Science of the Total Environment 832 (2022) 155017

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Low doses and lifecycle exposure of waterborne antidepressants in zebrafish

model: A survey on sperm traits, reproductive behaviours, and
transcriptome responses
Xiangsheng Hong a,c, Rui Chen a, Le Zhang a,d, Liang Yan a,d, Jiasu Li a, Jinmiao Zha a,b,d,
⁎
a
Key Laboratory of Drinking Water Science and Technology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
b
Beijing Key Laboratory of Industrial Wastewater Treatment and Reuse, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
c
State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
d
University of Chinese Academy of Sciences, Beijing 100049, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Venlafaxine and citalopram reduced egg

production, but did not impact fertiliza-
tion rate.
• Venlafaxine and citalopram did not affect
sperm quality.
• Venlafaxine and citalopram weakened re-
productive behaviour.
• Transcriptomic profiling of zebrafish
ovary reveals impaired ovarian regenera-
tion and energy metabolism.

A R T I C L E I N F O A B S T R A C T

Editor: Henner Hollert Venlafaxine and citalopram have been commonly found in surface water and may disrupt fish reproduction, yet the
long-term impact and the underlying mechanism are largely unknown. Here, zebrafish were exposed to 0.1–100
Keywords: μg/L venlafaxine and citalopram for their entire life cycle from embryo to adult, respectively. After exposure for 180
Reproductive toxicity days, the lowest observable effective concentration (LOEC) of venlafaxine and citalopram to significantly reduce the
Sperm quality
mean number of egg production in adults were 10 and 1 μg/L, respectively, whereas the fertilization rate displayed
Transcriptomic
SSRI
no significant changes. Further, we investigated the impacts of venlafaxine and citalopram in a reproductive context,
SNRI including sperm quality and reproductive behaviour. In contrast, venlafaxine and citalopram exposure did not affect
sperm quality but caused a reduction of reproductive behaviour (e.g., mating duration and mating interval) of adults
exposed to 1–10 μg/L of the antidepressant. Transcriptomic profiling of the whole ovary revealed that lifecycle
venlafaxine and citalopram exposure significantly affected the Na+/Cl− dependent neurotransmitter transporters sig-
naling. Moreover, immune system-mediated ovarian regeneration and creatine metabolism regulated energy metabo-
lism were proposed as the novel mechanism in the observed effects. Taken together, our results highlight the risk of
lifecycle venlafaxine and citalopram exposure to fish reproduction and provide novel perspectives for unveiling the
mechanism of female reproductive dysfunction.

⁎ Corresponding author at: Key Laboratory of Drinking Water Science and Technology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing Road,
Haidian District, Beijing 100085, China.
E-mail address: jmzha@rcees.ac.cn (J. Zha).

http://dx.doi.org/10.1016/j.scitotenv.2022.155017
Received 11 January 2022; Received in revised form 25 March 2022; Accepted 30 March 2022
Available online 5 April 2022

0048-9697/© 2022 Elsevier B.V. All rights reserved.

