Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations
1984 The comparative effects of three toxic substances on bluegill behavior Mary Gerard Henry Iowa State University
Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Ecology and Evolutionary Biology Commons, and the Environmental Sciences Commons
Recommended Citation Henry, Mary Gerard, "The ompc arative effects of three toxic substances on bluegill behavior " (1984). Retrospective Theses and Dissertations. 8996. https://lib.dr.iastate.edu/rtd/8996
This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. INFORMATION TO USERS
This reproduction was made from a copy of a document sent to us for microfilming. While the most advanced technology has been used to photograph and reproduce this document, the quality of the reproduction is heavily dependent upon the quality of the material submitted.
The following explanation of techniques is provided to help clarify markings or notations which may appear on this reproduction.
1.The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting through an image and duplicating adjacent pages to assure complete continuity.
2. When an image on the film is obliterated with a round black mark, it is an indication of either blurred copy because of movement during exposure, duplicate copy, or copyrighted materials that should not have been filmed. For blun 3d pages, a good image of the page can be found in the adjacent frame. If copyrighted materials were deleted, a target note will appear listing the pages in the adjacent frame.
3. When a map, drawing or chart, etc., is part of the material being photographed, a definite method of "sectioning" the material has been followed. It is customary to begin filming at the upper left hand comer of a large sheet and to continue from left to right in equal sections with small overlaps. If necessary, sectioning is continued again—beginning below the first row and continuing on until complete.
4. For illustrations that cannot be satisfactorily reproduced by xerographic means, photographic prints can be purchased at additional cost and inserted into your xerographic copy. These prints are available upon request from the Dissertations Customer Services Department.
5. Some pages in any document may have indistinct print. In all cases the best available copy has been filmed.
Universi^ MicrxSllms International 300 N. Zeeb Road Ann Arbor, Ml 48106
8423641
Henry, Mary Gerard
THE COMPARATIVE EFFECTS OF THREE TOXIC SUBSTANCES ON BLUEGILL BEHAVIOR
Iowa State University PH.D. 1984
University Microfilms
I nternstionsil 300 N. zeeb Road, Ann Arbor, Ml 48106
PLEASE NOTE:
In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V .
1. Glossy photographs or pages
2. Colored illustrations, paper or print
3. Photographs with dark background
4. Illustrations are poor copy
5. Pages with black marks, not original copy
6. Print shows through as there is text on both sides of page
7. Indistinct, broken or small print on several pages t/
8. Print exceeds margin requirements
9. Tightly bound copy with print lost in spine
10. Computer printout pages with indistinct print
11. Page(s) lacking when material received, and not available from school or author.
12. Page(s) seem to be missing in numbering only as text follows.
13. Two pages numbered . Text follows.
14. Curling and wrinkled pages
15. Other
University Microfilms International
The comparative effects of
three toxic substances
on bluegill behavior
by
Mary Gerard Henry
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of the
Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Major: Animal Ecology
Approved:
Signature was redacted for privacy.
Signature was redacted for privacy.
Signature was redacted for privacy.
For the Graduate College
Iowa State University Ames, Iowa
1984 ii
TABLE OF CONTENTS
Page
GENERAL INTRODUCTION 1
REFERENCES 4
EXPLANATION OF DISSERTATION FORMAT 5
SECTION I. BEHAVIORAL EFFECTS OF METHYL FARATHION 6 ON SOCIAL GROUPS OF BLUEGILL, LEPOMIS MACROCEIRUS
INTRODUCTION 7
MATERIALS AND METHODS 11
RESULTS AND DISCUSSION 19
REFERENCES 29
SECTION II. BEHAVIORAL EFFECTS OF COPPER ON 32 BLUEGILL, LEPOMIS MACROCHIRUS
INTRODUCTION 33
MATERIALS AND METHODS 35
RESULTS AND DISCUSSION 42
REFERENCES 55
SECTION III. BEHAVIORAL EFFECTS OF CHLORDANE ON 58 HIERARCHIES OF BLUEGILL, LEPOMIS MACROCHIRUS
INTRODUCTION 59
MATERIALS AND METHODS 60
RESULTS AND DISCUSSION 67
REFERENCES 76 iii
GENERAL SUMMARY 78
REFERENCES 83
ACKNOWLEDGEMENTS 84 1
GENERAL INTRODUCTION
Over the last decade, aquatic toxicologists have been searching
for new methods which embody the best features of acute lethality and
chronic sublethal toxicity tests, i.e. new tests that are short-term,
inexpensive, sensitive to low levels of contaminants, and provide
standardized protocols. Although many interesting approaches have
recently been developed, few have reached the standardization phase
and many produce results which are difficult to apply or extrapolate
to field situations. The current bottleneck in aquatic toxicology is
thus one of determining the ecological significance of an effect.
Can we first verify laboratory effects in the field, and secondly,
what do the results mean to the survival and reproductive capability
of the population?
This research was conceived in 1976 to address, in small part,
these issues. It was the original intent of this author to employ
behavioral changes as indices of toxicant effect at sublethal levels
of exposure. The underlying rationale to such an approach was
threefold: 1) behavior is the organismal level manifestation of a
physiological, biochemical, environmental influenced and motivational
state of an organism; 2) Selye (1974) indicated that behavioral
changes were very early warning indicators of diseased states in mammals, including humans, therefore demonstrated behavioral
sensitivity; 3} alterations in behavior due to contaminants might
provide more meaningful bridges for ecological interpretation. For 2
example, intensified aggression due to a toxicant could impact
survival and reproduction if social organization was degraded.
Few behavioral toxicology bioassays had been developed up to
1975. Most of the few tests conducted failed to address the
ecological/life history characteristics of the test animal. Sparks
et al. (1972) did recognize the dominant-subordinate hierarchy forming tendencies of bluegill, Lepomis macrochirus. but far from standardized an approach. Consequently, copious preliminary considerations had to be addressed in this author's research before standardization and testing could be completed. Optimal bluegill stocking density, the utility of plant cover, density and type of plant cover, feeding regime, photoperiod, fish size, etc. had to be determined. In addition, ground work had to be done to develop a bluegill ethogram, or inventory of behaviors, from which representative behaviors could be selected for monitoring under treated conditions. Recording devices, individual identification methods, and optimal observation period length were also developed prior to testing.
The first complete test was conducted in 1977-1978, using a cadmium and zinc mixture as the toxicant. These test results were published in 1979 (Henry and Atchison 1979a, 1979b). As with many research projects, a successful experiment raises more questions than it answers. Could this test be replicated; was it applicable across a range of chemically distinct compounds; could it be further standardized and streamlined? It was the purpose of the present 3
research effort to answer these questions. By using three compounds
belonging to different chemical families characterized by distinct
toxic modes of action, the general applicability of this approach
could be evaluated. Additionally, by using three different toxicants
the organismal level adaptive limitations of bluegill groups could be assessed.
The range of behaviors that have been monitored in this study attempt to address three ethogram subdivisions: 1} the respiratory process; 2) body maintenance and nervous system function; and 3) social interactions. All of these relate either directly or indirectly to survival through maintenance of body function or social organization. The concentrations which have been seen to cause behavioral alterations have also been compared in this text to classical toxicity effect concentrations as well as to concentrations designated as safe for aquatic life by the EPA. These comparisons of classical endpoints to behavioral endpoints have aided in the evaluation of the sensitivity of this test method. 4
REFERENCES
Henry, M.G., and G.J. Atchison. 1979a. Behavioral changes in bluegill, Lepomis macrochirus. as indicators of sublethal effects of metals. Environmental Biology of Fishes 4(l):37-42.
Henry, M.G., and G.J. Atchison. 1979b. Influence of social rank on the behavior of bluegill, Lepomis macrochirus Rafinesoue. exposed to sublethal concentrations of cadmium and zinc. Journal of Fish Biology 15:309-315.
Selye, H. 1974. Stress without distress. The New American Library, Inc., New York, NY. 193 pp.
Sparks, R.E., W.T. Waller, and J. Cairns, Jr. 1972. Effects of shelters on the resistance of dominant and submissive bluegills (Lepomis macrochirus) to a lethal concentration of zinc. Journal of Fisheries Research Board of Canada 29:1356-1358. 5
EXPLANATION OF DISSERTATION FORMAT
This dissertation is arranged in three sections. Each section describes a pair of experiments using a specific chemical but the same behavioral toxicity test technique. Each section should stand alone and it is my intention to send each section to three different scientific journals for possible publication. An overall introduction and summary provide the reader with the rationale for the research and a brief comparison of results from each set of experiments. 6
SECTION I. BEHAVIORAL EFFECTS OF METHYL
PARATHION ON SOCIAL GROUPS
OF BLUEGILL, LEPOMIS MACROCHHUS 7
INTRODUCTION
Organophosphate (OP) insecticides replaced most organochlorine
insecticides after the DDT controversy of the late I960*s. Most of
these first generation OF compounds, still widely used, seemed to
pose less hazard to nontarget organisms because they are less
lipophilic (Khan 1977) and less persistent in aquatic environments
than are organochlorine compounds. Although compounds such as
parathion, malathion, and methyl parathion are chemically ephemeral
(Apperson et al. 1976; Mulla and Mian 1981), they are nevertheless
toxic to fish and aquatic invertebrates and may do damage at recommended application rates, both over short time periods and after repeated exposures during a spraying season.
In producing a toxic effect, organophosphate compounds inhibit cholinesterase at synaptic and neuromuscular junctions. Severe inhibition of this enzyme results in hyperactivity, convulsions, muscular spasms, tetany, and eventually death (Brown 1978).
Inhibition of cholinesterase has been detected in fish from the laboratory (Coppage 1971; Coppage et al. 1975; Goodman et al. 1979;
Klaverkamp and Hobden 1980) and field (Coppage and Braidech 1976) that have been exposed to both lethal and sublethal concentrations of
OP compounds. More information is needed to evaluate how short-term, sublethal exposures relate to the organism's ability to function in its natural environment. 8
Methyl parathion was used in this study for these reasons: it is
widely applied to forests and cropland; it has been detected in
bluegill (Lepomis macrochirus) collected from a lake after spraying
(Apperson et al. 1976) indicating some potential for bioaccumulation;
and it is toxicologically representative of first generation OP
insecticides that induce the types of nervous system disruptions
previously mentioned.
An evaluation of the ecological significance of any
contaminant's impact on fish requires the use of an organized and
complementary array of measurements that demonstrate, directly or
indirectly, the impacts of contaminants on survival and reproduction.
When such measurements are examined in the laboratory, documented
changes can provide direction for development of indices useful for
field verification of toxicant effects.
