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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 .
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 . 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 . 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 . 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 , 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 firstname.lastname@example.org.
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 . 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.  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 . 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 . 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 . 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
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