Acute and chronic effects of diphenhydramine and sertraline mixtures in ceriodaphnia dubia

Environmental Toxicology and Chemistry, Vol. 32, No. 12, pp. xx–xx, 2013 ACUTE AND CHRONIC EFFECTS OF DIPHENHYDRAMINE AND SERTRALINE MIXTURES IN CERIODAPHNIA DUBIA ERIC W. GOOLSBY,y CHASE M. MASON,z JAMES T. WOJCIK,y ALEX M. JORDAN,y and MARSHA C. BLACK*y yDepartment of Environmental Health Science, University of Georgia, Athens, Georgia, USA zDepartment of Plant Biology, University of Georgia, Athens, Georgia, USA (Submitted 28 February 2013; Returned for Revision 21 April 2013; Accepted 25 August 2013) Abstract: Ceriodaphnia dubia were tested to evaluate the acute and chronic interactive effects of diphenhydramine and sertraline.
Observed effects were compared with 2 reference toxicity models, the concentration addition model and the independent action model.
Results indicate that the 2 drugs exhibit additive toxicity in C. dubia. In some cases, individually sublethal concentrations of the chemicalsresulted in 100% mortality when combined, demonstrating the potentially severe impact of trace environmental contaminants. EnvironToxicol Chem 2013;32:xx–xx. # 2013 SETAC which have been documented to be present in surface waters at In recent years, trace amounts of pharmaceuticals have been concentrations up to 0.1 mg/L [6–10], were selected for the detected in surface waters throughout the United States [1].
present study due to their pharmacologically distinct properties However, little information exists regarding the effects these yet similar mechanisms of action through inhibition of serotonin chemicals may have on the environment. It has been widely reuptake. The objective of the present study was to assess the assumed that low concentrations (<1 mg/L) pose little or no acute and chronic effects of combined diphenhydramine and threat to aquatic organisms, but recent studies indicate the sertraline exposure to Ceriodaphnia dubia. Acute mortality and potential for certain chemicals to adversely affect the health of reproductive effects of sertraline and diphenhydramine were ecosystems [2]. Particularly, drugs that are active on the central assessed and compared to predicted effects by 2 joint action nervous and endocrine systems may cause significant physio- reference models, the concentration addition model and the logical changes in aquatic organisms [3]. Additionally, the independent action model. The concentration addition model effects of multiple drugs with similar mechanisms of action may assumes a similar mechanism of action for toxicants, so result in additive or synergistic toxicity [4]. The complexity of individual effects from chemicals are assumed to contribute to drug interactions and the lack of data regarding the effects of toxic effects additively when combined [4,11,12]. The indepen- pharmaceuticals on aquatic organisms make it difficult to dent action model assumes dissimilar mechanisms of action in anticipate how aquatic environments may be affected by the which toxic effects from chemicals act independently, hypothet- ically resulting in a subadditive response [4,13–15].
Selective serotonin reuptake inhibitors are a class of prescription drugs prescribed to manage conditions such as depression, anxiety, obsessive–compulsive disorder, and other psychiatric pathologies. Sertraline (Zoloft; Pfizer) is one of themost frequently prescribed selective serotonin reuptake inhib- Ceriodaphnia dubia neonates were obtained from Aquatic itors in the United States. In humans, selective serotonin Biosystems and were acclimated for approximately 1 mo prior to reuptake inhibitors cause an increase in extracellular serotonin testing. Organisms were maintained in a stock culture consisting concentrations by blocking reuptake into neurons at the synaptic of 60 organisms cultured individually in 30-mL plastic cups cleft. Diphenhydramine (Benadryl; McNeil-PPC) is a widely filled with 15 mL moderately hard water, changed daily. In used over-the-counter antihistamine. The primary mechanism of accordance with US Environmental Protection Agency guide- action for diphenhydramine is antagonism of the histamine H lines for C. dubia culture maintenance and toxicity experi- receptor, which blocks histamine from binding to receptors and ments [16,17], the culture was maintained in an incubator at reduces histamine-mediated allergic responses. Diphenhydra- 25 8C with a 16:8-h light:dark photoperiod, and each individual mine is also an inhibitor of serotonin reuptake at the synaptic was fed daily with 100 mL of a mixture of yeast, cerophyl, and cleft [5], although it is not prescribed as an antidepressant.
