Environ. Sci. Techno/. 1995, 29, 613-621
Effects of Sublethal Exposure on lethal Body Burdens of Narcotic Organic Chemicals in Daphnia A N D R E W V. PAWLISZ*" A N D ROBERT H. PETERSi AVP Consultants Ltd., 351 St. Joseph Boulevard, 8th Floor, Hull, Quebec, K I A OH3 Canada, and Department of Biology, McGill University, 1205 Dr. Penfield Avenue, Montreal, Quebec, H3A 1B1 Canada
This work examines the hypothesis that exposure of Daphnia magna to sublethal levels of narcotic contaminants may affect subsequent sensitivity of the animals. Prior exposure (24 h) of Daphniato sublethal levels of acetone, acetophenone, benzene, benzoic acid, butanol, 1,2-dichIorobenzene, 2-methylnaphthalene, 1,2,4-trichlorobenzene, 1,1,2,2tetrachloroethane, and toluene had no effect on their sensitivity to effective levels of these chemicals in eight of 10 cases. Effective burdens (24-h acute exposure) were independent of the sublethal body burdens (24-h sublethal exposure) and of the sublethal water concentrations ( P < 0.025) in eight of 10 cases. Butanol and benzoic acid were the exceptions. The null hypothesis was then accepted in 80% of the cases. These results imply that animals from polluted sites should be no more resistantto high body residues of pollutants than those from clean sites and that the toxicity of narcotic organic contaminants may be independent of the time course of uptake.
Introduction Exposure to toxic levels of contaminants usually occurs after rare chemicalspills and near point sources of industrial effluents, so aquatic organisms seldom experience high concentrations of environmental pollutants. Most of the time, aquatic biota endure sublethal levels of contaminants (1). Consequently, examination of the effects of sublethal levels of contaminants on aquatic biota is often more relevant than the investigation of the effects of lethal contamination (2, 3). Experiments on nonlethal toxicity have revealed many detrimental effects of sublethal exposure includingreduction in body size, fecundity, mobility, suMval rate, induction of cancer, sensory impairment, and increase or decrease in tolerance (4-9). In this paper, we are concerned that sublethal exposure may make animals more or less sensitive so that bioaccumulation, ecological magnification,and toxicity on subsequent acute exposures change with the schedule of exposure to sublethal levels of pollutants. Whether sublethal exposures make organisms more or less sensitive, one can measure such changes as the amount of contaminant needed to narcotize animals. By change in sensitivity,we mean change in tolerance, which implies shift in the dose that can be withstood by animals indefinitely. If some of the sublethal burden is sequestered in metabolically inactive compartments or if the animals can somehow physiologically acclimate to a contaminant, sublethal exposure might result in higher effective body residues of contaminants. Examples of increased tolerance to toxins are frequent in medicine (10) and aquatic studies (8,11-131. The ecotoxicological implications of increased tolerance are that sublethally exposed animals would be less affected by pulses of acute toxicity from spills or industry and die with higher lethal burdens of pollutants in their tissues. Increased sensitivity, even if less common, is environmentally more threatening because it implies that low-level exposure may weaken the tolerance of organisms and, by extension, natural ecosystems. However, the null hypothesis of unchanged sensitivities is also tenable. There is evidence that animals die when body burdens of nonpolar, narcotic, organic chemicals reach a relatively constant level (14, 1 3 , and presumably this level is independent of the time course of narcotic accumulation. Although the whole body residue is a much better estimate of a dose than is the ambient concentration, it remains a surrogate and should be viewed as such. Narcotic contaminants are particularly important toxicants since some of these chemicals are widely used in industry and are released into the aquatic environment in large quantities, but at sublethal concentrations (16). The implication of the null hypothesis that the exposure history has little influence on the effective body burdens is that animals from locations affected by sublethal pollution should not differ in tolerance and should not die with differentquantities of contaminants in tissues than animals from unpolluted sites when challenged by high levels of pollutants. This consistency is thought to show that all * Corresponding author; Telephone: 819-953-0571;FAX: 819-9530461. +
AVP Consultants Ltd.