X. Hong et al.
1. Introduction
Depression is one of the most common neuropsychiatric disorders
worldwide. The number of depressive disorders worldwide increased
nearly 50% since 1990 (~258 million people in 2017) (Liu et al., 2020).
Currently, there has been a steady increase in prescription rates of antide-
pressants, which can almost entirely be explained by the increased prescrip-
tion rates of serotonin and noradrenaline reuptake inhibitors (SNRIs) and
selective serotonin reuptake inhibitors (SSRIs) (Ziegler et al., 2021).
These two drug groups accounted for 20% of the psychotropic drug market
in China, with the SNRI venlafaxine and the SSRI citalopram constituting
the majority of market leaders (https://www.menet.com.cn/). In
Germany, they even amount to about 30% of the market share in 2018,
which leads to a total consumption of 20.5 t of venlafaxine and 4.5 t of
citalopram (Schwabe et al., 2019). Given the high production volumes of
antidepressants containing venlafaxine and citalopram and poorly removal
efficiency in wastewater treatment plants (<30%) (Burns et al., 2018), sig-
nificant amounts have been frequently detected in the aquatic environ-
ment. Its concentrations in surface waters are in the range of ng/L to μg/L
(Zhang et al., 2020), and have reached tens of μg/L in urban sewage
(Mole and Brooks, 2019). High levels of antidepressants have also been
found in wild aquatic biota (Grabicova et al., 2017). Richmond et al.
(2018) estimated the daily intake for the top predators such as brown
trout (Salmo trutta) from streams in Australia are about 26% human daily
dose (203 μg/kg/day). Previous laboratory and field studies demonstrated
that antidepressants can be accumulated in various fish organs such as the
liver, brain, musculature, and gonads (Arnnok et al., 2017; Duarte et al.,
2022; Ziarrusta et al., 2017). However, their ecotoxicological data are com-
monly based on acute toxicity at relatively high concentrations, which are
considered to be much greater than actual concentrations in the environ-
ment (Bachour et al., 2020; Karakaya et al., 2021). Also, based on their ther-
apeutic functions and modes of action, certain kinds of antidepressants
have been reported to represent risks for non-target aquatic organisms,
even at concentrations of tens to hundreds of ng/L, particularly under con-
ditions of chronic exposure (Sehonova et al., 2018; Wiles et al., 2020).
Reproductive toxicity is an important direction in toxicology research,
which is directly linked to the quality and quantity of offspring. In human
and mammalian test systems, antidepressants have been shown to induce
reproductive damage (Beeder and Samplaski, 2020; Carvalho et al.,
2016). Indeed, there have been various reports on the effects of antidepres-
sants (mainly fluoxetine) on fish reproduction-related behaviours and phys-
iological processes, such as changes in nest-tending behaviours of male
fathead minnow (Pimephales promelas) (Weinberger and Klaper, 2014), in-
creased mating behaviours of the male guppy (Poecilia reticulata) and
mosquitofish (Gambusia holbrooki) (Fursdon et al., 2019; Martin et al.,
2019), and decreased fecundity and ovarian or circulating estradiol levels
of zebrafish (Danio rerio) and goldfish (Carassius auratus), respectively
(Lister et al., 2009; Mennigen et al., 2017, 2010). However, investigations
into the reproductive effects of exposure to other antidepressants are rela-
tively scarce compared to fluoxetine. Table 1 summarizes the reproductive
toxicity of venlafaxine and citalopram to teleost fish at field-realistic con-
centrations. These studies have shown that subacute or chronic exposure
(<30 days) to venlafaxine and citalopram caused no significant effects on
fish reproductive behaviours or spermatogenesis (Holmberg et al., 2011;
Olsén et al., 2014; Prasad et al., 2015a; Schultz et al., 2011). While repro-
ductive parameters, including egg production, spermatogenesis, and nest-
protecting behaviour were all impacted with increasing duration of expo-
sure (Galus et al., 2013; Parrott and Metcalfe, 2018, 2017; Prasad et al.,
2015a). As non-amniotes, fishes lay their eggs in water and have a lifetime
exposure to the external environment. At present, research on the reproduc-
tive toxicity of venlafaxine and citalopram mainly focused on chronic expo-
sure, but generally <30 days.
Here, using the zebrafish (Danio rerio) model, we investigated the repro-
ductive effects of 180-day continuous exposure (from the early embryonic
stage to adults) to environmentally relevant concentrations of venlafaxine
and citalopram on sperm quality and reproductive behaviour of zebrafish.
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Science of the Total Environment 832 (2022) 155017
Utilizing the RNA-seq, we also provide insights on the potential toxicologi-
cal mechanisms of venlafaxine and citalopram using transcriptional analy-
sis and specifically focused on signaling pathways modulating fish
reproduction.
2. Materials and methods
2.1. Chemicals
Venlafaxine hydrochloride (CAS number: 99300–78-4, purity >98.0%,
Cat number: V0110) and citalopram hydrobromide (CAS: 59729–32-7,
purity >98.0%, Cat number: C2370) were purchased from J&K Scientific
(Beijing, China). Stock solutions of venlafaxine and citalopram were
prepared in dechlorinated tap water (1 mg/mL) and no carrier solvent
was used.
2.2. Animals
Wild-type zebrafish (AB strain) were purchased from China Zebrafish
Resource Center (CZRC, Wuhan, China) and raised as previously described
(Hong and Zha, 2019). The animal study was approved by the Research
Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Lab-
oratory Animal Ethics Committee. Experiments were conducted following
the Laboratory Animal Ethics Committee (2015) #25 guidelines.
2.3. Experimental design
Zebrafish embryos collected within 2 h post-fertilization (hpf) and ex-
posed to waterborne venlafaxine and citalopram at nominal concentrations
of 0, 0.1, 1, 10 and 100 μg/L (three replicates/treatment, 50 embryos/
replicate) until 180 days post-fertilization (dpf). The exposure solutions
were renewed daily. Details of the exposure conditions are supplied in
the Supporting Information, Text S1. Water quality parameters (tempera-
ture, dissolved oxygen, pH, and conductivity) were measured once a
week in fish exposure aquaria (see Table S1).
2.4. Water sample analyses
The extraction and analysis of venlafaxine and citalopram from the ex-
posure solutions were performed as previous studies (Minguez et al., 2015).
Briefly, the actual concentrations of venlafaxine and citalopram were ana-
lyzed for each exposure aquarium at the beginning of the exposure (T0)
and 24 h before the next renewal (T24). During the entire 180-day exposure,
concentrations of both antidepressants were measured monthly in fish ex-
posure aquaria. Detailed protocols are provided in the Supporting Informa-
tion, Text S2.
2.5. Evaluation of sperm quality
Sperm from zebrafish were collected following established protocols
(Poli et al., 2019). Briefly, fish were dissected after anesthetized with MS-
222 (tricaine) and the sperm then was collected and immediately tested
for sperm traits. To evaluate the potential effects of venlafaxine and
citalopram on sperm traits, we measured sperm concentration, sperm via-
bility (the proportion of live sperm), and sperm motion. Sperm was appro-
priately diluted in Hanks' balanced salt solution (300 mOsmol/kg,
HBSS300) and its concentration was estimated using a cell counting cham-
ber (Marienfeld Superior, Lauda-Königshofen, Germany). Following manu-
facturer instructions, sperm viability was evaluated by dying sperm with
eosin-nigrosin stain (Sigma-Aldrich Co., MO St, Louis, USA), as previously
described (Chen et al., 2020). Using a light microscope (Olympus BX53,
Tokyo, Japan), 5 non-overlapping fields were photographed and the per-
centage of live sperm was then assessed by counting a minimum of 100
sperm cells per male. Sperm concentration and sperm viability were
assessed for 9 males per treatment, respectively, thus the final sample size
for each sperm trait parameter is n = 45.
X. Hong et al. Science of the Total Environment 832 (2022) 155017

