratum and S. vicarium) . They are common stream species
which cling to the rocks, scraping algae and organic debris
from the rock surfaces (Merritt and Cummins, 1978), These
two species, predominant among numerous species of mayflies
living in the stream, were found in riffle areas of Hayden
Run. S. vicarium, most abundant as large nymphs during the
winter, appeared to have one major adult emergence per year
during May and June. After this late spring emergence it
was difficult to find large nymphal instars. Coleman and
Hynes (1970) noted that S. vicarium in a small Ontario
stream appeared to have early egg hatches and that nymphs
grew steadily throughout the year. S. femoratum was the most
abundant species of mayfly at Hayden Run during the summer
months and large instars were present through the year.
This species appeared to have several distinct cycles of
emergence throughout the year. Such an emergence pattern would explain the abundance of large instars of S. femoratum
at Hayden Run over a longer period of time. 27
The two species of caddisflies (Trichoptera) used in the experiments, both in the family Hydropsychidae, are fil ter feeders. The two species, Hydropsyche sp. and Cheuma- topsyche sp., constructed nets at the open end of their tubes which were attached to the bottoms and sides of rocks.
Muller (1956) and Chutter (1963) noted that filter feeding organisms were generally present in dense popula tions below lake outlets, postulating that this was due to the large plankton populations in the lakes. In addition,
Chutter (1963) concluded that impoundments allow abrasive material such as sand to settle out before water is dis charged. Accordingly, large populations of these organisms were formed 200 meters below the outfall from Griggs Reser voir in Columbus. Evidently, these species have staggered cycles of emergence because large instars could be collected throughout the year. Adult emergence occurred throughout the summer with the peak in the spring. Hynes (1970) con cluded that all species of Trichoptera are univoltine.
Two species of flies (Diptera) were used in the experi ments. Chaoborus sp. is pelagic and predatory, feeding on the many small crustaceans present in their environment. It was found that they emerged from April through September with a peak in June at the Newtown, Ohio location. Large instars became scarce after the June peak emergence. However, 28
during the warm summer months, these organisms go through a
complete life cycle in 30 days. Therefore, by mid-summer
they were abundant once more.
The other Dipteran, Chironomus riparius, was reared in
the laboratory. This species burrows in the soft substrate
and is a detritus feeder. They complete a life cycle in 30
days under laboratory conditions. Large instars were always
available for experiments.
Physical Characteristics of Experimental Insects
Average wet weight (mg/insect) was determined for both
the winter and summer forms of each species. These data were acquired from weights taken during the course of the
other experiments and analyses (respiration, lipids, percent water and acetylcholinesterase activity). Weight measure ments were obtained by counting out from 3 to 100 insects,
depending on the species and experiment, drying them briefly on a paper towel and then weighing them on a Mettler analyti
cal balance. Generally each spec'es from a given season was
fairly uniform in size, particularly because of the selection process used by the investigator. Variance and standard
deviation of mean values were not calculated.
The average percent water of each organism was deter mined only once. Six to fifty organisms, depending on species and size, were weighed as previously described in a tared 29
aluminum foil tray which had been dried in an oven at 80°C.
The insects and tray were then placed in a drying oven at
80°C for 24 hours. The tray and dried organisms were then cooled and reweighed. Loss of weight from the insects was assumed to be water.
Average percent lipid of each species was determined by modifying the method developed by Bligh and Dyer (1959) to accommodate a smaller mass of organisms than the original technique. The procedure involves homogenization of the sample with a Virtis homogenizer in distilled water, methanol and chloroform. Using cod liver oil as a standard, 971 re covery of lipids was obtained. Approximately one gram of insects, weighed as previously described, was used in each trial. Duplicate weighings were performed but variance and standard deviation of mean values were not determined.
Metabolic Rate
Respiration rates were determined as ul oxygen/mg organism/hour using a Warburg respirometer. This standard manometric technique is described in Umbreit {1964). Tempera ture was maintained within + 1°C by submerging flasks con taining the insects into a recirculating water bath. Thermo barometer readings (flasks without the insects) were used to adjust for effects of changes in barmetric pressure or tem perature on experimental values. The glass flasks were 30
approximately 20 ml in size and contained a small glass well
in the center of the flask. Ten percent potassium hydroxide 2 (0.2 ml), placed into the center well with a 2.5cm fluted wick made from Watman # 1 filter paper, absorbed any carbon dioxide given off by the organisms during the experiment.
Preliminary experiments were conducted under light and dark conditions, with and without substrate, to determine the minimum respiratory rate of each species. Light conditions had little effect on respiratory rate, but the presence of substrate (1 cm by 2 cm strips of paper toweling) was needed to promote quiescence and reduce fluctuations in oxygen con sumption of the clinging and burrowing insects. /II of the species except Chaoborus sp. hid under or dinged to these s trips.
Three to eight insects, depending upon size, were placed in the flask with four ml of carbon-filtered water.
The flasks remained immersed in the water bath for fifteen minutes to allow the temperature in the flask to equilibrate and permit the insects to adjust to their new environment.
Each experiment ran for 2 to 6 hours depending on the tem perature and species. The insects were weighed at the end of each experiment.
These experiments were conducted at 22°C with insects collected in the summer and 4°C with those collected in the winter. In addition, tests were conducted at 4°C on summer 31
organisms and 22°C on winter organisms after a 24 hour accli mation period. Acclimation was accomplished by placing trays of organisms in an incubator at the temperature to which they were naturally acclimatized (4°C in the winter and 22°C in the summer). The incubator was then set at the alternate temperature and allowed to warm or cool for 24 hours. Experiments were conducted at the resulting tempera ture .
Three replicate flasks were tested during each experi ment and each experiment was repeated two to three times.
Mean and standard deviation values were determined for all experiments.
Toxicity Tests
These tests were conducted following guidelines es tablished in USEPA publications and standard methods (USEPA,
1975 5 1978; APHA, 1980) .
Tests were conducted using 500 ml of water in 1 liter beakers. Filtered, aerated tap water was prepared as pre viously described. Stock solutions of test chemicals were prepared from high purity stock and dissolved in reagent grade acetone. The acetone solutions were added in 0.5 ml quantities to the 500 ml of test water to achieve final test concentrations. Controls containing water only or water with 0.5 ml of acetone were utilized for all experiments. 32
All experiments were conducted for 48 hours in incubators at
designated temperatures and light cycles. Fiberglass screen
substrates (3 cm square) were utilized in each beaker for
the clinging and burrowing species.
Because of variations in morphology and habits among
the organisms, differing end points were used to measure the
toxic effects of chemicals for each species. However, all
end points were based on the ability of the organisms to
maintain normal equilibrium or posture. At the end of an
experiment the organisms and water were poured into shallow
trays for observations on toxic effects. Different numbers
of tests were conducted on each seasonal form of each species
depending on their abundance at that time. Data from all
similarly designed experiments were pooled and hand plots (% mortality vs. concentration) were made on standard log-probit paper. EC 50 values were interpolated from these plots.
Tests were conducted using parathion and paraoxon, each with
and without piperonyl butoxide (PBO). Animals were pre
treated with 0.5 mg/1 of PBO for four hours prior to addition of the insecticides. The concentration of PBO was maintained
at 0.5 mg/1 during the course of the experiment and an addi
tional control containing 0,5 mg/1 of PBO was utilized.
Summer forms of each species were tested at 22°C and winter forms at 4°C. In addition, winter forms of Chaoborus sp. and both warm and cold acclimated forms of C. riparius 33
were acclimated over 24 hours to the alternate temperature as previously described. They were then tested at this tem perature .
In vitro Acetylcholinesterase Activity
Several different tests were conducted to measure the in vitro acetylcholinesterase activity of each species. The assay utilized for all tests was a photometric technique developed by Ellman, et al (1961), employing an artificial substrate, acetylthiocholine (ATCH) and a trapping agent, dithiobisnitrobenzoate ion (DTNB). The Ellman technique is based on the following reactions:
acetylcholinesterase ATCH & thiocholine and acetate
thiocholine + DTNB 1> yellow color
The acetylcholinesterase data obtained with the arti ficial substrate, acetylthiocholine are similar to those obtained with the natural substrate, acetylcholine (Ellman, et al., 1961). Riddles, et al (1979) confirmed this test and established the molar absorption coefficient of the TNB dianion as 14,150 at 412 nm. The actual color is produced by the TNB dianion which is formed when a thiol group reacts with DTNB. The other product of this reaction is a mixed disulfide. However, DTNB reacts with any free thiol group, including endogenous thiols in homogenates. Analytical 34
artifacts due to endogenous thiols were eliminated by mixing excess DTNB with the enzyme homogenate 1 hour prior to use.
Any free thiol groups present (i.e., cysteine) would react with the DTNB prior to addition of the substrate and any endogenous chromogen so produced could be accounted for with a control (homogenate without the substrate).