* McGill University. 0013-936X/95/0929-0613$09.00/0
1995 American Chemical Society
VOL. 29, NO. 3,1995 / ENVIRONMENTAL SCIENCE &TECHNOLOGY 1613
TABLE 1
Source, Purity, log Partition Coefficient, 48-h lC50 for Daphnia magna and 10 Narcotic Organic Chemicals Used” specific activity chemical
source
purity (%)
KO,
48-h LC50 (mmol/L)
supplied (GBq/mmol)
adjusted (MBq/mmol)
acetone butanol acetophenone benzoic acid benzene toluene 1,1,2,2-tetrachloroethane 1,2-dichlorobenzene 2-methylnaphthalene 1,2,4-trichlorobenzene
Sigma Sigma Sigma Sigma Sigma Chemsyn Chemsyn Sigma Sigma Sigma
>98 ’98 > 98 > 98 298 > 98 > 86 > 98 > 98 > 98
-0.26 0.88 1.6 1.9 2.1 2.6 2.8 3.4 3.9 4.2
131 219 4.4 0.0016 0.035 0.055 0.095 0.00089 0.021 0.16
0.10 0.29 0.65 0.08 1.87 0.08 0.18 0.33 0.33 0.20
0.27 0.014 1.24 5.6 1867 13.3 13.8 0.28 325 0.27
”The supplied specific activity was adjusted by dilution with the unlabelled forms of these chemicals to yield the adjusted specific activity.
narcotic organic compounds are equipotent (17) and that all sites of toxic action are equisensitive (18). Because all narcotics affect organisms in the same way, conclusions reached for one set of narcotic contaminants should apply to the remaining thousands of chemicals that constitute this group. This study examines the effects of sublethal exposure to 10, narcotic organic chemicals (acetone, acetophenone, benzoic acid, benzene, butanol, 1,2-dichlorobenzene, 2-methylnaphthalene, 1,2,4-trichlorobenzene, 1,1,2,2-tetrachloroethane, and toluene) on the effective body burdens in Daphnia magna exposed to effective body burdens of the same compounds. If narcotic compounds are equipotential, animals should die with approximately constant levels of narcotic pollutants in their bodies (14,15), and sublethal exposure should have little effect on the effective levels of pollutants in Daphnia. In other words, the hypothesis of equipotentiality implies that the effective residues of contaminants in Daphnia exposed to lethal concentrations of the same chemicals will not be affected by sublethal exposure. Therefore, there should be no significant relationship between the effectiveburdens and the sublethal water concentrations or between the effective and the sublethal body burdens of the narcotic contaminants. We also expect that the mean effective residues of narcotic compounds in animals that were sublethally exposed should not differ from the mean effective residues in animals that were only exposed lethally.
Experimental Section Parthenogenetic female Daphnia were raised in glass beakers (1-L) containing standard reconstituted water (19). Animals were fed daily, and each beaker received enough algal suspension to keep the culture water slightly green (20). Beakers were incubated at 20 “C under constant photoperiod (16-h light: 8-h dark). Culture water was replaced weekly. All radiochemicals (14C) were purchased from commercial suppliers (Sigma,St. Louis, MO; Chemsyn, Lenexa, KS). The specific activities ranged from 0.080 (toluene] to 1.87 nBq/mmol (benzene), and the purities (supplierdetermined) ranged from more than 86% for 1,1,2,2tetrachloroethane to more than 98% for the other nine compounds (Table 1). Before testing, all radiochemicals were diluted with their chemical counterparts (Sigma, St. Louis, MO) to lower the specific activity (Table 1) and to increase the volume of the radiolabeled chemical. 014
ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29, NO. 3, 1995
Test solutions were formulated by introducing a known quantity of each radiochemical (48-h LC50; Table 1) into a volumetric flask (1-L) containing standard reconstituted water. The flask was sealed and wrapped in aluminum foil to reduce evaporation and the potential for photodegradation (21). After moderate agitation by hand for a few seconds, the vessel was placed on a magnetic heater-stirrer at 20 “C for 24-72 h. Sublethal solutions were derived by serial dilutions of 48 LC5O’s for each chemical with reconstituted water (nominal concentrations 50%, 25%, 12.5%,and 6.3% of 48-h LC50). The actual concentrations of radiochemicals in lethal and sublethal solutions were evaluated by placing a 50-pL sample of a given solution into a scintillation vial containing Ready Protein (Beckman, Ontario) scintillation fluid (2 mL) and measuring the radioactivitywitha scintillationcounter (Wallac LKB, Turku, Finland). The measured radioactivities were corrected for quench and background radiation and were divided by the specific activities of each radiochemical to estimate the actual concentrations of narcotics (mmol/L)in water. Water