trout chow (Aquatic Biosystems) and 100 mL algae (Selenas- Research in the area of combined chemical toxicity is trum spp.; Aquatic Biosystems). Moderately hard water was necessary to anticipate how multiple pharmaceuticals may affect prepared with 1.20 g MgSO4, 1.92 g NaHCO3, 0.080 g KCl, and aquatic environments. Diphenhydramine and sertraline, both of 1.20 g CaSO4 · 2 H2O added to 20 L Milli-Q water [16,17],which was aerated and conditioned for 48 h prior to use. Water-quality parameters were assessed prior to use of moderately hard * Address correspondence to mblack@uga.edu.
water to ensure that the following ranges were met: pH of 7.9 to Published online 2 September 2013 in Wiley Online Library 8.4, hardness of 80 mg/L to 90 mg/L (as CaCO of 60 mg/L to 80 mg/L (as CaCO3). The fecundity of each individual was recorded daily, and a new stock culture was tion addition model and the independent action model [19]. The initiated from the neonates of every fourth brood. The culture concentration addition model [4,11,12], defined below, assumes was allowed to acclimate to laboratory conditions for 5 similar mechanisms of toxicity between mixtures and states that generations prior to initiation of experiments. Culture quality for n chemicals exhibiting additive toxicity, the quotients of and sensitivity were verified with acute 48-h reference toxicity concentration c for chemical i and the concentration of chemical i tests using CuSO4, and acute median lethal mortality for CuSO4 that produces an x% response when applied individually sum to 1 was within acceptable laboratory ranges (34.8 mg Cu2þ/L).
Preliminary 48-h acute and 7-d chronic tests were performed to determine appropriate concentration ranges for experiments.
For acute experiments, 117 combinations of concentrations of mixtures of diphenhydramine (0.0–4.5 mg/L) and sertraline(0.0–0.6 mg/L) were prepared with 4 replicates per treatment.
The independent action model [4,13–15] assumes dissimilar For chronic tests, 24 combinations with varying diphenhydra- mechanisms of toxicity and states that for n chemicals, the mine (0.0–3.0 mg/L) and sertraline (0.0–0.5 mg/L) concentra- product of each chemical’s individual probability of nonre- tions were prepared with 10 replicates per treatment. Neonates sponse (1–E[xi]) subtracted from 1 equals the expected response younger than 8 h were collected from third or fourth broods and to a mixture of the chemicals E(x1,2…n) pipetted into 30-mL plastic cups, each containing 15 mL test solution (moderately hard water with concentrations of diphenhydramine and sertraline based on the treatment group); 100 mL yeast, cerophyl, and trout chow; and 100 mL algae(Selenastrum spp.). For acute tests, each cup received 5 to 8neonates (total number of C. dubia individuals ¼ 2466), and All models were compared using Wald 95% confidence mortality (indicated by lack of movement within 30 s of intervals, which were calculated by multiplying the standard observation) was recorded after 48 h. For chronic tests, each errors of regression coefficients by z0.025, the 97.5th percentile cup received a single neonate (total number of C. dubia point of the normal distribution ($1.96).
individuals ¼ 240), and the number of living offspring producedby each individual was recorded daily until more than 60% of the Logistic regressions of observed data significantly explained C. dubia acute and chronic effects in response to diphenhydra- mine and sertraline exposures (Wald x2, p < 0.0001). Acute 48-h Observed and predicted concentration–response relation- LC50 values (95% confidence interval) for diphenhydramine and ships were modeled for acute mortality and chronic reproductive sertraline were determined to be 3.94 mg/L (3.77–4.15 mg/L) and effects using logistic regressions in SAS (PROC LOGISTIC, Ver 0.433 mg/L (0.417–0.449 mg/L), respectively. Chronic EC50 9.2; SAS Institute) according to the following formula values (95% confidence interval) for reproduction were deter-mined to be 0.991 mg/L (0.525–1.52 mg/L) for diphenhydramine and 0.184 mg/L (0.101–0.274 mg/L) for sertraline. These resultsare comparable to results from similar studies involving C. dubia Logit(y) is the log of the probability of the expected effect and Daphnia magna [3,20,21], with the exception of the 48-h (mortality for acute tests, total offspring number for chronic acute LC50 for diphenhydramine in D. magna, which Berninger tests), and m, x, and b represent the slope of the regression, the et al. [8] determined to be 0.374 mg/L, approximately 1 order of concentration of the toxicant, and the regression intercept value, magnitude lower than diphenhydramine acute LC50 values for C.
