Dieldrin. Effects of Chronic Sublethal Exposure on Adaptation to Thermal Stress in Freshwater Fish Ellen K. Silbergeldl Stroud Water Research Center, Department of Limnology, Academy of Natural Sciences of Philadelphia, Avondale, Pa. 19311
The freshwater fish, Etheostoma nigrum, were pretreated with 2.3 ppb dieldrin for 30 days and then exposed to thermal stress, applied by heating the water a t a rate of l“C/hr to a maximum of 7-9°C above ambient temperature. Pretreated fish had significantly higher mortality than fish not pretreated with dieldrin but also exposed to the same thermal stress. Measurements of blood glucose, feeding, growth, oxygen consumption, opercular movement, whole body lipid content, and liver condition were made throughout the experiment. Pretreated fish demonstrated significant changes in all of the above parameters except opercular movement during the first 15 days of exposure to dieldrin. After 15 days, pretreated fish were found to have adapted to dieldrin exposure, as indicated by significant reversals of the above changes (except for liver damage, which was not reversed but progressed over time). In the adapted state, the pretreated fish remained different from untreated fish, in terms of these parameters. These differences provide grounds for predicting differential response to sublethal thermal stress for the pretreated and untreated fish. Such response is discussed as it may relate to the effects of chronic sublethal exposure to dieldrin.
The toxicity of a compound to organisms has been shown to be changed by alterations in the organism’s environment. Increases in water temperature, for example, lower the LC50 of insecticides and ionizing radiation for aquatic organisms (Macek et al., 1969, Blaylock and Mitchell, 1969). The effect of exposure to a specific toxicant on the susceptibility of organisms to environmental change has not been extensively studied. In one experiment, pretreatment with ABS (alkyl benzene sulfonate) detergent did not affect the abilities of bluegills (Lepomis macrochirus) to survive increases in water temperature (Cairns and Scheier, 1964). In another study, pretreatment with DDT potentiated the effects of lowered water temperature on brook trout (Saluelinus fontinalis), demonstrated by a rise in the temperature a t which cold block occurred (Anderson and Peterson, 1969). Knowledge of the interactions between intoxicating compounds and environmental change is important for predicting the effects of altering ecosystems already containing xenobiotic compounds. Organochlorine insecticides are probably the most extensive and persistent additives to the world environment (Miller and Berg, 1969; Rudd, 1966). The general nature of insecticide toxicology (O’Brien, 1967; Kraybill, 1969) suggests that chronic exposure to insecticides may indeed be equivalent to pretreatment for many organisms, including perhaps man (USDHEW, 1969; Deichmann and MacDonald, 1971). To investigate the concept of insecticide pretreatment
Present address, D e p a r t m e n t of E n v i r o n m e n t a l Medicine, School of Hygiene a n d P u b l i c H e a l t h , Johns H o p k i n s University, Baltimore, Md. 21205 1
846
Environmental Science 8, Technology
and its interactions with subsequent environmental changes, the following study was conducted. A fish population was pretreated by exposure to the cyclodiene organochlorine insecticide dieldrin (hexachloroepoxy-endo, exo-dimethanonaphthalene). The level of exposure used was well below the mean lethal concentration for the species of fish studied and within the range of dieldrin residues detected in United States watersheds (Henderson et al., 1971). After 30 days of dieldrin exposure, the fish were exposed to sublethal thermal stress, a t a rate and amount within natural fluctuations for the watershed from which they were collected. The thermal change exerted was within the limit of 10°C above maximum ambient levels suggested by the U.S. Department of Interior in 1968 (USDI, 1968). Experimental Two experiments were run, one in June and one in September 1971. Mature darters (Etheostoma nigrum) were selected from fish captured by seine in the White Clay Creek in southern Chester County, Pennsylvania. The darter is a small Percid, weighing a t maturity between 1.5 and 6.0 grams, with habitat preference and tolerance of environmental change similar to those of brown trout and other important freshwater fish (Love, 1970). Organochlorine insecticides are not known to have been used on the watershed of the upper White Clay Creek for a t least four years, largely because of cooperation by local residents with the ecological research of the Academy of Natural Sciences of Philadelphia. The organochlorine insecticides -lindane, o,p’-DDD, o,p’-DDE, p,p’-DDD, p,p’-DDE, and p,p’-DDT-were detected in stream water samples analyzed with electron capture gas chromatography, but the total concentrations of all these insecticides was below