samples were taken before and after each experimental run. Sublethal exposure was begun by placing 20 daphnids into four 20-mL screw-top vials containing 18 mL of four different sublethal solutions (50%,25%, 12.5%,and 6.3% of 48-h LC50). A fifth vial, filled with dilution water, served as the control. No food or replacement water was provided during the test. After 24 h, animals were removed with a pipet, rinsed with clean water, and all but five animals from each vial were transferred to another vial containing 18 mL of a toxic solution (48-hLC50; Table 1)ofthe same chemical and exposed for another 24 h. The five remaining animals from each vial are here termed “sublethally exposed”, whereas narcotized animals removed from the lethal solutions 24 h later are termed “previously exposed”. Each experimental run also included one vial containing LC50 solution of a given contaminant and 10, 24-h starved daphnids from the control. Animals in that vial were exposed only to lethal solutions (24h), and they are termed “24-hlethally exposed”. All sublethal and lethal vials were tightly capped to prevent possible volatilization of test compounds. Although 24 h of sublethal and 24 h of lethal exposure is short, substantially longer exposures of unfed Daphnia are not practical because such animals would die of starvation. Longer experiments would be more complicated because animals would require food that also takes up
contaminants. Nevertheless, because the lifespan of Daphnia is about 2 months (22), 24 h is a physiologically substantial exposure that is comparable to 1 week in the life of a fathead minnow or 1 year of a human. To determine whether 24-h lethal exposure leads to different body residues than does the standard 48-h exposure, body residues from narcotized Daphnia from 24-h exposures in this study were compared with those residues from 48-h exposures in a previous study (15).The animals from the earlier work are here termed “48-hlethally exposed”. Prior to residue analysis, the length of each daphnid (L; mm) was measured with an ocular micrometer, and the wet weight (W;pg) was determined using an allometric relationship log W = 3.39 log L
r = 0.93
+ log 1.70
P -= 0.0001
n = 94
At that time, Daphnia were also inspected for signs of narcosis. Animals were considered narcotized when they ceased to swim while maintaining the movement of the appendages, the digestive track, andlor the filtering apparatus. Only the narcotizedanimalswere analyzed further. Because some vials containing acetone, acetophenone, 1,2dichlorobenzene,and 1,2,4-trichlorobenzenesuffered 100% mortalities, they were not included in further analysis. In consequence, the number of body burden estimates in subsequent analyses varies across chemicals. The analysis of body residues of radiochemicals consisted of placing each daphnid individually in 2.0 mL of Beckman Ready Protein scintillation cocktail in a 7.0-mL glass scintillation vial. Later, all vials were left undisturbed overnight in a cool room to allow for digestion and subsidence of chemiluminescence. The next morning, each vial was vortexed for 15 s and assayed for radioactivity. Counting time was adjusted so the counting error was less than 2% of the mean. After correction for background radiation, the measured radioactivities were expressed as millimoles of contaminant per kilogram ofwet bodyweight. Each experimental run was replicated at least five times. Statistical Analysis. All body residues were logtransformed to stabilize variance and the individual estimates (subsamples) of body residues for each test vial were combined into log-meansto reduce pseudo-replication(23). An ANOVA was performed to examine the variance in the body burden measurements. General linear models [log y = b log x + log a] were constructed to relate the body burdens from sublethally exposed or previously exposed animals (j) to the sublethalwater concentrations (x). These were later tested for differences using a t-test (23). The models relating body burdens from previously exposed animals and sublethally exposed animals were constructed similarly. Body burdens from the previously exposed or 24-hlethally exposed Daphnia were pooled into means for each treatment and tested for differences using a t-test (23). The probability levels for all regressions and t-tests were adjusted for multiple comparisons using the Bonferroni correction (24) to allow for the increased likelihood of i d e n w n g significant relationship in multiple comparisons with standard statistical procedures. All statistical analyses used SAS analytical software (25).