Table 1
Overview of publications exploring reproductive toxicity of venlafaxine and citalopram on fish.
Chemical Fish species Life stage Exposure NOEC/LOEC Endpoints Effects References
length (μg/L)

Venlafaxine Fathead minnow Life-cycle 5.5 months 8.8/88 Egg production ↑ (Parrott and Metcalfe, 2017)
(Pimephales promelas) (Egg to adult)
88/>88 Egg quality, % No effect
Fertilization, %
Hatching
Fathead minnow Life-cycle 5.5 months −/88 Nest-protecting behaviour ↑ (Parrott and Metcalfe, 2018)
(Pimephales promelas) (Egg to adult)
Fathead minnow Adult male 21 days 0.31/− Vitellogenin (VTG) No effect (Schultz et al., 2011)
(Pimephales promelas) Spermatogonia
Spermatozoa abundance
Secondary sex characteristics
Reproductive behaviour (nest holding ability)
Zebrafish (Danio rerio) Adult 6 weeks −/10 Egg production ↓ (Galus et al., 2013)
Zebrafish (Danio rerio) Adult 20 days −/<1 Courtship behaviour ↓ (Tang et al., 2022)
Citalopram Guppy Adult male 6–7 days 1/− Mating behaviours No effect (Holmberg et al., 2011)
(Poecilia reticulata)
Endler guppies Adult male 28 days 1/− Courting behaviours No effect (Olsén et al., 2014)
(Poecilia wingei)
Zebrafish (Danio rerio) Adult male 2 weeks 100/− Spermatogenesis No effect (Prasad et al., 2015a)
1 month 4/40 Spermatogonium, % ↓
Secondary spermatocytes, %
Spermatozoa, %