Enzyme homogenates were prepared with whole insects that had been frozen prior to the experiment. Insects were homogenized in 0.05 M phosphate buffer (pH 7.5) and 0.05 M sodium chloride with a Virtis homogenizer in a beaker im mersed in ice. Quantity of buffer varied with the snecies used. The homogenate was then centrifuged at 2000 g in a refrigerated centrifuge for 20 minutes. The supernatant was decanted, mixed with an excess of DTNB (0.4 ml/ml homo genate) , then held at room temperature for one hour prior to use. It was recooled before using. The slight warming dur ing this period accelerated the reaction of the endogenous chemicals containing thiol groups with DTNB and did not harm the enzyme (Shank and Collins, unpublished).
The DTNB solution was prepared by mixing the powdered
DTNB with pH 7.0, 0.1 m phosphate buffer and a small amount of sodium bicarbonate. Substrate solutions were prepared by dissolving crystallized ATCH in distilled water. The number of insects used to prepare the homogenate varied 35
among species in order to achieve a measurable level of
acetylcholinesterase activity.
The pH and ionic strength of the Tris buffer were held
at 8.0 and 0.1 M, respectively, for all preparations. These values were based on other work conducted in our laboratory on C. riparius acetylcholinesterase (Randall Detra, personal communication) as well as the general literature. Although the optimum temperature for the acetylcholinesterase prepara
tions of each species was determined, most experiments were
conducted at nominal summer (22°C) and winter (4°C) tem peratures . The optimal substrate concentration for each
form of each species was determined at both 4°C and 22°C using a Beckman DU spectrophotometer and chart recorder.
Results from these experiments were converted to umoles
ATCH/organism/minute and analyzed graphically by plotting enzyme activity versus temperature or substrate concentra tion .
In vitro Inhibition of Acetylcholines-terase
Inhibition experiments were conducted by preparing homogenates as described previously and using the same con ditions and assay method (Ellman technique). However, in the inhibition experiments the enzyme homogenate was incu bated with buffer and inhibitor (parathion or paraoxon) for
1 minute prior to assay. Various in vitro concentrations 36
of the inhibitors were prepared by adding 0.1 ml of acetone solutions of chemicd.1 to the homogenate. An acetone control was used in each experiment. Results were plotted on log- probit paper, percent inhibition versus molar concentration of the inhibitor and I5Q values were determined for each form (summer and winter) of each species at 4°C and 22°C.
In vivo Inhibition of Acetylcholinesterase and Regeneration of Inhibited knzyme
In vivo inhibition experiments were conducted with the summer form of Chaoborus sp. using parathion. Twenty to thirty organisms were placed in 500 ml of carbon filtered water in 1 liter beakers and exposed to either 10 ug/1 of parathion for 5.25 hours or 1 ug/1 for 48 hours. Acetone controls were run concurrently. At specific time periods during exposure, all organisms were removed from one beaker, rinsed in distilled water and frozen. The frozen samples were homogenized in ATCH solution using the protective sub strate technique (Van Asperen, 1958 8 1960) and immediately assayed spectrophotometrically. Less than 45 seconds elapsed between mixing with ATCH until the assay was started. Per cent inhibition of the enzyme was determined by comparison to the acetone controls.
Experiments were also conducted on the summer form of
Chaoborus sp. to determine the regeneration rate of acetyl cholinesterase activity after exposure to an inhibitor. 37
Approximately 300 organisms were exposed to 1 ug/1 of para thion at 22°C for 24 hours in 2 liters of carbon filtered water. An identical exposure was conducted with acetone as a control. At the end of the exposure period dead and af fected animals were counted. Those animals appearing normal were sorted into smaller groups of 10 to 15 and placed into
1 liter beakers containing 500 ml of carbon filtered water.
One group of organisms from each exposure was selected at this time and assayed for acetylcholinesterase activity.
Thereafter, at 24 hour intervals, two beakers from each group of exposed animals were removed, rinsed in clean water, weighed and frozen for acetylcholinesterase assay. Dead and affected animals were counted and removed. The experi ments lasted 7 days. Two such experiments were done. Per cent inhibition of the enzyme was determined by comparing the activity of the organisms exposed to parathion with the acetone controls.
Statistical Analysis
Pearson's product moment correlation coefficient (r) was used to compare various data from the respiration, toxi city and enzyme experiments. This correlation coefficient is commonly used to measure the degree of linear association between two random variables. An r value of 1.0 indicates a perfect correlation (Li, 1964). RESULTS
Emergence Patterns and Seasonal Effects on Size
Weight differences between winter and summer forms of insects varied with each species (Table 2). Generally, to reduce experimental variation, compensation for size dif ferences between seasonal forms was accomplished by careful selection of uniform specimens on the part of the investiga tor. However, size and availability were greatly affected by the life cycles of the insects. The univoltine species, those having short, defined emergence periods were usually available only at certain times of the year. The most promi nent of these was Allocapnia sp. This species emerged in mid-winter, was only available for three months, and all specimens were uniform in size during this period. S^. vicarium emerged in late spring. During the summer only the tiniest instars were available, but large instars were avail able throughout the winter. Summer experiments were conducted on those specimens collected in late summer and early autumn.
Those species having a longer emergence period (i.e., emerged sporadically throughout spring and summer) exhibited no significant difference in body sizes between winter and
38 Table 2
Average Wet Weight, Water Content and Lipid Content of Experimental Animals
Avg. Wet Wtf Avg. % H 2O Avg. % mg/Insect (summer Lipids form) (form) Summer Winter
Allocapnia sp. - 5 73d 5.8 (W)
S. femoratum 20 20 77 3.0 (S)
S. vicarium 20 22 79 3.5 (W)
Cheumatopsyche sp. 9 14 66 9.6 (W)
Hydropsyche sp. 18 27 68 6.2 (S)
Chaoborus sp. 4 6 93 1.5 (W)
C. ripariuse 6 6 f 83 1.6 (S)
n Based on three or more replicate experiments (<201 variation)
■L Based on one test.
Based on two replicates.
^Winter form. 0 Laboratory culture. f Acclimated to winter conditions in laboratory. 40
summer forms. They were usually available in various sizes of instars at all times. S, femoratum appeared to emerge throughout the warm months and was always available. Chao- borus sp. also emerged throughout the warm months with a peak emergence in early spring. During the summer this species required 30 days or less to complete a life cycle. There fore, they were available throughout the year but were slightly larger in the winter.
The Trichopterans were also univoltine and exhibited the greatest size differential between winter and summer forms. However, they had several distinct emergence periods throughout the. spring and early summer. They were available throughout the year, but the summer forms were distinctly smaller than the winter forms.
Water temperatures remained high enough for continued growth of all species into late autumn and some species apparently continued to grow throughout the winter. Conse quently, large instars were available during the winter and they would be the first forms to emerge with the advent of warm weather in the spring.
Water Content
Considerable variation existed in water content (range of 66-931) among the various species (Table 2). Generally the Dipterans had the highest water content (83% and 93%), 41
•the mayflies the second highest (77% and 79%) and the Trichop-
terans the lowest (66% and 68%). The Dipterans were worm
like in appearance with thin integuments that were easily
damaged. Although the Trichopterans also were worm-like,
they had very thick, tough integuments that were not easily
punctured. The mayflies had hard integuments.
Lipid Content
The Trichopterans had the highest lipid content (6.2
to 9.6%) of the species examined and the Dipterans the lowest
(1.5 to 1.6%) (Table 2). The mayflies and stonefly fell in
between (3.0 to 5.8%). An inverse trend between body water
content and lipid content was evident, i.e., those insects
with the most body water had the least fat and vice versa.
No trends were noted among body size and lipid content.
Respiration Rates
Results of experiments conducted to determine base
respiration rates (measured as oxygen consumption) are sum marized in Table 3. Rates were determined in each form (sum mer and winter) of each species at the acclimatization tem perature (temperature to which the organism had naturally
adjusted through seasonal change) and the acclimation tem perature (temperature to which the organism had been Table 3
Respiration Rates (02 Consumption) of Experimental Insects
at Two Temperatures, ul 02/mg/hr (+ S.D.)
Winter Form Summer Form
4°ca 22° Cb 4° Cb 22° Ca
Allocapnia sp. 0.26 (.04) 0.61 (.09) - -
S. femoratum 0.065 (.003) 0.39 (.02) 0.07 (.01) 0.34 (.08)
S. vicarium 0.10 (.04) 0.49 (.08) 0.06 (.01) 0.33 (.08)
Cheumatopsyche sp. 0.13 (.02) 0.35 (.04) 0.08 (.01) 0.54 (.06)
Hydropsyche sp. 0.09 (.01) 0.37 (.05) 0.07 (.01) 0.47 (.04)
Chaoborus sp. 0.06 (.01) 0.275 (.03) 0.02 (.01) 0.30 (.05)
C. riparius* 0.085 (.03) 0.49 (.10) 0.03 (.01) 0.44 (.10)
laboratory Culture
aTemperature to which the organism had naturally adjusted through seasonal change (acclimatization).