Results and Discussion To determine whether exposure to a range of sublethal levels of narcotics has the desired effect of producing a range of sublethal body burdens, we plotted the sublethal body burdens against the sublethal water concentrations. If the tissue concentrations of narcotic contaminants in Daphnia vary accordingto the magnitude of sublethal water concentration, body burdens in the sublethally exposed animals and sublethal water concentrations should be positively correlated. The correlations for six chemicals agreed with anticipated results. Body burdens of butanol, benzoic acid, toluene, 1,1,2,2-tetrachloroethane, 1,2-dichlorobenzene, and 2-methylnaphthalene in the sublethally exposed animals were significantly and positively correlated with the sublethal water concentrations of the same compounds. The probability levels of these relationships ranged from 0.0001 (2-methylnaphthalene, 1,1,2,2-tetrachloroethane, and toluene) to 0.01 (butanol; mean = 0.003; SD = 0.004; Figure 11, and the correlation coefficients (r) ranged from 0.26 (butanol) to 0.95 (1,2-dichlorobenzene;mean = 0.71; SD = 0.55; Figure 1). Regression between the sublethal body burdens and the sublethal exposure concentrations for the remaining four chemicals (acetone, acetophenone, benzene, and 1,2,4trichlorobenzene) were not significant (P > 0.05). That lack of trend suggests that the body burdens in the sublethally exposed animals are independent of the sublethal water concentrations for those four chemicals. This is contrary to expectation and suggests that the variation in body residues was large relative to the 4-fold change in the external concentration. The latter is alikely explanation because the body burdens of narcotics in Daphnia vary over 2 orders of magnitude (26). This problem was compounded in four cases (acetone, acetophenone, 1,2dichlorobenzene, 1,2,4-trichlorobenzene)where high mortality reduced sample sizes. The hypothesis that prior exposure to the sublethal concentration of narcotics has no effect on the internal concentration needed to immobilize animals implies that prior exposure to sublethal concentrations of contaminants should not affect the body burdens of animals narcotized by subsequent lethal exposure. When the mean body burdens of previously exposed Daphnia were compared to the mean body burdens from24-h lethally exposed animals, no statistical differences at narcosis were identified in eight of 10 cases (Figure 2). Of the 10 possible relationships, only two (butanol and benzoic acid) were significant. Butanol and benzoic acid were exceptions in that the 24-h lethally exposed animals in both cases were narcotized at lower body burdens than the previously exposed animals. The desensitizing effect of sublethal exposure to butanol and benzoic acid is inconsistent with the hypothesis of equipotentiality (14,15,27,28)and suggeststhat the tissue residues of these two narcotics associated with narcosis and death vary with the schedule of exposure of Daphnia to sublethal levels of narcotic contaminants. Nevertheless, the hypothesis of no effect of prior sublethal exposure on effective body burden is supported by the remaining 80% of the data (Figure 2). These experiments suggest that daphnids can acclimate to butanol and benzoic acid. Similar observations have been made for fish, where sublethal exposure to metals resulted in acclimation to that contaminant, so sublethally VOL. 29, N O . 3, 1995 /ENVIRONMENTAL SCIENCE &TECHNOLOGY
615
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Sublethal water concentrations (mmoV1) FIGURE 1. Relationships (y= bx+ a) between the sublethal body burdens (v) and the sublethal water Concentrations (x)for Daphnia magna and 10 neutral narcotic compounds. Significant relationships (P= 5%) are marked by an asterisk. SE stands for the standard error of the slope, N stands for number of estimates composing each model, and r stands for the correlation coefficient. Vertical bars represent 1 SE of body burdens of all animals in each vial. 616 m ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29, NO. 3, 1995
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exposed fish were more resistant to subsequent lethal doses of these pollutants (8, 12, 13). Thus, the phenomenon of increased tolerance after sublethal exposure is not unknown, although the mechanisms involved may be very different because our study deals with organic chemicals whereas literature considers metals. The unexpected results for butanol and benzoic acid could also be explained if these two narcotic compounds are rapidly broken down so radiotracer burdens do not measure the body burdens of the parent compounds. Because butanol and benzoic acid contain hydroxyl and carboxyl functional groups, they can be readily oxidized by the mixed functions oxygenases (MFO) of Daphnia and made less toxic. Prior exposure may even have mobilized the MFO defense mechanism so that the MFO-active Daphnia, exposed to lethal levels of contaminants, could tolerate higher levels of contaminants than those that were not previously exposed. Thus, higher residues of butanol and benzoic acid in previously exposed animals (Figure 2) might indicate that Daphnia can become more resistant during the sublethal exposure and thus more tolerant of higher quantities of contaminants during the subsequent lethal challenge. Pawlisz and Peters (13 invoked a similar argument to explain the high effective body burdens of acetone and butanol relative to other chemicals. Daphnia showed no acclimation to acetone in these experiments,