respectively. The probability of the expected effect, y, for any dubia (3.94 mg/L). This discrepancy in sensitivities may be concentration, x, was calculated using the following formula: indicative of differences in sensitivities or distinct mechanisms oftoxicity between the 2 species.
Observed and predicted (both concentration addition and independent action) concentration–response relationships weremodeled with logistic regressions (Figure 1). Quantitative Acute and chronic median lethal and effective concentrations comparisons of regressions using likelihood ratio tests for acute (LC50 and EC50, respectively) for diphenhydramine and mortality showed that all 3 regressions differed significantly sertraline were estimated using logistic regressions generated from one another (Table 1). While neither the concentration from each chemical individually. Fieller’s procedure was used to addition nor the independent action model significantly calculate 95% confidence intervals for each LC50 and EC50 predicted acute mortality, the observed regression fell between value [18]. Concentrations for each chemical were converted the regressions for the 2 predictive models, possibly reflecting into toxic units based on LC50 or EC50 values for individual the drugs’ overlapping pharmacological properties (inhibition of toxicants [15]. Toxic units are calculated by dividing the serotonin reuptake) as well as each chemical’s distinct concentration for a specific toxicant (c mechanism of action. However, the ability of either model to predict toxic effects resulting from mixtures of differentsubstances does not necessarily support the hypotheses of similar or dissimilar mechanisms of toxicity respectivelyassumed by the models [14]. Furthermore, differences detected Using toxic units, logistic regressions were generated to by statistical tests should be interpreted with consideration to model observed mortality and reproduction. The observed biological relevance. For chronic tests, the observed regression regressions were then compared using a likelihood ratio test to was not significantly different from either the concentration compare observed effects to predicted effects by the concentra- addition or the independent action model (Table 1).
Toxicity of diphenhydramine and sertraline mixtures Figure 1. Logistic regressions of observed and predicated (concentration addition [CA] and independent action [IA]) concentration–response relationships for (A)48-h acute Ceriodaphnia dubia mortality and (B) 7-d reproduction in response to individual and mixed concentrations of diphenhydramine and sertraline. Shadedregions indicate 95% confidence interval for additive model (CA). Regions outside of the concentration addition confidence interval are designated as synergisticor antagonistic; regions within the concentration addition confidence interval are additive. Concentrations are given in toxic units (TU). For acute mortality, 1 toxicunit ¼ 3.94 mg/L diphenhydramine ¼ 0.433 mg/L sertraline. For chronic reproduction, 1 toxic unit ¼ 0.991 mg/L diphenhydramine ¼ 0.184 mg/L sertraline.
Observed data points represent the mean effect for each treatment (acute n ¼ 4, chronic n ¼ 10).
To qualitatively distinguish deviations from additive toxic toxicity was observed, the highest concentrations that produced interactions (synergism or antagonism), 95% confidence no acute mortality for single-compound exposures of diphenhy- intervals for the concentration addition model were used as dramine (2.5 mg/L) and sertraline (0.3 mg/L) resulted in 100% reference boundaries for interactive effects. Observed regres- mortality when combined. These results indicate that apparently sions for both acute and chronic experiments fell completely nontoxic concentrations of nonsynergistic chemicals can within the additive region (Figure 1). Although no synergistic drastically affect the health of exposed organisms when exposed Table 1. Likelihood ratio contrasts of regression models for observed 8. Berninger JP, Du BW, Connors KA, Eytcheson SA, Kolkmeier MA, Ceriodaphnia dubia and concentration addition (CA) and independent action Prosser KN, Valenti TW, Chambliss CK, Brooks BW. 2011. Effects of the antihistamine diphenhydramine on selected aquatic organisms.
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