^Temperature to which the organism had been artificially adjusted through a gradual temperature change in the laboratory during a 24 hour period (acclimation). 43
artificially adjusted through a gradual temperature change
in the laboratory during 24 hours).
The range of respiration rates for winter forms at either 4°C (acclimatization temperature) and 22°C (acclima tion temperature) varied not more than two-fold among all species with one exception. Allocapnia sp. had the highest rate among the summer and winter forms of all insects tested at 4°C and 22°C, despite only being available in the winter.
Its respiration rate at 4°C approached the 22°C rates of the summer forms of the other species. Moreover, when Allocapnia sp. was acclimated overnight to 22°C, its respiration rate more than doubled and was higher than the 22°C rates of the winter forms of all other species.
The range of respiration rates among the summer forms also did not vary more than two-fold among all species at each temperature with the exception of the two Dipterans,
Chaoborus sp. and C. riparius. All species could be accli mated overnight to a lower or higher temperature with no apparent adverse effect except for the Dipterans. The win ter forms of these two species could be acclimated to 22°C with no apparent problem, however, the summer forms exhibited mortality and extreme lack of mobility and response when acclimated to 4°C overnight. As a result, the summer form respiration rates at 4°C were barely measurable on the equip ment used in this experiment. 44
Other than Allocapnia sp., which was collected only in the winter, Cheumatopsyche sp. had the highest acclimatiza tion respiration rates in both forms of all species despite being somewhat dormant during the winter. This species does not build feeding nets during this period and remains in its case on the rocks. Chaoborus sp. had the lowest respiration rates among summer and winter forms of all species at both temperatures.
Respiratory quotients (Qj q * Table 4) were calculated for all species between winter forms at 4°C and summer forms at 22°C as well as within winter forms (4°C to 22°C) and summer forms (4°C to 22°C). The standard exponential equa tion was utilized to calculate Q^g values (Schmidt-Nielsen,
1979). The calculated Q^g values between winter forms (4°C) and summer forms (22°C) were all within the expected range of 2 to 3. S. vicarium had one of the highest winter res piration rates and lowest summer rates and therefore had a comparatively low Q1Q of 1.9.
The winter form Q^g values also fell generally within the expected range of 2 to 3. However, Cheumatopsyche sp.
(1.7) had a relatively high respiration rate at 4°C (0.13) and a low rate at 22°C (0.35) resulting in a low Q-^g. Allo capnia sp. had an extremely high rate at 4°C and despite a high 22°C rate still exhibited a low Q^g at 1.6. 45
Table 4
Respiratory Quotients (Q^q ) for the Experimental Animals
Winter Form 4°C and Summer Form Winter Summer 22°C______Form Form
Allocapnia sp. - 1.6
S. femoratum 2.5 2.7 2.4
S. vicarium 1.9 2.4 2.6
Cheumatopsyche sp. 2.2 1.7 2.9
Hydropsyche sp. 2.5 2.2 2.9
Chaoborus sp. 2.4 2.3 4.5
C. riparius 2.5 2.6 4.4
R = respiration rate
T = temperature, °C 46
Summer form Q^q values also fell between 2 and 3 with the exception of the two Dipterans. These high values (4.5 and 4.4) were the result of the extremely low 4°C respiration rates that were previously discussed.
The problem noted earlier regarding difficulties in acclimating the T'ipteran summer forms to 4°C also affects the respiration rate ratio at 4°C between summer and winter forms (winter form rate at 4°C/summer form rate at 4°C).
Whereas the other species exhibited respiration rate ratios from 0.9 to 1.7 (Table 5), the Dipterans had ratios of 3.0 and 2.8. However, their 22°C ratios between seasonal forms were 0.9 and 1.1 which fell in the range of the other species
(0.7 to 1.5). The respiration rate ratio is significant because it provides a means of comparing the respiration rates of the acclimatized form to the acclimated form at the same temperature. The closer the ratio is to unity, the less difference existed in the respiration rates, indicating the ability of the organisms to compensate for temperature changes over a short period of time (less than 24 hours). The Dip terans exhibited good winter form acclimation to 22°C with ratios of 1.1 and 0.9 but poor summer form acclimation with ratios of 3.0 and 2.8. The Trichopterans exhibited fairly high ratios (1.3 to 1.6) for both temperatures and had the highest respiration rates of all species at temperatures cor responding to their acclimatized form (4°C for winter forms Table 5
Comparison of Acclimatization Respiration Rates to Acclimation Respiration Rates in the Experimental Animals
Winter Form Rate, 4°C Summer Form Rate, 22°C Summer Form Rate, 4°C Winter Form Rate, 226C
Allocapnia sp. - -
S. femoratum 0.9 0.9
S. vicarium 1.7 0.7
Cheumatopsyche sp. 1.6 1.5
Hydropsyche sp. 1.3 1.3
Chaoborus sp. 3.0 1.1
C. riparius 2.8 0.9 48
and 22°C for summer forms). S. femoratum, the least uni-
voltine of the organisms tested, was just the opposite of
the more univoltine Trichopterans. S. femoratum had higher
respiration for each temperature in their acclimated form,
but more significantly, exhibited the least difference from
unity in their acclimation/acclimatization ratios (0.9 for
both temperatures). S. vicarium, the most truly univoltine
of the species, varied much more from unity (1.7 and 0.7),
but more importantly, the winter form had the highest res
piration rates at both temperatures.
Generally those species that were the most univoltine
had respiration rate ratios that deviated the greatest from unity and those species that were the least univoltine were
closest to unity with the exception of the dipteran winter
forms as already mentioned.
There were no observable trends between body size and
respiration rate among species. These two factors appeared to vary independently when respiration rates were indexed on a mg body weight basis.
Toxicity Experiments
Results for toxicity tests with summer and winter forms are summarized in Table 6 . Due to the necessity of pooling results to obtain EC50 values on many of the tests, it was not possible to calculate confidence intervals. However, all EC50 results would not be expected to vary by more than Table 6
Toxicity Data of Experimental insects (48 hour EC50) for Parathion (PT), Paraoxon (PO), /ith and Without PBO.(ug/L)
Summer Form3 Winter FormC b
PT PT + PBO PO PO + PBO PT PT + PBO PO PO +
Allocapnia sp. - 2.2 21.0 2.5 2.6
S. femoratum 1. 7 2.4 4.5 4.9 30.0 46.0 32.0 36.0
S. vicarium - 29.0 38 .0 18.0 23.0
Cheumatopsyche j 5 sp. 2.7 14.5 13.5 21.0 39.0 67.0 62.0
Hydropsyche sp . 1. 3 2.4 10.0 6.0 36.0 - 68.0 -
Chaoborus sp. . 48.0 56.0 450 .0 410.0 22° 1.0 5.9 22.0 26.0 0.8 4.2 21.0 -
C. riparius 4° 18.0 11.5 8.4 31.0 12.0 12.5 22° 1.6 10.0 3.0 2.5 1.8 6 2 2
aTested at 22°C except as noted.
Tested at 4°C except as noted. CD 50
50% if enough organisms had been available to conduct more testing. This should be kept in mind when reviewing the toxicity results. The EC50 values for parathion were very similar among the summer forms at 22°C of all species tested
(range of 1.0 for Chaoborus sp. to 2.5 for Cheumatopsyche
). This was also true for the winter forms at 4°C (21 to
48) with the exception of Allocapnia sp. (2.2) and C. riparius (8.4). The EC50 values for paraoxon exhibited a much wider range than parathion for summer forms at 22°C
(3.0 to 22.0) and also winter forms at 4°C (2.5 to 450.0).
Within one species, the difference between EC50 values
for summer forms at 22°C and winter forms at 4°C also varied considerably (Table 7). The greatest differences existed for Chaoborus sp. with parathion being 48 times more toxic in the summer form at 22°C than in the winter form at 4°C and paraoxon 20 times more toxic in the summer form. The species showing the least difference between the two forms was C. riparius which was only 5.2 times more sensitive to parathion and 4 times to paraoxon.
In the summer forms of the insects used in this study, parathion was 1.9 to 22 times more toxic than paraoxon at
22°C (Table 8 ). There was little difference in the toxicity of parathion and paraoxon among the winter forms at 4°C ex cept with the Trichopterans which were 1.9 to 3.2 times more 51
Table 7
Toxicity Comparison Between Winter and Summer Forms of the Experimental Insects
Winter EC50 (4°C) Summer EC50 (22°C
Parathion Paraoxon
Allocapnia sp.
S. femoratum 17.6 7.1
S. vicarium --
Cheumatopsyche sp. 8.4 4.6
Hydropsyche sp. 27.7 6.8
Chaoborus sp. 48.0 20.0
C. riparius 5.2 4.0 52
Table 8
Toxicity Comparison of Parathion and Paraoxon for Winter and Summer Forms of the Experimental Insects
Paraoxon EC50* Parathion EC50
Summer Winter Form Form
Allocapnia sp. - 1.1
S. femoratum 2.6 1.1
S. vicarium - 0.6
Cheumatopsyche sp. 5.8 3.2
Hydropsyche sp. 7.7 1.9
Chaoborus sp. 4° - 9,4
2 2 ° 22.0 26.2
C. riparius 4° - 3.7
22° 1.9 1.1
*Winter at 4°C and summer at 22°C unless otherwise specified. 5 3
sensitive to parathion than paraoxon and Chaoborus sp. , which was 9.4 times more sensitive to parathion.