but perhaps this is a result of limited sample size, even faster metabolism, or quick depuration of this compound. Yet another possibility for the observed discrepancy between the expected and the observed results for butanol and benzoic acid might be the low partition coefficients of the two compounds. Because both compounds are lipophobic, where the partition coefficients for butanol and benzoic acid are 7.6 and 79 (Table 11, the majority of wholebody residue would be located in the aqueous phase of Daphnia. Therefore, the measured body burdens would not be representative of the quantities of these chemicals found in the lipid phase of Daphnia where the sites of toxic action are usually found. Hence, the observed relationships might be an artifact of butanol and benzene partitioning into the response-inert, aqueous phase of Daphnia. Although benzoic acid was initially thought to be a neutral narcotic, evidence exists to show that it might be a polar narcotic. In a study by Geiger et al. (27),fathead minnow tests were complicated by the fact that benzoic acid ionizes in the presence of water and exhibits higher toxicity than would be expected from a narcotic compound. Therefore, comparisons with neutral narcotics were not possible. The same is probably true in our study. Benzoic acid, being a polar narcotic, behaves unlike the neutral narcotics and leads to the observed discrepancies. Perhaps benzoic acid should be omitted from future investigations VOL. 29, NO. 3, 1995 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 1617
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VOL. 29, NO. 3, 1995 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
619
unless it is used as a reference for the polar narcotics. In a previous study (13,we measured the effectivebody burdens in Daphnia exposed to the same narcotics for 48 h under otherwise similar conditions. The hypothesis of equipotentiality suggests that the body burdens from that study should also be similar to those exhibited by the 24-h lethally exposed and the previously exposed animals. Comparison of the two data sets revealed significant differences in body burdens with a schedule of exposure only for 1,2,4-trichlorobenzene(Figure2). In that case, the effective burdens in previously exposed animals were less than those in 48-h lethally exposed animals. Therefore, Daphnia first exposed to low levels of 1,2,4-trichlorobenzene for 24 h appeared to be more sensitive than those exposed to high levels for 48 h. No other narcotic behaved similarly, and for the present the observation is no more than an interesting anomaly. If sublethal exposure of Daphnia to narcotic compounds affects the effective body burdens, increasingly higher sublethal levels of pollutants should result in increasingly higher body burdens at narcosis. The rationale behind that hypothesis is that increasing sublethal levels of contaminants should result in higher levels of enzyme induction and increasingly resistant animals that have higher thresholds for a specific response. In consequence, there should be a correlation between the body burdens in previously exposed animals and the sublethal water concentrations. Ten such relationships were constructed, and eight agreed with the hypothesis in that they showed no significant relationship between effective and sublethal burdens (Figure 3). Butanol and benzoic acid produced significant relationships between these two variables, but the relationships were weak ( P = 0.02 0.01 and r = 0.41 and 0.26, respectively;Figure 3). Therefore, these results suggest that the sublethal exposure has little or no effect on the effective body burdens, and the hypothesis that sublethal exposure has no effect on effective body burdens is confirmed in at least 80%ofthe cases. Extendingthat conclusion to butanol and benzoic acid would not affect predictions about toxicity, because the relations for those chemicals are too weak to be predictive. Because the relationships for butanol and benzoic acid had rather low probabilities, large variance in body burden estimates, and low coefficients of determination, it seemed possible that the observed relationships could be due to a few outliers. However, re-analysis of these data without obvious outliers had little effect on the overall relationship. Thus, outliers did not have much effect on the overall relationship. If sublethal exposure affects the equipotentiality of narcotic compounds, there should be a consistent relationship between effectivebody burdens and the sublethal exposure concentrations for each chemical. In that case, body burdens in both sublethally exposed and previously exposed animals would be significantlyrelated to exposure concentration,and these two relationshipswould be parallel to one another. However, all but two cases (butanol and benzoic acid) did not have the necessary combination of positive relationships and parallel slopes that imply a consistent effect of sublethal exposure. Butanol and benzoic acid yielded significant(Figures 1and 3) and parallel relationships (P = 5%; t-test (23)). A more direct approach for detecting the effects of the sublethal exposure is to correlate body burdens in previously exposed animals with the body burdens in sublethally exposed animals. The hypothesis of equipotentiality leads 620
1
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29, NO. 3, 1995
one to expect that body burdens from previously exposed and sublethally exposed animals would not be correlated. Regression analyses for each chemical revealed that indeed in majority of the cases significant relationships were not found. There were significantrelationships only for butanol ( P = 0.0001; r = 0.591, benzoic acid ( P = 0.005; r = 0.59), and 2-methylnaphthalene ( P = 0.0095; r = 0.41; Figure 4), but once again these were weak. These results reinforce earlier conclusions that sublethal exposure has little or no effect on effective body burdens of narcotic chemicals in Daphnia.
Acknowledgments Many thanksto Andrea and Worth Everett for their technical assistance. This work was funded by GLURF, FCAR, and NSERC grants.
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Received for review April 28, 1994. Revised manuscript received November 7, 1994. Accepted December 9, 2994.@ E389402664 @
Abstract published in Advance ACS Abstracts, January 15, 1995.
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