Standard acute lethality tests are useful in providing
information on concentrations producing mortality. However, acute
yet sublethal exposures are more likely to occur in nature when
organophosphates are of concern. Full life cycle chronic tests,
while helpful in assessing sublethal effects of toxicants on growth,
reproduction, and survival over a long period of time, are not
applicable to OF compounds like methyl parathion which do not persist
long enough in aquatic systems to span the life cycle of even
short-lived invertebrates that might be used in classical lab
bioassays. Consequently, sensitive yet short-term tests are needed to evaluate the acutely sublethal toxicity of methyl parathion. 9
A bluegill behavioral bioassay was used since it provides rapid
yet sensitive test results on biological effects of low-level
exposures and also relates toxicant-induced changes to intraspecific
fish interactions (Henry and Atchison 1979a, 1979b). Bluegill form
social hierarchies under laboratory conditions as well as in the
field during the spawning season. Contaminant induced changes in the
social organization of this species could affect survival and
reproduction of individuals in a natural population if associated
courtship, territoriality, aggression, feeding, and comfort movements
are disrupted.
Changes in both individual responses and responses of social
origin between conspecifics were monitored during this study. An
individual's behavior can reflect the state of its overall health
prior to and after toxicant administration, evidenced principally as respiratory disruptions and increased frequency of comfort movements.
Differential sensitivity to the toxicant due to social rank, reflected in aggressive behaviors, is indicative of effects at the
population level.
Behavioral changes associated with OP insecticides have only
been observed in a few cases. Loss of locomotor control (Rand 1977), avoidance (Kynard 1974), failure to select optimal temperatures
(Domanik and Zar 1978), decreased feeding, increased aggression, elevated numbers of performed comfort movements and respiratory disruptions (Bull and Mclnerney 1974) have been observed for a variety of species in connection with various OF compounds. 10
Selective prédation of gulf killfish (Fundulus grandis) on treated
prey (Farr 1977, 1978), disruption of social displays in Siamese
fighting fish (Betta splendens) (Welsh and Hanselka 1972), and
decreased thermal tolerance and activity in mosquitofish Gambusia
affinis (Johnson 1978) are the only documented behavioral changes
associated with methyl parathion toxicity.
The objectives of this study were to: 1) use a social
organization behavioral bioassay to evaluate the acutely sublethal
effects of methyl parathion on bluegill and 2) compare the
sensitivity of this approach to previously reported procedures
described in the literature for detecting changes in growth, reproduction, survival, or behavior of fish exposed to methyl
parathion. 11
MATERIALS AïïD METHODS
Forty age II bluegill (11.5 to 15.0 cm total length; 28.0 to
51.6 g) were raised and tested at the Columbia National Fisheries
Research Laboratory. Twenty fish were used in each of two
replications of this experiment. For each replication, five fish
were randomly assigned to one of four 295-liter flow-through glass
test tanks (Figure 1). Each tank was layered with gravel and planted
with Elodea sp. to provide fish with substrate and cover.
Fish were maintained throughout the study at 17°C, under a 16
hr/8 hr light-dark photoperiod, and were fed Purina trout chow (#6)
twice daily until satiation. Water quality characteristics (Table I) remained constant during and between replications. The sex of all individuals was determined by dissection at the end of the experiment. In 7 of 8 hierarchies, the most dominant individual was male and the most subordinate was female.
Each replication was composed of three phases: 1) acclimation period (1 to 2 wk), 2) control (96 hr), and 3) treatment (96 hr).
Acclimation was judged to be complete when all fish were feeding regularly and once hierarchies and territories had been established.
Ten behaviors (described briefly in Table II) were observed daily for each test population during control and treatment periods.
Two hr time blocks (1/2 hr/tank) were randomized and rotated through the 16 hr light portion of the photoperiod so that observations on day 1 of both control and treatment periods took place from 4-6 pm. 117 cm.
figure 1. 295 liter glass aquarium. 13
Table I. Characteristics of test water; means and standard deviations based on biweekly analyses (N=28), for both replicates.
Total Hardness (mg/L CaCO]) 273.3 +. 4.1
Alkalinity (mg/L CaCOg) 235.7 +_ 3.6 pH -7 .4 +_ 0.8
Temperature (°C) 16.5 +.1,3
Dissolved Oxygen (mg/L) 9.8 +_ 2.4 14
Table II. Brief descriptions of the ten behaviors monitored in this study.
CATEGORY OF BEHAVIOR DESCRIPTION
I. RESPIRATORY DISRUPTIONS
cough rapid, repeated opening and closing of mouth and opercular coverings, accompanied by partly extended fins
yawn singularly occurring maximal opening of mouth and opercular coverings, accompanied by hyperextension of all fins
II. COMFORT MOVEMENTS
"s"-jerk whole body flinch, moving sequentially from head to tail
partial jerk head or tail flinch
fin flick rapid, repeated extension and contraction of spines and rays of fins
burst swimming sudden, rapid, nondirected spurt of forward movement
chafe brush of the body against an inanimate object
III. AGGRESSIVE INTERACTIONS
threat flare of opercular coverings accompanied by slight intention movement toward conspecific
nudge contact of closed mouth to body between fish, recipient is not displaced or bitten.
nip bite 15
day 2 = 8-10 am, day 3 = 8-10 pm, day 4 = 12-2 pm. This rotated
observation schedule was incorporated into the experimental design so
that diurnal fluctuations in activity would not be confounded with
toxicant effect. Tank observation order was also randomized within
each 2 hr time block.
Behaviors were observed directly by only one investigator (M.
Henry, Columbia National Fisheries Research Laboratory, Columbia,
MO), and the frequencies of each behavior and the fish performing it
were recorded by hand, using codes, recording forms, and
abbreviations to facilitate the process. Individuals were
distinguished from one another on the basis of differences in color,
size, and natural markings. No tags, brands, or fin clips were used.
Fish readily formed hierarchies and were assigned numbers from
1-5, designating their rank within the hierarchy (1 = most dominant;
5 = most subordinate). The dominant fish initiated the greatest
number of aggressive acts (threats, nudges, or nips) during the
control period. In addition, territories were established by the top
one or two ranking individuals and territorial boundaries were
regularly defended against interlopers.
A stock solution of [1^~C] methyl parathion was mixed in 0.5%
acetone in water and administered to treatment tanks with two micromedic pumps and a modified proportional diluter system (Mount and Brungs 1967). Acetone alone was added to the control tank to serve as a check on solvent effect. 16
A 10% geometric progression decreasing from a level approaching
one tenth of the 96 hr LC50 for bluegill exposed to methyl parathion
(4.38 mg/L) (Johnson and Finley 1980) was used to establish three
test concentrations (Table III). For each replicate, treatments and
fish were randomly assigned to tanks.
The [14-c] methyl parathion (MW 263.23; specific activity 27.98
mCi/mM) used was from Pathfinders Laboratories, Inc., St. Louis, MO.
Labeled material was combined with unlabeled methyl parathion
(obtained from Monsanto, St. Louis, MO) (77% active) for addition to
treatment tanks. The stock solution concentration was analyzed by
liquid scintillation but was also confirmed using gas-liquid
chromatography (GLC) (Derek et al. 1981) so that the calculated
proportion of radioactivity to total methyl parathion could be
substantiated. Thereafter, liquid scintillation was used to monitor
the buildup of the toxicant. Tanks reached their full concentrations
after approximately 8 hr (x flow rate = 1790 ml per 5 min per tank;
N=8). Water samples were prepared for scintillation counting by combining, in vials, 1 ml water with 4 ml scintillation cocktail
(Triton X and PBBO). Due to the low numbers of counts above background in the lowest concentration, water from that treatment tank was first partitioned using ethyl acetate (Nuclear-Chicago Corp.
1967) then combined with scintillation cocktail for improved counting efficiency. Following collection and preparation, all samples were immediately placed in a Beckman LS-3313T liquid scintillation counter. Scintillation counts were recorded as counts per minute 17
Table III. Nominal and mean actual methyl parathion concentrations for each treatment tank (N=4).
Nominal Concentrations (mg/L) Mean Actual Concentrations (mg/L)
0.35 High 0.31+0.002
0.035 Medium 0.03 +0.0014
0.0035 Low 0.003 +0.0001
0.00 Control 0.00 (No toxicant)
96 hr LC50 = 4.38 mg/L (Johnson and Finley 1980) 18
(cpm) and corrected for quench by subtracting the mean number of cpms
for blanks counted from each sample mean. Values were then converted
to concentrations after being compared to a constructed quench curve
which corrected for counting efficiency by giving a % recovery value
for each sample. Eight 5 minute counts were taken for each sample
over a period of 5 days in.order to get stable and persistent readings. Intermittent GLC analyses of water samples were conducted during the treatment period. Results of these analyses confirmed that methyl parathion was indeed present in desired concentrations and that 86-90% of the material persisted in the form of methyl parathion and not degradation products or metabolites such as methyl paraoxon.
Behavioral data were analyzed by three-way analysis of variance
(ANOVA) and Least Significant Difference (LSD) determinations. The
ANOVA tested for significance due to treatment, rank, and duration of exposure, along with all possible combinations of interactions between those three main factors. LSD values allowed for comparisons to be made for each behavior between treatment levels and fish of different social rank. Note, however, that since social rank was determined by using the sum of aggressive acts initiated by an individual during the control period, rank is confounded with the base-line frequencies of threats, nudges, and nips in the statistical model. 19
RESULTS AND DISCUSSION
The results of this study were analyzed from two perspectives:
1) population responses (total behavioral acts per tank) and 2) individual responses (total behavioral acts per fish reflecting differential sensitivity due to social position in the hierarchy).
Throughout the study, the form and posture of each behavior remained unchanged but the frequency of occurrence was altered with the addition of methyl parathion.
The experimental design of this study employed two types of controls: one for treatment effect (control vs. treatment period for each tank) and one for time effect (a control tank that received no methyl parathion during the treatment period). No statistically significant differences in behavioral frequencies were found between populations during the control period. This supports the supposition that when healthy fish of similar size are used, replication of experimental units (i.e., hierarchies) is possible.
Frequencies of behaviors in control tank populations during the treatment period were not significantly different from control period values for all tanks, indicating no time effect. Consequently, all control data (control period values for all tanks and control tank values during the treatment period) were pooled to represent baseline levels of response. 20
Hyperactivity was the most noticeable overall effect of methyl
parathion. This response, defined as almost continuous swimming
coupled with numerous s-jerks, partial jerks, and fin flicks,
occurred in fish exposed to all toxicant concentrations within 10-14
hr of toxicant addition.