1
2
3
4
5
6
7
8
9
DAY 5
Figure 1. Rate of temperature change applied during thermal stress for both experiments, measured at 8:OO AM, 12 noon, 4:OO PM, and 8 : O O PM every day Lower solid lines and closed circles indicate water temperature of untreated, unstressed groups; upper solid lines and open circles, water temperature of thermally stressed untreated groups; dotted lines and stars, water temperature of pretreated stressed groups
Table I. Water Temperatures in White Clay Creek for 1971a Month
May June July
August September
Day
Minimum
Maximum
1 10 20 1 10 20 1 10 20 1 10 20 1 10 20
7.0 9.8 13.0 11.6 14.2 15.2 17.9 17.4 16.7 17.8 16.8 15.8 15.7 17.2 16.7
16.9 18.9 19.5 18.7 20.9 20.4 22.9
22.5 19.4 21.3 21.8 20.1 17.5 20.3 19.1
Recorded continuously at the site where most of the fish used in this research were collected.
Table I I . Comparison of Mortality Rates by Treatment Deaths!total population/days Treatment
Experiment i
Experiment II
Controlsa Dieldrin Thermal stress Dieldrin and thermal Controlsb
1711 20130 23/60/30 412011 0 1511811 0 1/22/10
13/120/30 21 / 6 0 / 3 0 2/25/10 12/16/10 2/24/10
a During days 1 30
During thermal stress period
1 ppb a t any one time. Fish from the creek contained residues of lindane, dieldrin, DDT, and its analogs. The residue levels were similar to those found in fish from nonagricultural watersheds throughout the United States (Henderson et al., 1971; Morris and Johnson, 1971). In each experiment, 180 fish were selected and randomly divided into 3 equal groups: fish exposed to dieldrin, fish not exposed to dieldrin but exposed to thermal stress, and fish not exposed to dieldrin or thermal stress. The experimental design partitioned out the effects of confinement and experimental handling from effects of the insecticide and thermal stress. Each group of fish was subdivided into 2 subgroups and held in tanks filled with recirculated stream water. The tanks were made of marine plywood sealed with three coats of Fiberglas. The design provided a riffle area by means of a 3-ft inclined plane and a pool in a 1 x 2-ft area. The amount of water per fish was 10 liters. Except for the addition of insecticide and thermal stress, all parameters of water quality-trace metals, pH, nitrogen, temperature, and dissolved oxygen -were the same for all groups and the same as the White Clay Creek. Dissolved oxygen concentrations were always a t saturation. Fish were fed live caddisfly larvae collected daily from the White Clay Creek. Stomach analyses of wild darters had shown caddisflies to be the major part of their diet. Food was supplied a t 3% of the biomass in each subgroup, which has been found to support average growth rates estimated for the species (Silbergeld, 1972). Sixty fish were exposed to a mean concentration of 2.3 ppb dieldrin (range 2.0-4.0 ppb), less than one-fourth the LC50 for the species (96-hr test) (Silbergeld, 1972). Concentrations were monitored every other day by gas chromatography: the day-to-day variation was attributed to adsorption onto surfaces and to uptake by algae and fish in the tanks. Technical grade dieldrin was dissolved in
pesticide quality acetone and added to the water. Controls were exposed to equal amounts of acetone only. After 30 days, the exposed fish and one group of 60 unexposed fish were subjected to increases in water temperature. Thermal stress was applied a t a rate of 1°C a day, with fluctuations following natural diurnal variations (Figure 1). Total increase in water temperature was about 7°C above ambient for the White Clay Creek. Temperature variation for the Creek during the summer and fall of 1971 is shown in Table I. On several occasions, water temperatures increased over 9" in one day, and the diurnal temperature pulse during the summer is usually about 5.C (Vannote, 1971). Experimentally elevated temperatures were maintained for four days after achievement of maximum elevation. The experiments were terminated on the tenth day of thermal stress to retain live fish in all groups for the analyses described below. Several indicators of metabolism and condition were measured during the pretreatment period, after the application of thermal stress, and a t the end of the experiments. These were: growth rate, measured every 5 days as percent change in weight; feeding rate, the proportion of food provided which the fish ate during the same 5 days; whole blood glucose levels, measured every 5 days by a micromodification of the method of Chavin and Young (1970); oxygen consumption by the live fish, measured every 10 days; rate of opercular movement, measured in conjunction with oxygen consumption: whole body lipid content, measured every 5 days by an adaptation of the method of Dambergs; condition of the liver, defined as the ratio of liver weight to whole body weight; and liver histology, the incidence of hemorrhage, pyknosis, necrosis, and lipidosis.