Experiments were also conducted with winter forms of
C. riparius and Chaoborus sp. at 22°C and summer forms at
4°C by acclimation over a 24 hour period. Generally, data for 4°C in summer forms were similar to 4°C data in winter forms and data for 22°C in winter forms were similar to 22°C data in summer forms. However, due to the apparent stress of overnight acclimation of summer forms to 4°C described earlier, the two EC50 values for the summer form of C. riparius at 4°C must be viewed with some reservation.
Experiments were conducted using piperonyl butoxide
(PBO) in conjunction with parathion and paraoxon (Table 6 ).
Interactive toxicity ratios (ITR) were determined by compar ing the EC50 values for each form of each species at one temperature with and without PBO (EC50 without PBO/EC50 with
PBO). ITR values less than one would indicate an antagonis tic effect on toxicity when PBO was present; an ITR value greater than one, a synergistic effect on toxicity. The closer the ITR is to one, the less effect the PBO had on the toxicity of the insecticide.
The summer forms of the insects used in these experi ments at 22°C with parathion exhibited ITRs greater than 0.5
(Table 9) with the exception of Chaoborus sp. and C . riparius.
Chaoborus sp. exhibited an ITR of 0.17 and C_. riparius, 0.16, 54
Table 9
Interactive Toxicity Ratios3, of the Experimental Insects for Parathion and PBO
Summer Winter Form“ Formc
Allocapnia sp. - 0.10
S. femoratum 0.71 0.65
S. vicarium - 0.76
Cheumatopsyche sp. 0.93 0.54
Hydropsyche sp. 0.54
Chaoborus sp. 4°C - 0.86
22°C 0.17 0.19
C. riparius 4°C - 0.27
22°C 0.16 0.30
EC50, Parathion Ratio = EC50, Parathion + PBO
bAt 22°C
At 4°C which agrees with previous work by Estenik and Collins on the
latter species (1979). Interactive toxicity ratios in win
ter forms at 4°C with parathion were similar to summer forms at 22°C except for Chaoborus sp. which was now near unity
(0.86) and C. riparius which was increased to 0.27. How ever, Allocapnia sp., which maintains a high respiration rate and activity rate at low temperatures, exhibited an interac tive toxicity ratio of 0.10 with parathion which was much lower than the other insects tested in either winter or sum mer forms.
Interactive toxicity ratios for paraoxon were close to unity in both winter and summer forms of all species (Table
10). Some of the ITRs were greater than one and may indi cate a slight synergistic effect.
Allocapnia sp., which had the highest winter respira tion rate, was more sensitive to parathion and paraoxon than the winter forms of the other species. The winter EC50 val ues of this species at 4°C were similar to the summer EC50 values for the other species at 22°C and were much less than the winter EC50 values of the others at 4°C. In addition,
Allocapnia sp. had the lowest interactive toxicity ratio of all the insects tested in either winter or summer forms.
Chaoborus sp. was the most sensitive summer form and had the lowest interactive toxicity ratio at 22°C. However, it was the least sensitive organism to parathion among the 56
Table 10
Interactive Toxicity Ratios3 of the Experimental Insects for Paraoxon and PBO
Summer Winter Form*3 Form0
Allocapnia sp. - 0.96
S. femoratum 0.92 0.89
S. yicarium - 0.78
Cheumatopsyche sp. 1.07 1.08
Hydropsyche sp. 1.67
Chaoborus sp. 4°C - 1.10
22 °C 0.85
C. riparius 4°C - 0.96
2 2 °C 1.20 1.00
a ^ EC50, Paraoxon Kari° EC50, Paraoxon + PBO
bAt 22°C
cAt 4°C 57
winter forms at 4°C and its ITR was greatly increased. It also exhibited the greatest difference between parathion and paraoxon toxicity in both the summer and winter forms by nearly an order of magnitude over any of the other species.
This species was by far the least sensitive species to para oxon in both the winter and summer forms.
Acetylcholinesterase Activity
The optimal in vitro substrate concentrations were as certained for summer and winter forms of each species at 4°C and 22°C using acetylthiocholine as an artificial substrate.
The optimal substrate concentration, nearly identical among summer and winter forms of each species of insect examined, -4 was 6.4x10 mM for all species except Chaoborus sp. which was 3.2x10 - 4 mM. Although acetylcholinesterase activity was higher at 22°C than 4°C (which may be due solely to tempera ture) the curves for 4°C and 22°C were similar in shape
(Figure 1, using Chaoborus sp. as an example). Inhibition of the enzyme due to excess substrate was not observed within the range of concentrations used in this test (Figure 1).
The activity of acetylcholinesterase at various tem peratures was determined for summer and winter forms of all species. Both forms of all species demonstrated fairly high activity over the range of temperatures tested (Figure 2, using Cheumatopsyche sp. as an example); activity at 4°C was approximately 60°o that of 22°C. Temperatures above 30°C Fig. 1 Effect of substrate concentration on in vitro
acetylcholinesterase activity of Chaoborus sp. (summer
form) at A°C and 22°C.
58
ro j4m Substrate-j4m Converted/Organism/Min. o o o j r o
[s: ]x 10"4 mM Acetylthiocholine 6S Pi-. 2 Effect of temperature on in vitro_ acetylcholinesterase
activity of Cheumatopsyche sp,.
60 01 ro jqm Substrate-jqm Converted/Organism/Min. o ro oj o j CJi o o o O Temperature (°C) Fig. 2 62
drastically reduced enzyme activity for most species. How ever, due to excessive non-enzymatic reduction of the DTNB at higher temperatures, total enzyme activity appeared arti ficially high for some species. Therefore, enzyme activities are summarized for all species in Table 11 using only data for 40C and 22°C.
Among all species, winter form activity at 4°C varied from 0.11 to 0.36 (urn substrate/mg organism/minute) and from
0.21 to 0.73 at 22°C. Variation among the summer forms was much greater: a range of 0.10 to 0.67 at 4°C and 0.18 to
1.16 at 22°C. The mayflies and Chaoborus sp. had the high est enzyme activity at both 4°C and 22°C among the winter forms. Chaoborus sp. and S. femoratum had the highest acti vities among the summer forms; noteworthy was the much re duced rate of S. vicarium. The summer form activities of the multi-voltine S. femoratum and Chaoborus sp. were nearly double that of the winter form at either temperature. In contrast, the extremely uni-voltine S. vicarium actually had summer form activities that were nearly one-half of its win ter form rates for each temperature and Cheumatopsyche sp., which falls in between these species in terms of lifecycles, maintained fairly constant rates between forms at each tem perature. However, it should be noted that S. vicarium had a winter rate at 4°C that is nearly identical to the summer form rate at 22°C. 63
Table 11
Acetylcholinesterase Activity Rates (u moles substrate/mg organism/minute) at 4PC and 22°C for the Experimental Insects
Winter Form Summer Form
4°C 22°C 4°C 22°C
Allocapnia sp. 0,29 0.45 - -
S. femoratum 0.33 0.62 0.67 1.16
S. vicarium 0.36 0.73 0.17 0.39
Cheumatopsyche sp. 0.13 0 . 22 0.10 0.18
Hydropsyche sp. 0.11 0.21 --
Chaoborus sp. 0. 34 0.54 0.61 1.02 64
Using the formula for Q-^0’ temPerature effects on the acetylcholinesterase activity rates were calculated and ap pear in Table 12. The for acclimatization temperatures
(winter form at 4°C and summer form at 22°C) correspond closely to the enzyme activity rate comparisons made between summer and winter forms in that the uni-voltine S. vicarium has a Q1q of unity and the multivoltine S. femoratum and
Chaoborus sp. have Q^q of 2.0 and 1,8. Cheumatopsyche sp.
falls in between at 1 .2 . Q^q values among winter forms of the insects tested had little variation (range of 1.3 to
1.5). This was also true among summer forms (1.3 to 1.6).