With the exception of chafes, all behaviors within the category
of comfort movements showed significant changes in frequency after
methyl parathion was added (Table IV). Each concentration of methyl
parathion produced significantly different frequencies of s-jerks
and fin flicks, thus indicating that these behaviors provided good resolution of response between discrete and increasing
concentrations of the toxicant (Figure 2 and Table IV). Partial jerks were similarly influenced by different treatment levels
(P Hyperactivity provided the most sensitive population level reflection of methyl parathion effect. Hyperactivity is a principal sign of central nervous system disruption due to OP poisoning (Matsumura 1975) reflecting the physiological and biochemical toxic mode of action of these compounds. S-jerks appear to be nervous system disruptions without social connotation or maintenance value. Fin flicks have been associated with freeing fins of debris and also 21 Table IV. Mean population frequencies of 10 behaviors examined over three toxicant concentrations and a control during the treatment period. X METHYL PARATHION LSD ANOVA CONCENTRATIONS (mg/L) P < 0.05*/ control 0.003 0.03 0.3 0.01** vs. treatment RESPIRATORY (within tank) DISRUPTIONS P > 0.05*/0.01** cough 19.0 60.0 29.5 50.0 9.4/12.8 0.0001** (41.0**) (30.5**) (20.5**) yawn 7.0 17.5 8.5 6.5 4.0/5.2 0.01** (10.5**) (9.0**) (2.0) COMFORT MOVEMENTS s-jerks 12.5 23.5 47.0 82.5 8.4/11.6 0.0001** (11.0*) (23.5**) (35.5**) partial 3.0 24.5 15.5 28.0 8.2/11.0 0.0001** jerk (21.5**) (9.0*) (12.5**) fin 18.5 64.0 50.0 73.5 12.0/17.0 0.0001** flick (45.5**) (14.0*) (23.5**) burst 2.5 2.5 0.5 14.5 5.0/7.0 0.02* swimming (0.0) (2.0) (14.0**) chafe 1.0 0.0 0.5 0.0 1.2/1.8 0.7 NS (1.0) (0.5) (0.5) AGGRESSION threat 68.0 51.0 28.5 38.5 14.0/19.2 0.0001** (17.0*) (22.5**) (10.0) nudge 8.0 1.5 1.5 11.5 17.8/24.6 0.8 NS (6.5) (0.0) (10.0) nip 39.0 20.5 12.5 28.5 44.6/61.4 0.0001** (18.5) (8.0) (16.0) 22 COUGHS ] J 1 PUTWl JtRlQ in nais HCH ammtiui MEDIUM CONSHTMIDN lOW CONCENIRMON HO WSKMII 10 •V 2 tHRCAIS J 1 NIPS » I! n H 2t <£ s » « 77 a a n r, 34 CONTROL PFBOr. WHTMCNI PrPO? rpvTuni gcRio; TPHIMCKT oromn Figure 2. Mean frequencies of six behaviors monitored in four treatment populations (x = 2 replications). 23 for social displays during spawning (Noakes and Leatherland 1977). Miller (1963) noted that fin flicks in bluegill occur more frequently in situations of environmental or social stress. Such behavioral changes induced by methyl parathion can be detected rapidly, at low concentrations and in all members of the population thus providing a less time-consuming and less expensive alternative to sublethal toxicity assessment. The increased frequencies of s-jerks, partial jerks, and fin flicks occurred principally during the first 2 days of toxicant administration (Figure 2) and plateaued or declined during the last half of the treatment period. Concomitantly, as initial activity levels increased, aggressive behaviors generally increased and then followed the same pattern of decline (Figure 2). Increased activity increased the number of times fish were in proximity to one another; therefore, it is not unexpected that increased aggression would ensue. The aggressive behaviors that most clearly reflected toxicant effect on populations were threats and nips (P = 0.0001; Table IV). However, neither behavior consistently reflected differences between treatments as clearly as the comfort movements previously mentioned. The control levels of aggressive behaviors are also slightly more variable between populations than the control frequencies of comfort movements and respiratory disruptions (Figure 2). Both coughs and yawns were significantly (P = 0.0001 and 0.01, respectively) affected by the toxicant at the populations level (Table IV) and when trends 24 are examined (Figure 2). Significant differences in yawns were observed between the adjacent control and low levels, low and medium, and low and high concentrations. Coughs have been associated with clearing gill surfaces of particulate matter or mucus that forms in response to an irritant (Cairns et al. 1982) while yawns seem to be a more extreme attempt to do the same. Miller (1963) suggested that yawning can also be indicative of stress. In terms of response of individual fish, the most striking result was that behavioral responses of subordinate (5) and dominant (1) fish are more significantly affected over all concentrations than intermediately ranked bluegill (Figure 3). This differential response related to rank illustrates the importance of considering life history aspects of a test species, such as hierarchy formation in bluegill, when conducting toxicity tests. Several authors have suggested (Noakes and Leatherland 1977; Schreck and Lorz 1978) that, in a hierarchy, the dominant and subordinate individuals are under the greatest amount of social stress. The dominant expends effort to maintain its position while the subordinate is occupied trying to avoid attacks from other fish. The present study substantiates this idea by demonstrating the combined effect of social stress and toxicant stress through differential behavioral responses among hierarchy members. If the three most sensitive comfort movement behaviors (s-jerks, fin flicks, and partial jerks) are examined with respect to distribution of response among individuals in each population, the 25 COUCHS i T FEH 1 FSH 2 FBH 3 FBH 4 FEH 5 THREAIS NIPS i (lOÛÏ OCC'S J CONCENIRIION imyi! Î CONCfNîilMON (mz/ll Figure 3. Mean frequencies of six behaviors performed by individuals of differing social rank during the treatment period (x = 2 replications). 26 subordinate in each tank performed more s-jerks and fin flicks under control conditions. Once methyl parathion was added, significant (P = 0.0001) increases were observed in both dominant and subordinate fish of each treatment group. Partial jerks were performed most frequently by the 4th and 5th ranking individuals while dominant fish in all concentrations performed this behavior least often but at levels still elevated above control levels of response. The #3 ranking fish appears to be least influenced by the toxicant if s-jerks, fin flicks, and partial jerks are evaluated. It was usually the least active individual in a tank and also had the fewest social interactions. However, heightened levels of response occurred for comfort movements when control period values were contrasted with treatment period values (Figure 3). The rapid yet sensitive nature of this behavioral bioassay is reflected in the number of responses seen within 24 hr of toxicant addition (Figure 2). In addition, the entire test was conducted in 16-23 days. Differential response due to rank was detected in all concentrations. The behavioral parameters measured in this study were influenced by an absolute methyl parathion concentration (0.003 mg/L) lower than most reported in the literature and a level representing <1% the 96 hr LC50. Mosquitofish, Gambusia affinis. showed a decreased level of activity after exposure to 0.005 mg/L for 48 hrs and decreased thermal tolerance after exposure to 0.005 and 0.01 mg/L methyl parathion for 24 hrs (Johnson 1978). Carbohydrate metabolism was affected at 5.6 mg/L over 96 hrs in Indian catfish. 27 Heteropaeustes fossilis (Srivastava and Singh 1981). The 5.6-mg/L exposure concentration represented 0.8 of the 96-h LC50 for Indian catfish exposed to methyl parathion. However, the ecological significance of such a biochemical change is difficult to interpret. Gulf kilifish, Fundulus grandis. selectively preyed on grass shrimp (Palaemonetes pugio) over sheepshead minnows (Cvprinodon variegatus) at exposures of 0.024, 0.119 and 0.475 pg/L methyl parathion for 24 hrs (Farr 1978). Siamese fighting fish (Betta splendens) failed to notice and display to male conspecifics at 1.0 mg/L after 5 days of exposure (Welsh and Hanselka 1972). This exposure concentration represented approximately one^half the 120-hr LC50 for Betta splendens exposed to methyl parathion. Behavioral parameters are obviously sensitive indicators of toxicant effect. It is necessary, however, to select behavioral indices for monitoring that relate to the organism's behavior in the field in order to derive a more accurate assessment of the hazards a contaminant may pose in natural systems. If social hierarchies have developed evolutionarily as a mechanism to efficiently ration limited resources (food, mates, spawning sites, etc.) among individuals of a population (Krebs and Davies 1978) then disruption of such a system by a toxicant could affect survival and reproduction within a population under natural conditions. Therefore, rank-related responses should be considered for species forming social organizations. If social interactions are not considered, only a certain portion of a population may be protected, and the toxicity of 28 the contaminant may be underestimated. More work is needed in the field to verify hierarchy related effects seen in the laboratory. Additional effort regarding impacts on reproduction as it relates to rank is also imperative if population maintenance in the presence of contaminants is to be assessed. 29 REFERENCES Appersoii, C.S., R. Elston and W. Castle. 1976. Biological effects and persistence of methyl parathion in Clear Lake, California. Environmental Entomology 5:1116-1120. Brown, A..W.A. 1978. Ecology of pesticides. John Wiley and Sons, Inc., New York, N.Y. 525 pp. Bull, C.J., and J.E. Mclnerney. 1974. Behavior of juvenile coho salmon (Oncorhvnchus kisutch) exposed to sumithion (fenitrothion), an organophosphate insecticide. Journal of Fisheries Research Board of Canada 31:1867-1872. Cairns, M.A., R.R. Carton, and R.A. Tubb. 1982. Use of fish ventilation frequency to estimate chronically safe toxicant concentrations. Transactions of the American Fisheries Society 111:70-77. Coppage, D.L. 1971. Characterization of fish brain acetylcholin esterase with automated pH stat for inhibition studies. Bulletin of Environmental Contamination and Toxicology 6:304-310. Coppage, D.L., E. Matthews, G.H. Cook, and J. Knight. 1975. Brain acetylcholinesterase inhibition in fish as a diagnosis of environmental poisoning by malathion, o,o-dimethyls- (1,2,-dicarbethoxyethyl) phosphorodithioate. Pesticide Biochemistry and Physiology 5:536-542. Coppage, D.L., and T. Braidech. 1976. River pollution by anticholinesterase agents. Water Research 10:19-24. Derek, C., G. Muir, N.P. Grift, and J. Solomon. 1981. Extraction and cleanup of fish, sediment and water for determination of triaryl phosphates by gas-liquid chromatography. Journal of the Association of Analytical Chemistry 64:79-84. Domanik, A.M., and J.H. Zar. 1978. The effect of malathion on the temperature selection response of the common shiner, Notropis cornutus (Mitchill). Archives of Environmental Contamination and Toxicology 7:193-206. Farr, J.A. 1977. Impairment of antipredator behavior in Palmonetes pugio by exposure to sublethal doses of parathion. Transactions of the American Fisheries Society 106:287-290. 30 Farr, J.A. 1978. The effect of methyl parathion on predator choice of two estuarine prey species. Transactions of the American Fisheries Society 107:87-91. Goodman, L.R., D.J. Hansen, D.L. Coppage, J.C. Moore, and E. Matthews. 1979. Diazinon chronic toxicity to, and brain acetylcholinesterase inhibition in, the sheepshead minnow, Cvprinodonvariegatus. Transactions of the American Fisheries Society 108:479-488. Henry, M.G., and G.J. Atchison. 1979a. Behavioral changes in bluegill (Lepomis macrochirus) as indicators of sublethal effects of metals. Environmental Biology of Fishes 4:37-42. Henry, M.G., and G.J. Atchison. 