Results The incidence of mortality by comparison of mortality rates clearly shows the impact of dieldrin on the ability of fish to survive thermal stress (Table 11). During the pretreatment period, an average of 12.5% (13-17) of control fish died in both experiments. This mortality may be attributable to confinement and possibly to a diet restricted to caddisfly larvae. During the same period, 36.77~(21-23) of the fish exposed to dieldrin died. This is only slightly higher than the number expected from prior tests for LC50. During the 10 days of thermal stress, 4.4% (1-2) of control fish died. The decline in control mortality from the first 30 days probably results from adjustment to experimental conditions. During thermal stress, 1 3 7 ~(2-4) of the fish exposed to thermal stress died. This is not significantly different from the mortality of the group during the first 30 days. It is, however, significantly higher than the mortality for the controls during the same period. Finally, of the fish pretreated with dieldrin and then exposed to thermal stress, 76.570 (12-15) died during the time they were subjected to thermal stress. The mortality of this population was significantly higher than either that of the same group exposed only to dieldrin or that of the group exposed only to thermal stress. The time course of deaths is important in understanding the results of the measurements mentioned above. During the pretreatment period, the incidence of mortality is shown in Table 111. ?\;early half the deaths in the pretreated group (43.5% in the first experiment and 52.4% in the second) occurred between days 11 and 15. Between days 11 and 15, the greatest alteration occurred in many metabolic and condition measurements of pretreated fish. Whole blood glucose levels peaked a t day 10 (Figure 2 ) . By day 15, the levels of blood glucose in Volume 7. Number 9, September 1973 847
Table 1 1 1 . Comparison of Incidence of Mortality, Days 1 Through 30, for Pretreated Fish and Controls Number dead Days Treatment
Controls, Experiment I Experiment I I Dieldrin, Experiment I Experiment I I
15
610
1115
2 1
2 2 4 4
4
0 2
1 10 1
1620
2125
2630
4
3
2
1 1
2 0 3
17 3 6 23 0 2 1
6 3 1
Total
1
T 110
100 90
s
80 I
\
P 70
\
60 50 1
2
5
1
+*-
10
15
* *** *
A
20 DAY 5
30
40
Figure 2. Means and standard deviations of blood glucose levels, in mg/100 ml Solid line indicates controls: dotted line, fish pretreated with dieldrin; starred line, fish exposed to thermal stress with no pretreatment
pretreated fish fell to approximately initial levels for all fish. The mean level of the pretreated fish was still significantly higher than the mean for controls. Blood glucose levels in controls declined over the experiment toward the mean of 37 mg/100 ml found in wild fish. The elevation in initial values probably resulted from the stress of confinement, as fish have the same adrenal-pituitary-glucocorticoid stress reaction as mammals (Chester Jones, 1960). The biphasic pattern of response to dieldrin was also seen in oxygen consumption. Until day 15, pretreated fish consumed significantly less oxygen per gram of body weight than did controls. After day 15, the consumption of oxygen by pretreated fish increased, but remained significantly different in slope and intercept from the consumption of oxygen by controls (significance determined by comparison of correlation coefficients, mg oxygen consumed/gram of fish/hour regressed against the whole body weight of the fish; all differences significant a t p