Inhibition of Acetylcholinesterase
In vitro inhibition experiments were conducted with acetylcholinesterase preparations of summer and winter forms of all species at 4°C and 22°C (Figures 3-13). Paraoxon was used as the primary inhibitor. Inhibition of acetyl cholinesterase, expressed as the Ij-q (molar concentration predicting 50% inhibition) was similar between summer and winter forms of each species at the same temperature. Experi ments conducted with parathion demonstrated that it was a much less effective inhibitor than paraoxon (Figures 3-5, 8 ,
9, 11-13). Paraoxon inhibition was greater at 22°C than at
4°C in both seasonal forms of all species (Table 13). Table 12
Q10 Values for Acetylcholinesterase Activity Rates of the Experimental Insects
Winter Form at 4°C and Summer Form Winter Summer at 22°C Form Form
Allocapnia sp. - 1.3
S. femoratum 2.0 1.4 1.4
S. vicarium 1.0 1.5 1.6
Cheumatopsyche sp. 1.2 1.3 1.4
Hydropsyche sp. - 1.4
Chaoborus sp. 1.8 1.3 1.3 66
Table 13
In vitro Inhibition of Acetylcholinesterase by*Faraoxon in Experimental Insects: I50, Molar (X 1 ( T 5 )
Summer Form Winter Form
4PC______22°C 4 °C______22°C
Allocapnia sp. - - 0.23 0.03
S. femoratum 1.6 0.6 2.5 0.8
S. vicarium 3.2 0.7 4.0 1.2
Cheumatopsyche sp. 4.3 1.2 4,6 1.6
Hydropsyche sp. 5.8 1.1 5.6 0.7
Chaoborus sp. 1.9 0.4 2.3 0.35 Fig. 3 Winter form of Allocapnia sp.: In vitro inhibition of
acetylcholinesterase by paraoxon at 4°C and 22°C and by
parathion at 22°C.
67 <0 Ol
i d tn x 1-1 01 n ooooooo o< o ^ at O) o Ol % % Inhibition (Probit Scale)
Inhibitor Cone., Molar (Log Scale) 89 Fig. 4 Summer form of Stenonema femoratum: In vitro inhibition
of acetylcholinesterase by paraoxon at 4°C and 22°C and
parathion at 22°C.
69 % Inhibition (Probit Scale) 80 - 90 30 95 60 50 20 40 70 2 C 22 r6 4 4 C niio Cn. Mlr Lg Scale) (Log Molar Cone., Inhibitor xIO i. 4 Fig. aaho (2 C) (22 Parathion -4-5 Fig. 5 Winter form of Stenonema femoratum: In vitro inhibition
of acetylcholinesterase by paraoxon at 4°C and 22°C and
parathion at 22°C.
71
00 M Ol O l OI m 01 ^ ai a) a) ai ^ o m 01 toco OOOOOOO O 01 O OOOOOOO — CJIO Inhibition ( Probit Scale) % I I O l
Inhibitor Cone., Molar (Log Scale) ZL Fig. 5 Fig. 6 Summer form of Stenonema vicarium: In vitro inhibition
of acetylcholinesterase by paraoxon at 4°C and 22°C.
73 «
95
90
^ 8 0 I 50= 0 .7x I0 '5 M 0) a 7 0 60 50 x> o 40 £ 30 2 2 W 20 c o UI 10 la !c 5 c H
J 1 1 L.J-I.L.Lf J 1------1___ |__ 1—1 1 I ______| L -6 10 I0"5 Inhibitor Cone., Molar (Log Scale) Fig. 6 Fig. 7 Winter form of Stenonema vicarium; In vitro inhibition
of acetylcholinesterase by paraoxon at 4°C and 22°C.
75 % Inhibition (Probit Scale) 90 5 r 95 60 80 70 30 40 50 20 niio Cn. Mlr Lg Scale) (Log Molar Cone., Inhibitor i. 7 Fig. Fig. 8 Summer form of Cheumatopsyche sp.: In vitro inhibition
of acetylcholinesterase by paraoxon at 4°C and 22°C and
parathion at 22°C.
77 % Inhibition (Probit Scale) 90 80 95 40 70 30 60 50 20 10 -6 2 C 22 4°C 0 12x 0 M 1.2 10x 50= I niio Cn. Mlr Lg Scale) (Log Molar Cone., Inhibitor xIO 5 i. 8 Fig. aaho (2 ) (22 Parathion x!0 4 - Fig. 9 Winter Form of Cheumatopsyche sp.: vitro inhibition
of acetylcholinesterase by paraoxon 4°C and 22° and
parathion at 22°C.
79 <0 m 1 1 ' <0 o ------1 03 o ---- oi O l b> °, O l T— i— I—I— I T o ro T o o o o o o o T r„ 0 1 — r\5 Ol Ol
acetylcholinesterase by paraoxon at 4°C and 22°C.
8.1 Ol — ro w ^ 01 o) ->i oo co co Oio Oio O O O O O O OOl O % % Inhibition (Probit Scale) I I I Ol
Inhibitor Cone., Molar (Log Scale) Fig. 11 Winter form of Hydropsyche sp.: In vitro inhibition of
acetylcholinesterase by paraoxon at 4°C and 22°C and by
parathion at 22°C.
83
acetylcholinesterase by paraoxon of 4°C and 22°C and
parathion at 22°C.
85 59 o> 2 O O O O O O o u» 01 01 a 01 o) ->i ® — i— i— I— I I T ro 1 ro o \ o o =3 ro ro o O O l % % Inhibition (Probit Scale) I I I Ol o> o o o Inhibitor Cone., Molar (Log Scale)
Fig. 12 §8 Fig. 13 Winter form of Chaoborua sp.: In vitro inhibition of
acetylcholinesterase by paraoxon at 4°C and 22°C and parathion at 22°C.
87 % Inhibition (Probit Scale) 90 r 95 70 60 40 30 80 50 20 Inhibitor Cone.,Molar (Log Scale) i, 3 “ 13 Fig, Parathion, 89
Ij-q values among the insects were well within an order
of magnitude of each other (4x or less) for each temperature
with the exception of Allocapnia sp. which had values
that were an order of magnitude less than the other insects
at each temperature. There were no correlations of acetyl
cholinesterase inhibition data with respiration rates (r=0.55)
or toxicity data (r=0.65 and 0.46) among species except for
summer acetylcholinesterase inhibition values and summer res piration rates at 22°C (r=0.97). Also of note was Allocapnia
sp. which had the highest respiration rate (Table 3), was the most sensitive organism to the toxicants (Table 6, had the
lowest interactive toxicity ratio (Table 9) and had the most
sensitive acetylcholinesterase (Table 13).
In vivo Inhibition of Acetylcholinesterase in Chaoborus sp.
In vivo inhibition experiments were also conducted with the summer form of Chaoborus sp. using parathion. A linear increase of acetylcholinesterase inhibition occurred as time of exposure to parathion was increased. The graph (Figure
14) exhibits the results of two replicate experiments (lOOug/L parathion) and the tabular results are found in Table 14,
The organisms exhibited 6 and 191 inhibition of their choli- nesterase after a one-half hour exposure and 20 and 40% in hibition after one hour. However, they demonstrated no visi ble toxic effects due to parathion exposure at these times Fig. 14 In vivo inhibition of acetylcholinesterase in Chaoborus
sp. (summer form) exposed to 100 ug/L parathion at 22°C.
A and B are duplicate experiments conducted at different
times.