1979b. Influence of social rank on the behavior of bluegill, Lepomis macrochirus Rafinesque, exposed to sublethal concentrations of cadmium and zinc. Journal of Fish Biology 15:309-315. Johnson, C.R. 1978. The effects of sublethal concentrations of five organophosphorus insecticides on temperature tolerance, reflexes and orientation in Gambusia affinis affinis (Pisces:Poeciliidae). Zoological Journal of the Linnean Society 64:63-70. Johnson, W.W., and M.T. Finley. 1980. Handbook of acute toxicity of chemicals to fish and aquatic invertebrates. U.S. Department of the Interior Fish and Wildlife Service Resource Publication 137. 98 pp. Khan, M.A.Q. 1977. Pesticides in aquatic environments. Plenum Press, New York, N.Y. 257 pp. Klaverkamp, J.F., and B.R. Hobden. 1980. Brain acetylcholinesterase inhibition and hepatic activation of acephate and fenitrothion in rainbow trout (Salmo gairdneri). Canadian Journal of Fisheries and Aquatic Sciences 371:1450-1453. Krebs, J.R. and, N.B. Davies. 1978. Behavioral Ecology: an evolutionary approach. Sinauer Associates, Inc., Sunderland, MA. 494 pp. Kynard, B. 1974. Avoidance behavior of insecticide susceptible and resistant populations of mosquitofish to four insecticides. Transactions of the American Fisheries Society 103:557-561. Matsumura, F. 1975. Toxicology of Insecticides. Plenum Press, New York, N.Y. 503 pp. 31 Miller, H.C. 1963. The behavior of the pumpkinseed sunfish, Lepomis gibbosus (Linneaus), with notes on the behavior of other species of Lepomis and the pygmy sunfish, Elassoma evergladei. Behaviour 22:88-151. Mount, D.I., and W.A. Brungs. 1967. A simplified dosing apparatus for fish toxicology studies. Water Research 1:21-29. Mulla, M.S., and L.S. Mian. 1981. Biological and environmental impacts of the insecticides malathion and parathion on nontarget biota in aquatic ecosystems. Residue Review 78:101-143. Noakes, D.L.G., and J.F. Leatherland. 1977. Social dominance and interrenal cell activity in rainbow trout, Salmo gairdneri. Environmental Biology of Fishes 2:131-136. Nuclear-Chicago Corporation. 1967. Handbook for Preparation of Liquid Scintillation Samples. Searle & Co., Des Plaines, 111. 171 pp. Rand, G.M. 1977. The effects of exposure to a subacute concentration of paratbion on the general locomotor behavior of the goldfish. Bulletin of Environmental Contamination and Toxicology 18:259-266. Schreck, C.B., and H.W. Lorz. 1978. Stress response of coho salmon (Oncorhvnchus kisutch) elicited by cadmium and copper and potential use of Cortisol as an indicator of stress. Journal of Fisheries Research Board of Canada 35:1124-1129. Srivastava, A.K., and N.N. Singh. 1981. Effects of acute exposure of methyl parathion on carbohydate metabolism of Indian catfish (Heteropneustes fossilis). Acta Pharmacology and Toxicology 48:26-31. Welsh, M.J., and C.W. Hanselka. 1972. Toxicity and sublethal effects of methyl parathion on behavior of Siamese fighting fish (Betta splendens). Texas Journal of Science 23:520-529. 32 SECTION II. BEHAVIOHAL EFFECTS OF COPPER ON BLUEGILL, LEPOMIS MACROCEIRUS 33 INTRODUCTION Copper has frequently been detected in freshwater ecosystems- Environmental concentrations above 5 yg/L copper (Alabaster and Lloyd 1980) can often be linked to direct application of copper sulfate used as an pesticide for aquatic plants and mollusc control. Copper has also been associated with point and non-point industrial inputs, especially with mining operations. There is a significant amount of information available on the fate and chemical speciation of copper under both aquatic field and laboratory conditions (Nriagu 1979). Dominant chemical form(s) and subsequent toxicity associated with the Cu2+ and Cu(OH)+ forms (Pagenkopf et al. 1974) depend largely on hardness (Brungs et al. 1976; Howarth and Sprague 1978; Mount and Stephan 1969), pH (Waiwood and Beamish 1978), humic acid content and suspended solid concentration (Wiener and Giesy 1979). Its widespread occurrence and variable toxicity have led to concern regarding the impact of copper on fish and aquatic invertebrates and make water quality standard setting a more difficult task. Consequently, toxicity test approaches are still needed which enable investigators to rapidly evaluate the sublethal effects of copper under many different water quality conditions. The objectives of this study were to: 1) compare copper levels which induce behavioral changes in bluegill (Lepomis macrochirus) hierarchies to those sublethal concentrations known to change growth rate, reproduction and survival (Benoit 1975; Swigert and Morgan 34 1977; Thompson et al. 1980) and 2) contrast concentrations causing behavioral changes in bluegill with levels designated by the USEPA (1980) as safe for aquatic life. 35 MATERIALS AND METHODS Forty age II bluegill (12.8-15.0 cm total length; 35.6-62.3 g) were raised and tested at the Columbia National Fisheries Research Laboratory, Columbia, MO. Twenty fish were used in each of two replications of this experiment. For each replication, five fish were randomly assigned to one of four 295 liter flow-through glass test aquarium (Figure 1). Each tank was layered with gravel and planted with Elodea sp. to provide fish with substrate and cover. Fish were maintained throughout the study at 170C, under a 16 hr/8 hr light-dark photoperiod and were fed Purina trout chow (#6) until satiation twice daily. Water quality characteristics (x ^ SD; N = 28), analyzed biweekly remained constant both during and between replications (total hardness, 273.3 +_ 4.1 mg/L CaCOg; alkalinity, 235.7 i 3.6 mg/L CaCOg; pH, 7.4 +. 0.8; temperature, 16.5 +_ 1.3°C; dissolved oxygen 9.8 +. 2.4 mg/L). The sex of all individuals was determined by dissection at the end of the experiment. In 6 of 8 hierarchies, the most dominant individual was male while the most subordinate was female in 5 of 8 test populations. Each replication had three phases: (1) acclimation period (1-2 wk); (2) control period (96 hr); and (3) treatment period (96 hr). Acclimation was judged completed once all individuals were feeding regularly and all hierarchies and territories were established. 117 cm. figure 1. 295 liter glass aquarium. 37 Ten behaviors (described briefly in Table I) were observed daily for each test population during control and treatment periods. Two hr time blocks (1/2 hr/tank) were randomized and rotated through the 16 hr light portion of the photoperiod so that observations on day 1 of both control and treatment periods took place from 4-6 pm, day 2 = 8-10 am, day 3 = 8-10 pm, day 4 = 12-2 pm. This rotated observation schedule was incorporated into the experimental design so that diurnal fluctuations in activity would not be confounded with toxicant effect. The order of tanks observed was also randomized within each 2-hr time block. Behaviors were observed directly by only one investigator (M. Henry, Columbia National Fisheries Research Laboratory, Columbia, MO) and the frequencies of each behavior and the individual fish performing them were recorded by hand, using codes, recording forms and abbreviations to facilitate the process. Individuals were distinguished from one another on the basis of differences in color, size and natural markings. No tags, brands or fin clips were used. Fish readily formed hierarchies and were assigned numbers from 1-5, designating their rank within that hierarchy (1 = most dominant, 5 = most subordinate). Criteria used to determine dominance were the initiation of the greatest number of aggressive acts (threats, nudges or nips) during the control period. In addition, territories were established by the top one or two ranking individuals and territory boundaries were regularly defended against interlopers. 38 Table I. Brief descriptions of the ten behaviors monitored in this study. CATEGORY OF BEHAVIOR DESCRIPTION I. RESPIRATORY DISRUPTIONS cough rapid, repeated opening and closing of mouth and opercular coverings, accompanied by partly extended fins yawn singularly occurring maximal opening of mouth and opercular coverings, accompanied by hyperextension of all fins II. COMFORT MOVEMENTS "s"-jerk whole body flinch, moving sequentially from head to tail partial jerk head or tail flinch fin flick rapid, repeated extension and contraction of spines and rays of fins burst swimming sudden, rapid, nondirected spurt of forward movement chafe brush of the body against an inanimate object III. AGGRESSIVE INTERACTIONS threat flare of opercular coverings accompanied by slight intention movement toward conspecific nudge contact of closed mouth to body between fish, recipient is not displaced or bitten nip bite 39 Reagent grade anhydrous copper sulfate (CUSO4) was used as the copper source in the toxicant stock solution. It was mixed in doubly distilled water and administered to treatment tanks using two micromedic pumps and a modified proportional diluter (Mount and Brungs 1967). Toxicant concentrations in stock solutions and concentrations in test tanks were verified using atomic absorption-flame ionization spectrophotometry. The rationale behind selection of the three nominal concentrations was that they would approach the 96 hr LC50 (Johnson and Finley 1980) and upper and lower limits of the Maximum Acceptable Toxicant Concentration (MA.TC) for bluegill exposed to copper (Benoit 1975). Using these concentrations to evaluate effects on behavior would subsequently allow comparisons to be made with classical bioassay endpoints monitoring changes in survival, growth, and reproduction. Nominal and mean actual concentrations are summarized in Table II. Tanks reached full concentrations after approximately 7 hr (x flow rate = 1795 ml/5 min/tank; N = 8). Behavioral data were analyzed using analysis of variance (ANOVA) and Least Significant Difference (LSD) determinations. The ANOVA tested for significance due to treatment, social rank and time along with all possible combinations of interactions between those three main factors. LSD values allowed for comparisons of behaviors between treatment levels and also between fish of different rank. Note, however, that since social rank was determined using the sum of aggressive acts initiated by an individual during the control period, 40 Table II. Nominal and mean actual copper concentrations for each •treatment tank over two replications (N=4). Nominal Concentrations (mg/L) Mean Actual Concentrations (mg/L) 0.031 0.034 + 0.01 0.050 0.057 + 0.005 1.00 1.30 +0.002 0.00 0.002 + 0.002 41 rank is confounded in the statistical model with baseline frequencies of threats, nudges and nips. 42 RESULTS AND DISCUSSION The results of this study were analyzed from two perspectives: 1) population responses (total behavioral acts per tank) and 2) individual responses (total behavioral acts per fish reflecting differential sensitivity due to social position in the hierarchy). Throughout the study, the form and posture of each behavior remained unaltered but the frequency of occurrence did change once copper was added. The experimental design of this study employed two types of controls; one for treatment effect (control vs. treatment period for each tank) and one for time effect (a control tank that received no copper during the treatment period). No statistically significant differences in the frequencies of all behaviors were found between tank populations during the control period. Therefore, all control data (control period data for all tanks and treatment period data for the tank not receiving copper) were pooled and mean values represent normal levels of response in the absence of the toxicant (Table III). The most significant (P = 0.0001) (Table III) overall effect of copper was on respiration, as indicated by the increased frequency of respiratory disruptions. Coughs, described in Table I, increased ( P = 0.0001) in every treatment group once copper was introduced. Yawns also increased significantly (P = 0.0001) in all treatment tanks. The most dramatic increase in coughs and yawns occurred within the 43 Table III. Mean population frequencies of 10 behaviors examined over three toxicant concentrations and a control during the treatment period. X COPPER CONCENTRATIONS (me/L) LSD ANOVA P=0.05*/ control 0.0 0.034 0.057 1.30 0.01** vs. treatment RESPIRATORY (within tank) DISRUPTIONS P > 0.05*/0.01*' cough 9.0 49.5 72.0 109.5 9.4/12.8 0.0001** (40.5)** (22.5)** (37.0)** yawn 6.0 22.5 31.0 40.0 6.4/8.8 0.0001** (16.5)** (8.5)** (9.0)** COMFORT MOVEMENTS s-jerk 5.5 12.0 15.5 15.5 6.0/8.2 0.32 NS (6.5)* (3.5) (0.0) partial jerk 1.5 10.0 10.5 10.0 4.2/5.84 0.10 NS (8.5)** (0.5) (0.5) fin flick 18.5 27.5 32.5 39.0 9.0/12.2 0.008** (9.0)* (5.0) (6.5) burst 0.0 1.0 0.5 0.5 1.6/2.4 0.56 NS swimming (1.0) (0.5) (0.0) chafe 1.0 1.0 2.0 3.0 1.4/2.3 0.07 NS (0.0) (1.0) (1.0) AGGRESSION threat 52.5 72.0 90.0 81.0 16.6/22.8 0.009** (19.5)* (18.0)* (9.0) nudge 4.0 14.5 9.0 1.0 6.8/9.4 0.06 NS (10.5)** (5.5) (8.0)* nip 31.0 53.0 71.5 54.5 10.2/14.0 0.02^ (22.0)**(18.5)**(17.0)** 44 first 24 hr of exposure (Figure 2) and the absolute number of each remained elevated above control levels throughout the treatment period in the medium and high concentration groups. Carlson and Drummond (1978) were the first to propose that an automated system for cough response monitoring could be used to evaluate the toxicity of complex effluents. Other studies confirm the sensitivity of cough responses to the presence of low levels of contaminants, especially metals (Cairna and VanderSchalie 1980; Henry and Atchison 1979a, 1979b; Sparks et al. 1972). This behavioral response, whether observed directly or electronically, correlates with the morphological and physiological changes resulting from copper exposure. Baker (1969) determined that 1 mg Cu/L produced morphological changes in the gill lamellae of exposed fish. Anderson and Spear (1980) found that pumpkinseed sunfish, Lepomis gibbosus accumulated copper in gill tissue itself. O'Hara (1971) concluded that sublethal levels of copper increased oxygen consumption in bluegill and that the maximum increase occurred between 3-6 hrs of initial exposure. Drummond et al. (1973) found that 6 yg Cu/L increased coughs in brook trout (Salvelinus fontinalis). The increased frequency of both coughs and yawns observed in this study verify copper's interference with respiratory processes which occurred within 24 hrs of exposure. These two behaviors were sensitive enough to reflect differences in response between concentrations (P = 0.01) as well (Table III). 45 COUGHS K T NO incMn 0J334 mt/l LOW J 0JB7 mi/i ueoniM. I JO mi/l HGM - •A/S NIPS 1 1 H 1 isP ^nwToj^^çthnj^ 221—ilZ-SlSiSZ •Figure 2. Mean frequencies of six behaviors monitored in four treatment populations (x = 2 replications). 46 Comfort movements were not particularly sensitive indices of copper effect. The only comfort movement that reflected change (P = 0.008) once the toxicant was added was fin flicks (Table III; Figure 2). Fin flicks increased most during the first 24 hrs of exposure (Figure 2). However, the frequency of fin flicks remained elevated above control levels only among fish of the high concentration tank for the duration of the treatment period. The frequencies of fin flicks among controls and the low and medium test groups were not statistically distinguishable from one another (Table III). Two aggressive behaviors were affected by copper. Threats increased (P = 0.009) above control levels in all concentrations, with the largest change noticed in the first 24 hr of exposure (Figure 2). Nips, the most intense manifestation of aggression examined, were influenced by copper at all treatment levels. Nips occurred often enough during treatment periods to produce large absolute numbers which decreased observational error and improved the sensitivity of statistical analyses. Nips were also most affected within 24 hrs of copper administration in every concentration group (Figure 2). The largest increase observed occurred in the medium concentration test population, followed by the high then the low concentration tanks. Differences between treatment groups were also significantly different (P = 0.01) (Table III). Despite their sensitivity to copper, it should be pointed out that control levels of nips and nudges (Figure 2) are slightly more variable between populations than control frequencies of coughs and 47 yawns. Replicability during control periods between populations as well as absolute frequency should therefore be considered when selecting the most suitable behavior for monitoring the effect of a specific toxicant. Not all fish within a hierarchy were similarly influenced by copper. The most striking result was that the most subordinate (5) and most dominant (1) individuals were the most affected in every treatment group for the majority of behaviors observed than were intermediately ranked bluegill (2,3,4) (Figure 3). Differential response due to rank demonstrates the importance of considering life history aspects such as social organization of test species when conducting toxicity tests. Several investigators (Noakes and Leatherland 1977; Schreck and Lorz 1978) have suggested that the most dominant and subordinate individuals in a hierarchy are under the greatest amount of social stress. The dominant must expend effort maintaining its alpha position while the subordinate continuously attempts to avoid attacks from other fish. The differential impact of copper on hierarchy members of various rank clearly illustrates the combined stresses of toxicant effect and social position. Copper induced changes in other behavioral responses were also differentially influenced according to social rank. An examination of the distribution of respiratory disruptions throughout each test population revealed that the most subordinate individual performed more coughs and yawns during treatment than the rest of the test population. The dominant fish was the next most affected. Both fish Figure 3. Mean frequencies of six behaviors performed by individuals of differing social rank during the treatment period = 2 replications). 49 COUGHS 301- I f THRtATS, fBH 1 - tBH J • fBH 3 - fBH I • fBH 5 - J 1 1 1 r 003: T CONCÎNIMID* imt'l! Î CONa*'M'DN imt/i: 50 5 and 1 coughed and yawned more in each concentration than other fish. These elevated levels were well above those observed during control periods or on any given day in the control tank. Miller (1963) hypothesized that yawns might be indicative of general physiological stress. When physiological stress from a contaminant which affects gill function and structure is combined with social stress, elevated numbers of respiratory disruptions occur. The automated ventilation and cough response monitoring systems mentioned previously require that single individuals be tested. Information regarding the differential responses of fish within social groups would be lost using this system. Additionally, gill purges or coughs appear on chart recordings as breaks in the respiratory rhythm. Yawns, although occasionally mentioned in the literature, have not been evaluated as extensively and may be indicative of a more extreme response. Fin flicks, the only sensitive comfort movement observed, were most frequently performed by the most subordinate individual, followed by the most dominant in all treatment groups (Figure 3). However, the absolute numbers of fin flicks were never as large as those reported for coughs and yawns. The most dominant fish in each tank became even more aggressive once copper was added (Figure 3). Threats and nips were particularly affected, with the largest absolute increase observed for the 1 and 2 ranked individuals in the medium concentration tank (Figure 3). Recall, however, that rank was initially assigned on the basis of 51 aggressive acts performed during control period observations. Consequently, it is not surprising that dominant fish were most affected in the behavioral area of their highest activity. Fish ranked 3 and 4 were generally least affected by copper at any concentration. Under control as well as treatment conditions, they were the least active, swimming only intermittently, and subsequently experiencing fewer social interactions. The social stress on these individuals seemed less than levels experienced by dominants and the lowest ranked subordinates. Despite this social buffer, coughs and yawns increased significantly over every concentration in fish 3 and 4 (Table III) indicating that respiratory disruptions are widely sensitive indices of copper effect, even in differentially ranked individuals. Benoit (1975) reported that a chronic exposure (22 mo.) to 162 yg Cu/L retarded growth and reproduction and lowered survival of adult bluegill. By contrast, 34 Ug Cu/L used in this study produced significant changes in respiratory disruptions, fin flicks, threats and nips in adult bluegill exposed in water of much greater hardness (x = 273.3 mg/L vs. 45 mg/L, Benoit 1975). The increased hardness of the water used here should decrease copper toxicity. In addition the MA.TC determined for larval bluegill exposed to copper was 21-40 Pg/L (Benoit 1975). Larval fish are generally more sensitive to toxicants, including copper, than adult fish. However, the lowest concentration of copper used in this study produced behavioral changes in adults at levels falling within the MATC and below the 52 copper concentration that influenced growth in larval bluegill exposed in softer water. Although the literature contains no other behavioral studies exposing bluegill to copper, several studies exist that have monitored copper induced changes in spontaneous activity, feeding, swimming stamina or avoidance in other species. For example, Waiwood and Beamish (1978) observed reductions in the spontaneous activity of rainbow trout, Salmo gairdneri. exposed to 5 yg Cu/L in soft water. In the same study, 5 yg Cu/L was found to also reduce swimming stamina in rainbow trout. These changes occurred at concentrations equivalent to 13% of the 96 hr LC50 for rainbow trout exposed to copper sulfate. The behavioral changes in this study occurred at a level approximately equal to 3% of the 96 hr LC50. The most sensitive behavioral index of copper effect previously examined appears to be avoidance (Folmar 1976) occurring in rainbow trout at 0.1 pg/L. These comparisons indicate that behavioral changes can be as sensitive or more sensitive to copper than classically measured bioassay endpoints even when classical endpoints are examined during the most sensitive life stage of the test species. If the 1980 EPA Water Quality Criteria for copper are examined, 5.6 pg/L, expressed as a 24 hr average, should be sufficient to protect freshwater aquatic life forms. Using the formula e(0.94 [log (hardness)] - 1.23) to calculate the copper level which should never be exceeded in water with the hardness of our test water (x = 273.3 mg/L CaCOg), the calculated value is 57 yg Cu/L. Behavioral 53 responses examined in this study, although significantly altered at 34 yg/L, were most affected for all behaviors at 57 yg/L. Therefore, the results of this study suggest that deleterious effects can occur at or below 57 y g Cu/L in exposed bluegill. Behavioral bioassays such as avoidance or feeding tests should be further employed to evaluate the impact of the 57 y g Cu/L protection limit set by the EPA for water of this designated hardness. Behavioral parameters are obviously sensitive indicators of toxicant effect, which in the present study, equalled or surpassed the sensitivity of chronic test endpoints. When selecting behavioral indices for monitoring, however, it is important to select behaviors that relate to the organism's survival in its natural habitat. If this is accomplished, then toxicant induced behavioral changes can be predictive of the potential hazard posed by a contaminant in the field. Social hierarchies have developed evolutionarily as mechanisms for the efficient distribution of limited resources (food, mates, desirable spawning sites, etc.) in individuals of a population (Krebs and Davies 1978), and disruption of such a system by a toxicant could ultimately affect survival and reproduction. Rank related responses need to be considered in species forming social organizations. If this aspect is neglected, then only a small portion of the population may be protected. More field studies are needed to verify whether hierarchy related effects of toxicants which have been observed in the laboratory actually occur in the field. Additional testing, using this bioassay approach in combination with 54 even lower copper concentrations than were utilized in this study could also help establish the lowest possible sensitivity limit of this approach which detects behavioral change. Analytical chemistry detection limits, for some toxicants, could be surpassed by the sensitivity of behavioral detection limits, as was shown by Pearson et al. (1981) using the Dungeness crab. Cancer magister. exposed to petroleum hydrocarbons. Behavioral methods could provide a rapid yet sensitive means of assessing toxicity, especially when dealing with chemically complex effluents or materials such as petroleum products which are comprised of thousands of individual compounds. 