90 % Inhibition, Acetylcholinesterase (Probit Scale) o ro o> 00 cd tn - m x TD 0Q CO o * 16 92 Table 14 Inhibition of Acetylcholinesterase in Ghaoborus sp. (Summer Form) Exposed to Parathion at 22°C conc. = 100 ug/L Exposure Time % Dead % Inhibition, % Inhibiti (Hrs) or Affected Normal Insects All Insec AC Bc A B A B 0.5 0 0 6 19 6 19 1.0 0 0 20 40 20 40 2.0 2 11 41 64 48 68 3.5 39 54 76 53 82 73 5.25 91 95 59 83 83 94 conc . = 1 ug/L 1 0 0 0 0 0 0 12 0 - 15 0 15 0 24 4 37 21 - 24 34 36 12 - 3 - 13 - 48 31 67 24 17 73 69 aNot visibly affected. Normal and affected. CA and B are duplicate experiments 93 despite relatively high inhibition of the enzyme. The first visible signs of toxic effects appeared at two hours and was associated with 41% and 64% inhibition of acetylcholinesterase. Organisms with visible impairment of their ability to move always exhibited 90% or greater inhibition and therefore the percent inhibition in the middle column of Table 14 is always lower than in the last columns where dead or affected animals are present. At 5-1/4 hours, 91% and 95% of the organisms were affected or dead while the few insects that appeared nor mal exhibited 59% and 83% inhibition of their acetylcholines terase . In a companion experiment, organisms were exposed to a high concentration (100 ug/1) of parathion for 1 hour and then transferred to clean water. Although they were showing no effect at this time, 85% were dead within 24 hours. Additional experiments were conducted with a lower con centration of parathion (1 ug/1) for a longer period of time (48 hours) (Table 14). None of the organisms exhibited any visible effect or demonstrated any measurable cholinesterase inhibition after 1 hour of exposure. Minimal inhibition (15%) was observed at 12 hours with no visible effect. Visibly affected organisms were observed after 24 hours; unaffected organisms at this time exhibited 20% inhibition. At 48 hours 31% and 67% of the insects were dead or affected; the portion of the exposure group that were visibly unaf fected still exhibited only 17% and 24% inhibition of their 94 cholinesterase. Thus, there appeared to be two distinct groups after 48 hours of exposure, those that were dead or affected with 90% or greater cholinesterase inhibition and a group that appeared normal with 20% or less cholinesterase inhibition. Experiments were conducted by exposing the summer form of Chaoborus sp. to 1 ug/1 of parathion for 24 hours and then transferring the normally appearing organisms to clean water. Dead and affected organisms were counted every 24 hours thereafter and cholinesterase inhibition was periodically determined for representative samples. Parathion is an ir reversible inhibitor. The enzyme must be regenerated in order to achieve normal activity ( B r a d y et al 1966). These experiments were intended to determine the length of time for enzyme regeneration to occur. After 24 hours exposure to the parathion, 17% and 23% of the organisms in two replicate experiments were either dead or visibly affected (Table 15). At this time, the nor mally appearing organisms had 8% and 22% inhibition of their cholinesterase. These apparent normal organisms were trans ferred to clean water and monitored during the next 6 days. An additional. 12% and 19% died or became affected, respec tively, within the next 48 hours but affects did not increase beyond this time. In experiment A, where only 8% inhibition of cholinesterase was observed after the initial exposure, 95 Table 15 Acetylcholinesterase Inhibition in Chaoborus sp. (Summer Form) Held in Clean W a t e T -a F _ 2TTrC-"^ After Exposure to Parathion Holding Time % Dead % Inhibition , % Inhibition (Hrs)a or Affected Normal Insects All Insects Ad Bd ABAB 0 17 23 8 22 23 39 24 11 8 0 <14 7 14 48 19 12 0 11 15 22 72 4 4 0 <11 0 11 96 19 8 0 19 23 27 120 4 8 22 0 25 9 144 8 12 17 0 24 13 aTime held in clean water after 24 hr exposure to Parathion (1 ug/L) ^Not visibly affected cNormal and affected. ^A and B are duplicate experiments 96 the organisms exhibited no inhibition after 24 hours in clean water and remained unchanged until 120 hours had elapsed, at which time the normally appearing organisms had 1 2 % inhibition. In experiment B, there was 22% inhibition at the start and it decreased to 0% inhibition within 120 hours. DISCUSSION Respiration Allocapnia sp. exhibited the highest respiration rates of all species. Their winter rates at 4°C were comparable to the summer rates of the other species at 22°C (Table 3). This might be expected because this species emerged mated and deposited eggs during the winter. In addition, most of its growth occurred from November to January. A high basal respiratory rate at cold temperatures is probably related to its growth and activity levels in much the same way as it is to the other species during the warm months when they are growing and emerging to adult forms. The higher respiratory rate of Allocapnia sp. would therefore be expected to re sult in a greater susceptibility to organophosphate toxicants as will be discussed later. Chaoborus sp. exhibited the lowest respiration rates in both winter and summer. Although the rates measured were basal metabolism and should not be affected by physical ac tivity, it should still be noted that Chaoborus sp. was the least physically active organism of the insects tested. Some physical movement was apparent in all of the respira tion experiments conducted on all of the species tested. 97 98 The Trichopteran species exhibited the highest summer respiration rates at both temperatures and, next to Allocap- nia sp., the highest winter rate at 4°C. These species are filter feeders that build cases and nets during the warm months and an enclosed case during the cold months. They were removed from their cases for testing. This may have resulted in a higher rate of activity by these organisms. Artificial substrates were provided during testing and the organisms attempted to reconstruct cases against the sub strates during the tests. This activity certainly added to the respiratory rates observed. Although most growth and activity of the Trichopterans occurs in the summer months, the winter forms appear to be semi-dormant in enclosed cases and therefore would be expected to exhibit high metabolic rates. Danks (1971) noted that several species of north temperate Chironomids also ceased feeding during the winter. These species as well as some of the others that ex hibited reduced respiratory rates during the winter may have been in a semi-dormant state or a condition of torpor. How ever, diapausing organisms which also exhibit reduced ac tivity and respiratory rates cannot resume normal activity respiration rates overnight by simpiy raising the tempera ture (Wigglesworth, 1972). Due to the general activity in the winter and the ability of the insects tested to achieve respiration rates equal to their summer forms with overnight 99 acclimation to summer temperatures, I feel that it is un likely that any of these species were exhibiting a state of diapause. All species tested appeared able to acclimate within 24 hours to temperatures much higher or lower than the tem perature at which they were collected. This would indicate that these species had some ability to compensate immedi ately to temperature changes. This resulted in nearly equal respiratory rates at 4PC between forms within each species and also at 22°C. This same effect was found by McFarlane and McLusky (1972) working with four species of midge lar vae. However, Edwards (1958) found seasonal differences in oxygen consumption in C. riparius. He found that these lar vae had a higher respiratory rate at 20°C in the summer than at 20°C in the winter. The summer forms of the two Dipteran species exhibited mortality and they appeared moribund when the temperature was reduced overnight to 4°C. This would indicate that these species could not compensate immediately to large temperature decreases and that seasonal acclimatization from summer to winter was extremely important to their life cycle. Q10 for Respiration Generally a rise in temperature of 10°C causes a 2 to 3 fold increase in oxygen consumption in poikilothermic 100 organisms (Schmidt-Nielsen, 1979). A Q^q of 2 demonstrates the van't Hoff effect of an in vitro biochemical reaction (an exponential increase in rate) (Gordon, 1972). A of 1 would indicate that the organism was a perfect metabolic rate compensator to temperature changes (Hazel and Prosser, 1974). Stenonema vicarium had one of the highest winter respiration rates (only Allocapnia sp. was higher) and lowest summer rates (only Chaoborus sp. was lower) of all the species and a resultant low of 1.9 between seasons, very close to the van't Hoff effect and significantly lower than the other species. More importantly, S. vicarium had higher winter respiration rates (Table 4) at both temperatures than its summer rates. Higher than expected winter rates have also been found in fish (Gordon, 1972). This effect allows these organisms to be as active at lower temperatures in the winter as they are at higher temperatures in the summer. This is extremely important to S. Vicarium which does much of its growing in the winter months in order to be ready to emerge as adults in the early spring. The other species do not exhibit this effect with the exception of C. riparius which could not be acclimated effectively to a lower tem perature overnight in its warm acclimated form. The other species exhibited cross - seasonal Q^q values of 2.2 to 2.5 which indicated that they had biochemical adaptions to in crease overall metabolism beyond that expected by only 101 temperature effects (van’t Hoff effect). This would indi cate that these species were more dependent on the warm months to complete their life cycles than S. vicarium and in fact the Dipterans and femoratum are multi-voltine and the Trichopterans, despite being uni-voltine, only feed in the warmer months. It should be noted that these Q^q values were calcu lated from an actual measurement over an 18°C difference in temperature. Actual Q-^q values for smaller intervals within this range will vary due to the exponential effect of the equation. The values given are more of a mean over the 18°C range. In fact Q^q values for general activity rate are usually higher at lower experimental temperatures and lower at higher experimental temperatures (Newell, 1973), Qjq values for winter forms of all species were be tween the expected 2 and 3 with the exception of Cheumato- psyche sp. and Allocapnia sp. which were below 2. Allocap nia sp. had the lowest winter form Q^q (1.6). Aquatic poiki- lotherms are generally not exposed to rapid temperature changes as are terrestrial species due to the high specific heat of water and resultant slow changes in temperature. Therefore, aquatic species can be more dependent on slow biochemical changes associated with seasonal rate tempera ture compensation and Q^q values greater than 2. The im mediate rate compensators, such as intertidal organisms, 102 cannot be as dependent on slow biochemical changes because they are periodically exposed to the rapid and drastic tem perature changes of a gaseous atmosphere during tidal cycles. The low Q Q of Allocapnia sp. apparently aids this animal during its winter emergence which occurs in January and February. This animal may be exposed to a wide range of air temperatures after living in a very narrow range of aquatic temperatures at that time of year. The winter form of Cheumatopsyche sp. also exhibited a low Q^q (1.7). Despite being semi-dormant in the winter, this species exhibited a relatively high respiration rate compared to the other species at 4DC although