55 REFERENCES Alabaster, J.S. and R. Lloyd. 1980. Water quality criteria for freshwater fish. Butterworth and Comp. Ltd., London, England. 297 pp. Anderson, P.D. and P.A. Spear. 1980. Copper pharmaco-kinetics in fish gills - II. Body size relationships for accumulation and tolerance. Water Research 14(8):1107-1111. Baker, J.T.P. 1969. Histological and electron microscopical observations on copper poisoning in the winter flounder (Pseudopleuronectes americanus). Journal of Fisheries Research Board of Canada 26:2785-2793. Benoit, D.A. 1975. Chronic effects of copper on survival, growth and reproduction of the bluegill (Lepomis macrochirus). Transactions of the American Fisheries Society 104(2):353-8. Brungs, W.A., J.R. Geckler and M. Cast. 1976. Acute and chronic toxicity of copper to the fathead minnow in a surface water of variable quality. Water Research 10(l):37-43. Cairns, J, and W.H. VanderSchalie. 1980. Biological monitoring Part I - Early warning systems. Water Research 14:1179-1196. Carlson, R.W. and R.A. Drummond. 1978. Fish cough response - a method for evaluating quality of treated complex effluents. Water Research 12:1-6. Drummond, R.A., W.A. Spoor and G.F. Olson. 1973. Some short-term indicators of sublethal effects of copper on brook trout, Salvelinus fontinalis. Journal of Fisheries Research Board of Canada 30:698-701. Folmar, L.C. 1976. Overt avoidance reaction of rainbow trout fry to nine herbicides. Bulletin of Environmental Contamination and Toxicology 15:509-514. Henry, M.G. and G.J. Atchison. 1979a. Behavioral changes in bluegill, Lepomis macrochirus. as indicators of sublethal effects of metals. Environmental Biology of Fishes 4(l):37-42. Henry, M.G. and G.J. Atchison. 1979b. Influence of social rank on the behavior of bluegill, Lepomis macrochirus Rafinesque. exposed to sublethal concentrations of cadmium and zinc. Journal of Fish Biology 15:309-315. 56 Howarth, R.S. and J.B. Sprague. 1978. Copper lethality to rainbow trout in waters of various hardness and pH. Water Research 12:455-462. Johnson, W.W. and M.T. Finley. 1980. Handbook of acute toxicity of chemicals to fish and aquatic invertebrates. U.S. Fish and Wildlife Service, Resource Pub. 137:21-22. Krebs, J.R. and N.B. Davies. 1978. Behavioral Ecology: an evolutionary approach. Sinauer Associates, Inc., Sunderland, MA. 494 pp. Lett, P.P., G.J. Farmer, and F.W.H. Beamish. 1976. Effect of copper on some aspects of the bioenergetics of rainbow trout (Salmo gairdneri). Journal of the Fisheries Research Board of Canada 33:1335-1342. Miller, H.C. 1963. The behavior of the pumpkinseed sunfish, Lepomis gibbosus (Linneaus), with notes on the behavior of other species of Lepomisand the pigmy sunfish, Elassoma evergladei. Behavior 22:88-151. Mount, D.I. and W.A. Brungs. 1967. A simplified dosing apparatus for fish toxicology studies. Water Research 1:21-29. Mount, D.I. and C.E. Stephan. 1969. Chronic toxicity of copper to the fathead minnow, Pimephales promelas. in soft water. Journal of Fisheries Research Board of Canada 26(9):2449-2457. Noakes, D.L.G. and J.F. Leatherland. 1977. Social dominance and interrenal cell activity in rainbow trout, Salmo gairdneri. Environmental Biology of Fishes 2:131-136. Nriagu, J.O. 1979. Copper in the Environment. John Wiley and Sons, Inc., New York, New York. 522 pp. O'Hara, J. 1971. Alterations in oxygen consumption by bluegills exposed to sublethal treatment with copper. Water Research 5(6):321-327. Pagenkopf, O.K., R.C. Russo, and R.V. Thurston. 1974. Effect of complexation on toxicity of copper to fishes. Journal of Fisheries Research Board of Canada 31:462-465. Pearson, W.H., P.C. Sugarman, D.L. Woodruff and B.L. 011a. 1981. Impairment of the chemosensory antennular flicking response in the Dungeness crab. Cancer magister. by petroleum hydrocarbons. Fishery Bulletin 79(4):641-647. 57 Schreck, C.B. and H.W Lorz. 1978. Stress response of coho salmon (Oncorhvnchus kisutch) elicited by cadmium and copper and potential use of Cortisol as an indicator of stress. Journal of the Fisheries Research Board of Canada 35(8);1124-1129. Sparks, R.E., J. Cairns, Jr. and A.G. Heath. 1972. The use of bluegill breathing rates to detect zinc. Water Research 6:895-911. Swigert, J. and E.L. Morgan. 1977. Mortality of bluegills, Lepomis macrochirus. exposed to successive doses of copper and zinc. Association of Southeast Biology Bulletin 24(2):89-93. Thompson, K.W., A.C. Hendricks and J. Cairns. 1980. Acute toxicity of zinc and copper singly and in combination to the bluegill (Lepomismacrochirus). Bulletin of Environmental Contamination and Toxicology 25(1):122-129. U.S. Environmental Protection Agency. 1980. Water Quality Criteria Document. Federal Register 45, No. 231:79318-79379. Waiwood, K.G. and F.W.H. Beamish. 1978. Effects of copper, pH and hardness on the critical swimming performance of rainbow trout (Salmogairdneri) (Richardson). Water Research 12(8):611-619. Wiener, J.G. and J.P. Giesy. 1979. Concentrations of cadmium, copper, manganese, lead and zinc in fishes in a highly organic softwater pond. Journal of the Fisheries Research Board of Canada 36(3):270-279. 58 SECTION III. BEHAVIORAL EFFECTS OF CHLORDAME ON HIERARCHIES OF BLUEGILL, LEPOMIS MACROCHIRUS 59 INTRODUCTION Over the last 30 years, chlordane has been extensively used for home and garden insect control, commercial termite control and agricultural pest control. However, its use was recently restricted to only commercial application (Water Quality Criteria 1980). Technical chlordane, a mixture of various chlorinated hydrocarbons, is the most frequently used form of this pesticide. It is relatively insoluble in water bioconcentrâtes in lipids, is quite toxic to aquatic organisms, is persistent in the environment (National Research Council of Canada 1974), and is commonly detected in fish and waterbodies across the country (Hall et al. 1977; Miles 1977; Veith et al. 1981). For the above reasons it was chosen for use in this study. Scant toxicological information is available on the effects of chlordane on fish at sublethal levels. Previous work by Henry and Atchison (1979a, 1979b) suggests that behavioral changes in bluegill are sensitive to low levels of some toxicants. Consequently, the objectives of this study were to: 1) assess the impact of sublethal levels of technical chlordane on bluegill social groups; 2) compare concentrations affecting behavior to those which have induced changes in survival, growth or reproduction (classically measured bioassay endpoints); and 3) comment on the sensitivity of this method in view of levels recommended as safe for aquatic life by the Water Quality Criteria (1980). 60 MATERIALS AND METHODS Forty age II bluegill (13.3-18.0 cm total length; 35.4-60.1 g) were raised and tested at the Columbia National Fisheries Research Laboratory, Columbia, MO. Twenty fish were used in each of two replications of the experiment. For each replication, five fish were randomly assigned to each of the four 295 liter flow-through glass test aquaria (Figure 1). Each tank was layered with gravel and planted with Elodea sp. to provide fish with substrate and cover. Fish were maintained throughout the study at 170C, under a 16 hr/8 hr light-dark photoperiod and were fed Purina trout chow (#6) twice daily until satiation. Water quality characteristics (x +_ SD; N = 28), analyzed biweekly remained constant both during and between replications (total hardness, 275.3 +_ 2.8 mg/L CaCOg; pH 7.7 +.0.6; temperature, 17.1 +.0.3OC; dissolved oxygen 9.3 +.2.8 mg/L). The sex of all individuals was determined by dissection at the end of the experiment. In 5 out of 8 hierarchies, the most dominant individual was male, and the most subordinate was female. Each replication was comprised of three phases: (1) acclimation period (1-2 wk); (2) control period (96 hr); and (3) treatment period (96 hr). Acclimation was judged as complete once all individuals were feeding regularly and once all territories and hierarchies were established. 117 cm. figure 1. 295 liter glass nqiiarliim. 62 Ten behaviors (described briefly in Table I) were observed daily for each test population during control and treatment periods. Two hr time blocks (1/2 hr/tank) were randomized and rotated through the 16 hr light portion of the photoperiod so that observations on day 1 of both control and treatment periods took place from 4-6 pm, day 2 = 8-10 am, day 3 = 8-10 pm, day 4 = 12-2 pm. This rotated observation schedule was incorporated into the experimental design so that diurnal fluctuations in activity would not be confounded with toxicant effect. The order of tanks observed was also randomized within each 2 hr time block. Behaviors were observed directly by only one investigator (M. Henry, Columbia National Fisheries Research Laboratory, Columbia, MO) and the frequencies of each behavior and the individual fish performing them were recorded by hand, using codes, recording forms and abbreviations to facilitate the process. Individuals were distinguished from one another on the basis of differences in color, size and natural markings. No tags, brands or fin clips were used. Fish readily formed hierarchies and were assigned numbers from 1-5, designating their rank within that hierarchy (1 = most dominant, 5 = most subordinate). Criteria used to determine dominance was the initiation of the greatest total number of aggressive acts (threats, nudges, and nips) during the control period. In addition, territories were established by the top one or two ranking individuals and territory boundaries were regularly defended against interlopers. 63 Table I. Brief descriptions of the ten behaviors monitored in this study. CATEGORY OF BEHAVIOR DESCRIPTION I. RESPIRATORY DISRUPTIONS cough rapid, repeated opening and closing of mouth and opercular coverings, accompanied by partly extended fins yawn singularly occurring maximal opening of mouth and opercular coverings, accompanied by hyperextension of all fins II. COMFORT MOVEMENTS "s"-jerk whole body flinch, moving sequentially from head to tail partial jerk head or tail flinch fin flick rapid, repeated extension and contraction of spines and rays of fins burst swimming sudden, rapid, nondirected spurt of forward movement chafe brush of the body against an inanimate object III. AGGRESSIVE INTERACTIONS threat flare of opercular coverings accompanied by slight intention movement toward conspecific nudge contact of closed mouth to body between fish, recipient is not displaced or bitten. nip bite 64 Technical chlordane (obtained from Velsicol Chemical Company, Chicago, IL) was used to prepare the toxicant stock solution. It was mixed in 0.5% acetone to improve solubility and administered to treatment tanks using two micromedic pumps and a modified proportional diluter system (Mount and Brungs 1967). Acetone alone was added to the control tank as a check on solvent erfact. Toxicant concentrations in stock solutions and concentrations in test tanks were verified by gas-liquid chromatography. The nominal concentrations of chlordane were selected with the rationale that they would approach or duplicate the 96 hr LC50, the level equal to the Water Quality Standard not to be exceeded and the lowest level chemically detectable (Water Quality Criteria 1980). Using these concentrations to evaluate effects on behavior would subsequently allow comparisons to be made with classical bioassay endpoints and Water Quality Standards aimed at protection of aquatic life. Nominal and mean actual concentrations are summarized in Table II. Tanks reached full concentrations after approximately 7.3 hrs (x flow rate = 1781 ml/5 min/tank; N = 8). Behavioral data were analyzed using an analysis of variance that included partitions for replication, treatment, social rank and time with their interactions. Least Significant Difference (LSD) determinations were also made to allow for behavioral comparisons between treatment levels and fish of different rank. Note, however, that since social rank was determined using the sum of aggressive acts initiated by an individual during the control period. 65 Table II. Nominal and mean actual chlordane concentrations for each treatment over two replications (N = 4). Nominal Concentrations (yg/l) Mean Actual Concentrations (yg/l) 0.00 0.001 + 0.02 0.10 0.15 +_ 0.06 2.40 1.91 + 0.28 52.00 44.30 + 1.01 96 hr LC50 = 57 yg/L (Johnson and Finley 1980) Standard not to be exceeded = 2.4 yg/L (Water Quality Criteria 1980) 66 differences between ranked fish in the treatment period are confounded with baseline frequencies of threats, nudges and nips. 67 RESULTS AND DISCUSSION The results of this study were analyzed from two perspectives; 1) population level response (total number of behaviors performed per tank) and 2) individual fish response (total number of behaviors per fish reflecting differential sensitivity due to social position in the hierarchy). Throughout the study, the form and posture of each behavior remained unaltered but the frequency of occurrence changed once chlordane was added to test tanks. The experimental design of this study employed two types of controls: one for treatment effect (control vs. treatment period for each tank) and one for time effect (a control tank that received no chlordane during the treatment period). No statistically significant differences in the frequencies of all behaviors were found between tank populations during the control period. Therefore, all control data (control period data for all tanks and treatment period data for the tank not receiving chlordane) were pooled and mean values (N = 10) represent normal levels of response in the absence of the toxicant (Table III). Significant (P = 0.01) changes in frequency were observed for eight of the ten behaviors monitored for each concentration of chlordane used. The largest absolute increase was seen in the number of nips performed once the toxicant was administered. Aggressive interactions, the most intense of which were nips, became so numerous 68 that the most subordinate fish in the high concentration of replication #1 died within the first day of exposure from hemorrhages resulting from receipt of numerous and successive bites. Consequently, note that the mean values expressed for all behaviors during the treatment period (Table III, Figure 2, Figure 3) were based on frequencies performed by only a total of 39 fish from two replications, not 40 individuals as planned. As nips increased in all treatment groups, threats decreased. Non-contact displays were thereby replaced, as the toxicant was added, with more extreme forms of physical contact. There was also a significant difference (P = 0.01) in the number of nips performed by each population exposed to the different chlordane concentrations when compared in any combination (Table III). This delineation of response seen when comparing nips from control vs. treated as well as between concentrations indicate that nips were, in general, the most sensitive indicators of chlordane's effect. Since chlordane is suspected to function as a neurotoxin, subsequent hyperactivity (Bansal et al. 1979) could be related to the increase in the number of intense manifestations of aggression displayed. Increased swimming put fish in closer proximity to one another and irregular movements (i.e. coughs, s-jerks, etc.) seemed related to the stimuli that elicited aggressive interactions, since threats and nips were often initiated after erratic movements by a recipient. Respiratory disruptions were also significantly affected (P = 0.0001) by chlordane (Table III). Coughs and yawns increased most 69 Table III. Mean population frequencies of 10 behaviors examined over three toxicant concentrations and a control during the treatment period. X CHLORDANE CONCENTRATIONS (ug/D LSD ANOVA P=0.05*/ control 0.0 0.15 1.91 44.3 0.01** vs. treatment RESPIRATORY (within tank) DISRUPTIONS P 2 0.05*/0.01* cough 13.5 27.0 48.0 75.0 17.4/24.0 0.0002** (13.5) (21.0)* (27.0)* yawn 7.0 26.5 41.0 39.5 8.4/11.6 0.0001** (19.5)**(14.5)** (1.5) COMFORT MOVEMENTS s-jerk 6.0 17.0 20.5 25.5 6.0/8.2 0.0006** (11.0)**(3.5) (5.0) partial jerk 2.0 10.5 12.5 17.5 4.24/5.84 0.0001** (8.5)** (2.0) (5.0)* fin flick 17.0 19.0 24.5 29.0 6.0/8.2 0.001** (2.0) (5.5) (4.5) burst 1.0 2.5 0.5 0.0 1.2/1.6 0.009** swimming (1.5)* (2.0)** (0.5) chafe 0.5 0.5 0.0 0.0 0.8/1.2 0.807NS (0.0) (0.5) (0.0) AGGRESSION threat 66.5 48.5 23.0 28.0 11.4/15.8 0.002** (18.0)**(25.5)**(5.0) nudge 6.5 3.0 1.0 1.0 6.4/8.8 0.322NS (3.5) (2.0) (0.0) nip 31.0 62.5 78.5 111.0 9.8/13.4 0.0001** (31.5)**(16.0)**(32.5)** 70 COUGHS YAWNS UJ S-JERKS FIN FLICKS - HIGH CONC CO ^ MEDIUM CONC -LOW CONC -NO TOXICANT T . THREATS NIPS v ii CONTROL TREATMENT CONTROL PERIOD PERIOD PERIOD Figure 2. Mean frequencies of six behaviors monitored in four treatment populations (x = 2 replications). 71 YAWNS S-JERKS FIN FUCKS — FISH 1 FISH 2 ta 26- — FISH 3 FISH 4 FISH5 THREATS 0.0001 0.15 44J .0001 0.15 1°J. 44i X CONCENTRATION (ug/I) î CONCENTRATION (ug/I) Figure 3. Mean frequencies of six behaviors performed by individuals of differing social rank during the treatment period (x = 2 replications). 72 dramatically in the first 24 hrs of exposure (Figure 2) in all treatment groups. Dalela et al. (1980) noted that respiratory changes (monitored as VO2 changes) were good indicators of sublethal and lethal chlordane stress in Saccobranchus fossilis. Other oxygen uptake data have shown that there is a critical threshold time for respiratory and hyperactivity responses existing between 6-24 hrs of exposure to chlordane (Bansal et al. 1979). The increase in respiratory disruptions could also be relate to the demonstrated development of anaerobic conditions in chlordane stressed S. fossilis due to inhibition of acetylcholinesterase, succinic dehydrogenase and pyruvic dehydrogenase in several tissues, including gill tissue (Verma et al. 1981). The significant increase (Table III, Figure 2) seen in the comfort movements, especially s-jerks and fin flicks, may also be related to the hyperactivity observed. Recent findings on dieldrin, a related cyclodiene insecticide, indicate that it acts on the terminals of the presynaptic membrane, increasing the release of acetylcholine at the pre-synapse producing hyperactivity (Shankland, 1979). Behavioral changes were distributed in each toxicant treated hierarchy specific to rank. Subordinate fish (5) were the most affected, when compared to fish of different rank, in areas of respiration and comfort movements, while dominant fish exhibited the largest increases in nips (Figure 3). Coughs, yawns and fin flicks increased during treatment for the dominant individuals of each group 73 to frequencies just below the most affected subordinates. These responses, along with evidence put forth by other investigators (Noakes and Leatherland 1977; Schreck and Lorz 1978), indicate that the most dominant and most subordinate individuals of a hierarchy are under the most social stress. The most dominant must expend effort maintaining its alpha position while the most subordinate attempts to avoid harassment from other fish. Territory defense disappeared for fish 2 and 3 of each replication in the highest treatment concentration. Conversely, the most dominant fish became more territorial, defending expanded borders and driving all other hierarchy members into the vegetated area at the far left side of the aquaria. Fish in the high concentration, including the most dominant, also had greatly diminished appetites relative to controls. Fishes 2, 3, and 4 were least affected by chlordane. The social stress on these individuals was probably less than that on the most dominant and most subordinate individuals. However, increases in coughs for fish of each of these ranks were large in medium and high concentration groups. Therefore, that social stress, combined with chlordane stress, seemed to produce differential frequencies of response in fish of varying rank. The literature reveals a paucity of information available on the sublethal effects of chlordane on fish. Even fewer studies have examined changes in behavior of fish exposed to chlordane or any specific effects on bluegill. The literature that does exist 74 confirms the suspicion that chlordane bioconcentrates in the lipid of exposed fish (Verma et al. 1981). Also, chlordane has been detected in fish and water of natural systems (Hall et al. 1977; Miles 1977; Veith et al. 1981) exceeding permissible or recommended levels. Under laboratory conditions, bluegill rapidly bioconcentrated (in 48 hrs) 5 yg/L cis-^^C chlordane and very slowly eliminated it (Sundershan and Khan 1980). Consequently, the levels of exposure used in this study are realistic in view of expected environmental concentrations of chlordane and bluegill are ubiquitous enough to be affected and apparently vulnerable both physiologically and behaviorally. If the most conservative interpretation is adopted, it is clear from Table III that there is a significant difference (P >. 0.01) between the frequencies of five behaviors when control values are compared to values from the lowest test concentration. Consequently, it is also apparent that 0.15 yg/L chlordane can cause significant changes in behavior monitored in bluegill social groups. Using avoidance to evaluate chlordane effect, Summerfelt and Lewis (1967) found that green sunfish (Lepomis cvanellus) avoided 5 mg/L of chlordane. This concentration represents 25% of the 96hr LC50 as they determined under their laboratory conditions. Little (personal communication, Columbia National Fisheries Research Laboratory, Columbia, MO) found that feeding was impaired in rainbow trout (Salmo gairdneri) exposed to a chlordane concentration equal to 5% (2.1 yg/L) of the 96hr LC50. Comparing the results of the research 75 reported here to the 96hr LC50 of 57 yg/L determined for bluegill exposed to tech chlordane (Johnson and Finley, 1980), statistically significant behavioral changes occurred at a concentration of 0.15 yg/L, less than 0.1% of the LC50. This level of sensitivity is below 2.4 yg/L, the EPA level set which is "not to be exceeded". Furthermore, chronic exposure studies showed first effects on bluegill as reductions in number of young produced by spawning adults exposed for 6 mo. to 1.22 yg/L technical chlordane. The behavioral changes observed in this investigation were seen at a level well below those classically representing lowest effect concentration or that deemed safe by the EPA. In addition, if hierarchies are disrupted by low level exposure to chlordane, population maintenance and reproduction could subsequently be affected. Confirmation of these effects under field conditions could help improve in situ interpretation of this laboratory assessment. 