it was rela tively lower at 22°C. In addition this species exhibited the highest respiration rate at 22°C in the summer forms but a 4°C summer form rate that was close to the other species. Hydropsyche sp. (winter form) exhibited a similar but less pronounced effect. Both species were removed from cases and therefore were highly active. This may have resulted in the higher acclimatization respiration rates. Cooling of the summer forms drastically reduced the Trichopteran res piration rates resulting in fairly high Q^q values of 2.9. Warming of the winter forms did not produce comparable in creases in respiration rates. This is also reflected in the comparison of acclimatization respiration rates to 103 acclimation respiration rates (Table 5). This ratio is relatively high at 4°C and 22°C for both Trichopteran species. These high ratios, as well as the high summer form and low winter form Q^q values, may be explained by the ab normally high respiration resulting from the removal of these organisms from their cases. The summer form Q^q values were also between 2 and 3 except for the Dipterans. The higher summer form values of these species occurred due to the inability of the Dip terans to effectively acclimate within 24 hours to 4°C. These two species apparently are quite reliant on the slow biochemical adjustment associated with seasonal rate compen sation to temperature. This is also apparent in the high ratios exhibited when comparing respiration rates of winter and summer forms at 4°C (Table 5). However, the same com parison at 22°C would indicate that the Dipterans are quite capable of acclimating to higher temperatures in their win ter forms. Thisphenomenon may, in effect, reduce the ability of these multi-voltine species to continue emergence late into the autumn when air temperatures are lower than water temperatures but promote their ability to emerge early in the spring when air temperatures are warmer than water temperatures. The latter effect can also be seen in the mayfly species tested. 104 S. vicarium, which is highly dependent on growth in the winter, also exhibited a higher 4°C respiration rate ratio than at 22°C. This is due to the higher winter res piration rate of this species. All of the insects tested exhibited some ability to compensate immediately (<24 hours) to sudden changes in temperature. However, it was apparent that all species de pend to varying degrees on longer seasonal acclimatization strategies. Toxicity Parathion and paraoxon were several times more toxic to the insects at 22°C than at 4°C. This would indicate that stream insects are more sensitive to organophosphate insecticide run-off during the warm months and that summer forms of these insects are appropriate for establishing stream water quality criteria or standards. Although results of parathion toxicity experiments were similar among all species in the summer form, Allocap nia sp. and C. riparius exhibited EC50 values that were con siderably lower than the other species in the winter form (Table 6). Allocapnia sp. is at the peak of its annual ac tivity cycle during the winter months and exhibited respira tion rates comparable to summer form rates of the other 105 species. Due to their elevated respiration rates they would be more susceptible to phosphorothioates during the winter months. The winter form of C. riparius was actually a labora tory acclimated colony of organisms. This may have resulted in incomplete acclimation of all physiological systems with in the organism, although its winter form respiration rate was not high in comparison to the other species. Overall parathion was significantly less toxic in the winter forms of the insects tested at 4°C than in the sum mer forms at 22°C. Danks, in a review article (1978), noted that several authors found that many substances, including respiratory poisons like cyanide, have less effect on aqua tic insects in the winter when respiration and temperatures are reduced. Historically, water quality criteria has been based on testing at warmer temperatures (>15°C). My re search supports that approach for parathion and probably other phosphorothioate insecticides. Stream insects would therefore be more susceptible to run-off of organophosphate insecticides during the warm months. Much more variation existed in EC50 values of paraoxon between species in both winter and summer forms than existed with parathion. This may be due in part to the fact that paraoxon is approximately 100 times more soluble in water than parathion due to its greater polarity (Bigley, I960)- 106 This probably results in a decreased ability of paraoxon to penetrate the insect integument due to the non-polar com ponent of the integument and the hydrophilic nature of the compound. This phenomenon may occur to varying degrees among the insects tested with paraoxon. Parathion, being more hydrophobic, apparently has less problem penetrating through the integument. Other facts such as metabolism and excretion also af fect the overall toxicity of paraoxon as well as parathion. Differences between the toxicity of parathion and paraoxon within each species also varied (Table 8) , par ticularly in the summer. Paraoxon is the active, toxic form of parathion by virtue of its stronger potency as an acetyl cholinesterase inhibitor. Parathion is converted to para oxon in most organisms by the mixed function oxidase system (Brattsten et al, 1973). In experiments with terrestrial insects, paraoxon often may be more toxic than parathion (Bigley, 1966). However, in my experiments, parathion was considerably more toxic than paraoxon, especially in the summer form. In experiments with terrestrial insects the compound is applied directly to the organism by topical ap plication and not exposed to an aqueous solution, such as herein. As already noted, parathion is much more hydro- phobic, and thus, much less soluble than paraoxon. This apparently results in much faster penetration of the insect 107 integument by parathion. Loehner (M.S. thesis, 1983) found that uptake of chlorinated organic insecticides increased with decreasing solubility of the insecticides using C. riparius. This may explain the greater toxicity of para thion to these aquatic insects. Much less difference in toxicity between parathion and paraoxon existed in the win ter forms of each species. This was probably due to de creased respiration and reduced biochemical activity as a result of lower temperature. Although the main degradation reaction of parathion is preceded by oxidation to paraoxon and hydrolysis to para-nitrophenol (Aizawa, 1982), in addi tion, hydroxylation of the ethyl groups or the aryl moeity can occur. The lower winter temperatures would certainly lower the reaction rates of these metabolic schemes. How ever, each reaction rate might be affected to differing de grees. However, it seems apparent that any transformation of parathion to paraoxon in the environment prior to expo sure of stream insects would decrease the toxicity of the compound. Experiments were conducted using piperonyl butoxide (PBO) to inhibit the mixed function oxidase (MFO) system and compare the effect of MFO on conversion of parathion to paraoxon. Interactive toxicity ratios (ITRs) were then cal culated from these data (Table 9). PBO acts as an inhibitor of MFO (Brattsten and Metcalf, 1973) and, therefore, 108 antagonizes the toxic effect of parathion by preventing for mation of paraoxon. It can be assumed that ITR values are therefore a good indicator of insect MFO activity or utili zation of MFO to metabolize a toxic compound. Generally ITR values below 0.5 would indicate fairly active MFO systems for activation of phosphorothioate compounds. However, it should be noted that the MFO system also acts to detoxify compounds such as parathion by hydroxylation of the ethyl groups and the aromatic ring moieties. The summer forms of Chaoborus sp. and C. riparius had low ITR values and were the most sensitive summer forms to parathion. This would indicate that these species have ex tremely active MFO systems and would be susceptible to any xenobiotics that require activation by MFO. All winter forms had similar ITR values with the excep tion of Chaoborus sp. which now had an ITR of 0.86. This would indicate that this species had a much reduced MFO activity in the winter and would be less sensitive to para thion. In fact, it was the least sensitive winter species to parathion and exhibited the greatest difference in toxi city between its summer and winter forms (Table 7). C. riparius exhibited little change in the ITR value of its "winter" form and was therefore still quite sensitive to parathion. 109 Allocapnia sp. , which was the most sensitive species to parathion, exhibited the lowest ITR of all species (0.10). Interactive toxicity ratios for paraoxon were all close to unity (Table 10). This indicates that the MFO system has little effect on the toxicity of paraoxon and therefore may not greatly affect its metabolism. Correlation of ITR values of all species in the summer with summer toxicity data resulted in a correlation coeffi cient (r) of 0.78 for parathion and -0.81 for parathion with PBO. This reversal of r, in effect the slope of the regres sion line, is due to the close relationship of ITR and MFO activity. Parathion with PBO was always less toxic than parathion alone and, therefore, the closer the ITR values were to unity (less MFO activity) the less difference exis ted between EC50 values for parathion and parathion with PBO. The largest difference existed with C. riparius and Chaoborus sp. which were at the lower end of the curve (lowest ITR values). The extreme shift in toxicity of these two species from low ITR values and low EC50 values with parathion alone to low ITR values and relatively high EC50 with parathion and PBO resulted in a shift of the regression line to a negative correlation despite little change in the results obtained with the other species. Correlation of winter form toxicity and ITR values was much different. ITR and parathion toxicity resulted in an 110 r of 0.96 and parathion with PBO resulted in r = 0.91. MFO was less active in the winter and although there was a wider numerical range between EC50 values for parathion and para thion with PBO, the relative range was not as great as in the summer and the difference within each species was more uniform. Interactive toxicity ratio for parathion did not cor relate well with summer respiration rates (r = 0.54). This is due to the greater variation of ITR values and respira tion rates in the summer species. However, the same com parison among winter species resulted in r = -0.75. It appears that as respiration increases, the ITR decreases. Apparently the MFO becomes more active with increasing res piration rate among winter species. The poor correlation in the summer forms due to greater variation of data may also be due to higher temperatures resulting in greater variation of other biochemical systems that affect penetra tion, metabolism and excretion. Winter respiration correlated to winter toxicity re sulted in r = -0.72 for parathion and r = -0.81 for para thion with PBO. This indicates that increased respiration among species results in increased toxicity. As already noted, increased respiration also resulted in increased MFO activity which would increase toxicity. Ill However, in comparing summer form respiration to toxi city of parathion a positive correlation exists (r = 0.072). This indicates that increased respiration results in de creased toxicity. The higher temperatures in the summer and the more active state of the insects tested may result in more rapid metabolism of paraoxon and parathion which would