76 REFERENCES Bansal, S.K., S.R. Verma, Â.K. Gupta, S. Rani and R.C. Dalela. 1979. Pesticide-induced alterations in the oxygen uptake rate of a freshwater major carp, Labeo rohita. Ecotoxicology and Environmental Safety 3(4):374-382. Dalela, R.C., S.K. Bansal, A.K. Gupta and S.R. Verma. 1980. Short-term stress on the oxygen consumption of a fresh water teleost, Saccobranchus fossilis. following lethal and sublethal levels of chlordane metasystox and Sevin. International Journal of Environmental Studies 15(3):229-235. Hall, R.W., Jr., R.H. Plumb, Jr., K.W. Thornton, R.L. Eley and A.S. Lessem. 1977. Arcadia Lake Water-Quality Evaluation. Army Engineer Waterways Experiment Station Final Report WES-TR-Y-77-2 (Vicksburg, Miss.) Henry, M.G. and G.J. Atchison. 1979a. Behavioral changes in bluegill, Lepomis macrochirus. as indicators of sublethal effects of metals. Environmental Biology of Fishes 4(l):37-42. Henry, M.G. and G.J. Atchison. 1979b. Influence of social rank on the behavior of bluegill, Lepomis macrochirus Rafinesque. exposed to sublethal concentrations of cadmium and zinc. Journal of Fish Biology 15:309-315. Johnson, W.W. and M.T. Finley. 1980. Handbook of acute toxicity of chemicals to fish and aquatic invertebrates. U.S. Fish and Wildlife Service Resource Publication 137:20-21. Miles, R.L. 1977. Commercial Fishery Investigations. National Marine Fisheries Service Final Report NOAA-04-4-043-409 (Charleston, West Virginia). Mount, D.I. and W.A. Brungs. 1967. A simplified dosing apparatus for fi'h toxicology studies. Water Research 1:21-29. National Research Council of Canada. '1974. Chlordane: Its effects on Canadian ecosystems and its chemistry. NCRR No. 14094 (Ottawa, Canada). Noakes, D.L.G. and J.F. Leatherland. 1977. Social dominance and interrenal cell activity in rainbow trout, Salmo gairdneri. Environmental Biology of Fishes 2:131-136. 77 Schreck, C.B. and H.W. Lorz. 1978. Stress response of coho salmon (Oncorhvnchus kisutch) elicited by cadmium and copper and potential use of Cortisol as an indicator of stress. Journal of the Fisheries Research Board of Canada 35(8):1124-1129. Shanklord, D.L. 1979. Action of dieldrin and related compounds on synaptic transmission in Neurotoxicology of Insecticides and Pheromones. Plenum Press, New York, 342 pp. Summerfeltj R.C. and W.M. Lewis. 1967. Repulsion of green sunfish by certain chemicals. Journal of the Water Pollution Control Federation 39:2030-2038. Sundershan, P. and M.A.Q. Khan. 1980. Metabolism of cis carbon-14 labeled chlordane and cis carbon-14 labeled photo chlordane in bluegill fish, Lepomis macrochirus. Journal of Agricultural Food Chemistry 28(2): 291-296. Veith, G.D., D.W. Kuehl, E.N. Leonard, K. Welch and G. Pratt. 1981. Polychlorinated biphenyls and other organic chemical residues in fish from major United States watersheds near the Great Lakes. U.S. Environmental Protection Agency, EPA-600/J-81-549. Verma, S.R., I.P. Tonk, A.K. Gupta and R.C. Dalela. 1981. In-vivo enzymatic alterations in certain tissues of Saccobranchus fossilisfollowing exposure to 4 toxic substances. Environmental Pollution Series A: Ecological Biology 26(2):121-128. Water Quality Criteria. 1980. Ambient Water Quality Criteria for Chlordane. U.S. Environmental Protection Agency, EPA-440/5-80-027. 78 GENERAL SUMMARY Behavioral changes occurred in all bluegill exposed to sublethal concentrations of methyl parathion, copper sulfate and chlordane. Hyperactivity was noticeable in fish exposed to methyl parathion. S-jerks and fin flicks were the individual behaviors whose frequencies were most altered once methyl parathion was administered. Conversely, respiratory disruptions, coughs and yawns, increased In the presence of sublethal levels of copper sulfate. Chlordane produced elevated levels of aggression, reflected as increased numbers of nips. The most dramatic changes in these frequencies occurred within approximately the first 24 hrs of toxicant administration. Responses generally declined or plateaued during the period from 48-72 hr possibly indicating some degree of acclimation to the chemicals. However, increases often began again around the 72 hr mark until the end of the treatment period. An indeterminate treatment period length would elucidate whether acclimation does occur and would provide an excellent question for future research. Differential responses associated with social rank of individuals occurred in every treatment group for each toxicant examined. The most subordinate individuals were more sensitive to contaminants than other fish in the behavioral areas of respiration and comfort movements. The most dominant individuals were generally next most affected in these areas and showed the greatest degree of change with regard to aggressive behaviors. The combination of 79 social stress and toxicant stress was apparent in the uneven distribution of response throughout hierarchies exposed to each chemical. Once the contaminant addition ceased, behaviors were continually monitored (5 min/tank/hr) for 24 hrs. General trends in the raw data suggest that elevated frequencies of the .^ost changed behaviors returned to pre-treatment control levels in all populations exposed to copper. However, every fish in the high chlordane concentration continued to behave abnormally while only the subordinate in the high methyl parathion treatment group continued to show irregularities during the post-treatment period. The largest shift in hierarchy organization took place in the high chlordane treatment group. The most dominant fish became more aggressive and dissolution of territorial boundaries for fish 2 and 3 reflected this change. Feeding trials were initiated on each day of the treatment period for every tank exposed to each of the chemicals. Examination of the raw data reflects hierarchial influence as well as the individual's state. In the low and medium concentrations of each chemical exposure, the most dominant fish fed first. The alpha fish in the medium copper treatment exhibited some lack of coordination in capturing the fathead minnow prey. Successful strikes but spitting by the dominant occurred in this case and was also observed for the #2 and 3 ranked fish in the high concentration group. Feeding order was rearranged in the methyl parathion high treatment group. The #2 fish fed more often than the #1 fish. 80 Erratic swimming and jerky movements made capture less successful. Chlordane treated fish generally exhibited suppressed appetites. The highest treatment group never ate all 10 fathead minnows presented on any given day. These data will be statistically analyzed and written up for future publication. Maki (1979) used ventilation rate to assess the impact of different concentrations of surfactants on bluegill. One of the advantages of the method that he cited was the proportional linear relationship between frequency and concentration. In other words, there was a direct dose-response correlation in this instance. None of the behaviors observed in this experiment always showed a direct linear dose-response trend. However, coughs come the closest for all three chemicals and are especially good indicators for copper and chlordane effect. In the case of methyl parathion, s-jerks approximate the best linear correlation between frequency and concentration but it is not totally linear. Further testing would be necessary, with use of an expanded treatment time period, to assess the linear dose-response relationship potential of the behaviors monitored in this approach. Several advances have been made in an effort to standardize this method. Use of healthy similar size and age adults, all introduced to their respective tanks within minutes of each other, hastened the adjustment process and the formation of hierarchies. Tanks were identical, in both biotic (plants) and abiotic respects. Maintenance food regimes during the test were the same as during the culture of 81 the fish and water type and temperature were also identical during rearing and test periods. Careful handling of fish, low density stocking practices and good water quality streamlined acclimation, accomplished in 7-10 days. The control observations and test observations took 8 days (4 days each) and post-treatment examinations took 24 hr. Consequently, this test approach can be completed in 16-19 days, a very manageable time frame from a labor standpoint. This type of behavioral test is adequate for species which form hierarchies at least during part of the year. However, it is not an appropriate test for solitary predators, schooling fishes, etc. This brings to light my recommendation that several behavioral approaches should be developed and utilized for examination of fish of various lifestyles. For example, swimming stamina or avoidance tests are ideal for anadromous species who rely heavily on their swimming performance and detection of nursery streams for spawning. Conversely, bluegill are not strong swimmers due to lateral compression and swimming stamina tests would not be the most appropriate type of behavioral assessment for this species. Behavior is sensitive to perturbation and can therefore be variable or unpredictable. Thus, my second recommendation, regarding use of this and other behavioral bioassays, is to use it in conjunction with other measurements such as changes in growth, the hemoglobin content of the blood or tissue residue levels. If used in conjunction with both classical partial chronic measurements and with 82 physiological, biochemical and field assessments, it could provide ecologically valuable information on how natural assemblages of fish are affected by toxicants. Additionally, field verification of hierarchy formation in bluegill would increase the validity of this approach-and further laboratory tests could be conducted to determine the lowest possible level of a toxicant which causes behavioral alterations. 83 REFERENCES Maki, A.W. 1979. Respiratory activity of fish as a predictor of chronic fish toxicity values for surfactants. In Aquatic Toxicology: Proceedings of the Second Annual Symposium. ASTM, Philadelphia, PA. 303 pp. 84 ACKNOWLEDGEMENTS Recognition of financial support for this research goes to the U.S. Fish and Wildlife Service - Columbia National Fisheries Research Laboratory and the Iowa State University Agricultural Experiment Station. I would like to sincerely thank Ed Little and Rick Archeski for their technical assistance throughout this project. Much appreciation is extended to Dave Cox, Bruce Menzel, Leslie Perham and Mark Ryan for their major contributions, both intellectual and emotional. I am extremely indebted to Gary Atchison for eight years of academic guidance, interest and friendship. None of this research could have been completed without his consistently thoughtful technical input and personal patience. I would lastly like to acknowledge the most significant contributor to this project - John Pacini. The costs were significant but the project is now complete. Without his encouragement and sacrifice these would be blank pages. To him I am most grateful.