negate the more rapid conversion of parathion to para oxon by MFO. No correlation existed between respiration and parathion and PBO in summer forms (r = -0.16). This was due to the great decrease in toxicity to C. riparius and Chaoborus sp. when PBO was present to inhibit their MFO systems. Correlation of respiration data with paraoxon toxi city was very poor (winter r = -0.39 and summer r = -0.20). These poor correlations were probably due to the great vari ation in paraoxon penetration due to its high polarity as well as other metabolic systems. Acetylcholinesterase Activity Data for substrate concentration vs. acetylcholines terase activity rates were very similar between winter and summer forms (Figure 1). This would indicate that the same enzymes were present and active in both winter and summer forms. In addition, acetylcholinesterase activity rates were high over a wide range of temperatures (Figure 2) and 112 were similar between winter and summer forms of each species. This would indicate that the acetylcholinesterase of these species was capable of immediate rate compensation to tem perature. This was also indicated by the values for acetylcholinesterase activity of the winter and summer forms (Table 12). The values were very close to unity (1.3 to 1.6), which is associated with immediate rate compensa tion (Hochachka and Somero, 1973). The multi-voltine species, S. femoratum and Chaoborus sp. had the greatest increase in acetylcholinesterase acti vity rate from winter to summer forms (Table 11). In addi tion they exhibited interseasona1 Q^q values for acetyl cholinesterase activity rates near 2 (1.8 and 2.0). The fact that these species emerge throughout the warm months makes, this period very important to their life cycles. They are not dependent on one large emergence such as the uni- voltine species. The winter period is not crucial for growth, particularly for Chaoborus sp. which can go through a complete life cycle within 30 days during the warm months. Therefore, overall activity can be greatly reduced during the colder months when food may not be as abundant as during the warm months. These organisms would need a higher acetyl cholinesterase activity rate during the warm months to main tain high activity and lower rates would be acceptable 113 during the winter. Compensation to temperature is not cru cial and these species therefore exhibit a seasonal Q^q close to the van't Hoff effect. The uni-voltine Trichopterans, and especially S. vicarium, are more dependent than other species on winter growth in preparation for an early spring emergence. They therefore maintain a constant acetylcholinesterase activity rate between summer and winter which would allow them to maintain activity high enough for feeding during the winter. This was also observed with the respiration rates of S. vicarium. This species was active throughout the winter and increasingly larger instars were observed as spring ap proached. The Trichopterans also exhibited a constant acetylcholinesterase activity rate from winter to summer. However, these species fed actively throughout the warm months but withdrew their filtering nets during the winter when food was less abundant. They were much larger in the winter than in the summer and may have been converting their large fat stores into growth for spring emergence. The uni-voltine species exhibited interseasonal Q^q values for acetylcholinesterase near unity (1.0 and 1.2). This would be expected for a species that maintains a con stant activity rate. 114 Acetylcholinesterase Inhibition Paraoxon is the activated form of parathion and, therefore, the main inhibitor of acetylcholinesterase. Parathion was found to be a very poor cholinesterase in hibitor compared to paraoxon despite the fact that parathion was several times more toxic to the insects tested than was paraoxon due to its greater ability to penetrate the integu ment. The inhibition experiments were performed in vitro and, therefore, penetration of the integument and other metabolic systems was not present in these experiments. There was no correlation between the toxicity data and the inhibition data. This result is similar to what Lovell found in mammals (1963) and Morrallo and Sherman with flies (1967) . However, there was correlation between summer respira tion at 22°C and summer cholinesterase inhibition (r = 0.97), summer respiration and summer cholinesterase activity (r = -0.71) as well as summer cholinesterase activity and summer inhibition (r = -0.82). The correlation between cholines terase activity and inhibition seems logical. However, the cholinesterase activity and inhibition experiments were done in vitro and it is difficult to explain why they should cor relate with the in vivo respiration experiments. Also, these same correlations did not exist with the winter data. The correlation between the inhibition data and the 115 cholinesterase activity data may be real in that when choli- nesterase is most active in breaking down the substrate, it would also be most vulnerable to combining with the para- oxon which competes directly with the substrate. The result would be lower 150 values associated with higher cholines- terase activity rates. Therefore, it would require less paraoxon to achieve 501 inhibition within a fixed time period when the acetylcholinesterase was more active rather than less active. Although the correlation between winter cholinesterase activity and winter inhibition data was not as good (r = -0.63), general cholinesterase activity was lower in the winter and may have resulted .in the poorer correlation. Allocapnia sp. had the most sensitive acetylcholines terase by an order of magnitude over the other species. It also had the most active MFO, was most sensitive to the toxicant and had the highest respiration rate. Plecopteran species are well known to be extremely sensitive to pollu tion (Hart and Fuller, 1974). These findings concur with that general observation. Overall, there was little difference in the inhibition of the cholinesterase by paraoxon between winter and summer forms of each of the species tested. This adds to the con clusion that the same enzymes were present in both seasonal forms. 116 In vivo Inhibition of Acetylcholinesterase in Chaoborus sp. Inhibition of the acetylcholinesterase occurred ra pidly in the summer form of Chaoborus sp. when exposed to high concentrations of parathion (100 ug/1) (Table 14). High levels of inhibition were required for the organisms to be visibly affected (>80%) and levels greater than 90% were associated with death. These organisms apparently have a surplus of acetylcholinesterase. It is also apparent that some accumulation of parathion occurred after a short exposure (1 hour) to the toxicant which acted during the following 24 hours of exposure. Other investigators have also noted the need for high levels of inhibition in order to achieve visible results (Metcalf et al, 1949; Chamberlain et al, 1951; and Bigley, 1966). At a lower concentration (1 ug/1) of parathion, Chaoborus sp. exhibited a very gradual increase in inhibi tion over a 24 hour period with the greatest increase in inhibition of the enzyme occurring during the second 24 hour period (Table 14). Approximately 90% inhibition was still associated with visible impairment of the organism, however after 48 hours, one group of organism still exhibited less than 20% inhibition of the enzyme and had no increase in inhibition during the second 24 hours. There appears to be some organisms in the population that can cope with low 117 concentrations of the toxicant. This may occur through re duced penetration or alteration in their metabolic scheme and may be hereditary in nature. Regeneration of Acetylcholinesterase Parathion/paraoxon is considered to be an irreversible inhibitor due to its permanent bonding with the acetyl cholinesterase molecule. Experiments were conducted to determine the rate of cholinesterase regeneration in the summer form of Chaoborus sp. The enzyme apparently regen erates within 5 days at initial inhibition levels of ap proximately 20o. These experiments were conducted without feeding the organisms. The addition of food may expedite the regenerative process. These results would tend to indicate that Chaoborus sp. could experience accumulative reduction in acetylcholi nesterase activity if organophosphate pesticides were intro duced into their environment sporadically over a short period of time (several days or weeks). CONCLUSIONS Whereas, the overall basal respiration of the insects tested indicates the need for some seasonal acclima tization, there is some ability of the insects tested to compensate immediately to sudden temperature changes. Parathion is significantly less toxic in winter forms of aquatic insects at 4°C than in summer forms at 22°C. This information would support the use of toxi city data acquired at warmer temperatures as the basis for establishing water quality criteria for parathion and other organophosphate insecticides. It appears that the insects tested would be more susceptible to run-off of organophosphate insecticides during the warmer months. Paraoxon is less toxic in winter forms of aquatic insects at 4°C than in summer forms at 22°C, but sea sonal toxicity differences are less marked for paraoxon than for parathion. Parathion is more toxic than paraoxon in summer forms at 22°C, but in most winter forms, parathion and para oxon are approximately equitoxic at 4°C. Conclusions numbers 3 and 4 suggest that any environmental trans formation of parathion to paraoxon prior to exposure of the insects would be beneficial to aquatic insects. The high polarity and thus high water solubility of paraoxon appears to reduce toxicity by impeding pene tration of the insect integument in aquatic species. This results in lower toxicity than the more lipid soluble parathion. Development of high polarity pes ticides that would be effective on terrestrial insect pests but relatively non-toxic to aquatic organisms might be desirable in the future. Oxidative metabolism as reflected by interactive toxi city ratios correlated directly with toxicity of para thion in both summer and winter forms of the insects tested. However, oxidative metabolism appeared to have little effect on paraoxon toxicity and thus meta bolism. These data suggest that any cocontaminant in natural waters that reduce the oxidative metabolism of parathion would benefit the aquatic organisms. Increased respiration results in increased oxidative metabolism and increased toxicity of parathion among winter forms of the insects tested. However, in creased respiration in summer forms results in de creased toxicity of parathion probably due to increased paraoxon metabolism as a result of higher temperatures. 120 Respiration, oxidative metabolism and toxicity in crease within a species from winter (4°C) to summer (22°C) forms. This is related directly to temperature, however other physiological effects associated with life cycles also are important. 8) The acetylcholinesterase activity of the insects tested exhibited an immediate rate compensation to temperature changes. However, the uni-voltine species appeared to make substantial changes in their cholines- terase in order to maintain constant activity rates between seasons. The multi-voltine species appeared to adjust their cholinesterase activity to higher levels in the summer. However, there was little dif ference in inhibition of the enzyme by paraoxon be tween winter and summer forms. 9) High levels of acetylcholinesterase inhibition (>80%) must occur before insects exposed to parathion are noticeably affected. 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