Sperm motion was observed using an Olympus BX53 light microscope
and analyzed by a free Computer Assisted Sperm Analysis (CASA) plugin
for the software ImageJ (v15.1). The CASA input parameters used in this
study are shown in Table S2, with slight modifications based on a previous
investigation (Wilson-Leedy and Ingermann, 2007). A minimum of 500
sperm cells were tracked per male (n = 9). Six measures of sperm motion
parameters (Table 2 for definitions) were obtained by these tracks: motility
(MOT, %), straight line velocity (VSL, μm/s), average path velocity (VAP,
μm/s), curvilinear velocity (VCL, μm/s), linearity (LIN, %), and wobble
(WOB).
2.6. Evaluation of reproductive success and reproductive behaviour
In the first reproductive success experiment, 9 pairs consisting of 9
males and 9 females were randomly selected from the same treatments (un-
exposed, venlafaxine, or citalopram) and placed in mating tanks (ESEN,
Beijing, China) and separated by dividers, respectively (one pair of fish
for each tank). The divider was removed and collected all eggs on the
next morning, the total number of released eggs and fertilized eggs (devel-
oped past the epiboly stage) were counted. The reproductive testing was
performed between 0830 and 0930. Between 181 dpf and 190–192dpf, re-
peated reproductive trials (n = 3 for each pair of fish) were conducted to
assess whether venlafaxine or citalopram exposures affected fish reproduc-
tive capacity. Egg production (mean number of released eggs per mating
pair) and fertilization rate (the number of fertilized eggs divided by that
of spawned) were used to evaluate reproductive success.
Table 2
Measured sperm motility parameters with accompanying definitions.
Parameters Unit Definition
Motility (MOT) unitless Percentage of sperm that are moving actively either
linearly or in a large circle regardless of speed.
Straight line μm/s The average point-to-point velocity along the Straight
velocity (VSL) line between its first and last detected position.
Average path μm/s The average point-to-point velocity of sperm along its
velocity (VAP) average path.
Curvilinear μm/s The average point-to-point (total distance traveled)
velocity (VCL) velocity of sperm along its track.
Linearity (LIN) unitless The ratio of VSL/VCL describes path curvature.
Percentage linearity of the sperm path, calculated by the
departure of the actual sperm track from a straight line.
Wobble (WOB) unitless The ratio of VAP/VCL describes side to side movement
of the sperm head.
In the second reproductive behaviour experiment, both males and fe-
males were randomly subjected to the plexiglass cylindrical arena (outside
dimensions: diameter, 20 cm; height, 10 cm) adapted from our previously
described (Hong and Zha, 2019) and separated by dividers (9 pairs, n =
18 for each treatment; one pair for each arena). The behavioral testing
was performed between 0830 and 0910 h. Briefly, the test tank filled
with tepid water (27 ± 1 °C) and its reproductive behaviour was recorded
for 30 min by a video camera FDR-AX60 (SONY Handycam, Tokyo, Japan),
positioned to capture a vertical view of the test tank. All evaluation exper-
iments were conducted in the absence of the camera operator. Male per-
forming chase, tail-nose, encircle, quiver and zig-zag, and distance
between male and female fish does not exceed 1.5 cm, and the movement
state lasts for more than 5 s were defined as mating behaviour (Darrow
and Harris, 2004). The mating-related metrics were used to evaluate repro-
ductive behaviour (Table. S3), which was analyzed with a video tracking
software, using the manual scoring setting module (EthoVision XT® 14,
Noldus Inc., the Netherlands). To avoid any potential carryover effects,
each pair of fish was only used in a single trial. The parameters of mating
rate (percentage of the total time fish spent in mating, %), mating duration
(total time fish spent in mating, s), and mating interval (average time inter-
val between each mating behaviour, s) were used to evaluate the reproduc-
tive behaviour. For both of the reproductive assays, fish were acclimated in
testing rooms for at least 12 h before testing. All reproductive behaviour
was recorded for 30 min.
2.7. Transcriptomic analyses
RNA sampled from 9 pooled zebrafish ovaries (0 and 10 μg/L) were iso-
lated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The mRNA
Quality check, Library Preparation, and Sequencing were conducted as in
previous studies (Yuan et al., 2019). More details are provided in the
Supporting Information, Text S3. The differentially expressed genes
(DEGs) between treatment groups and controls were identified by
DESeq2 software (v1.62). Genes with absolute fold change ≥2, false dis-
covery rate (FDR) < 5%, and average read count in at least one sample ≥
10 were considered as DEGs. All DEGs for each group were used to map
and identify enriched Gene Ontology (GO) terms (http://www.
geneontology.org/). Pathway enrichment analysis was performed using
the PANTHER, Reactome, and KEGG pathway database. FDR at the thresh-
old below 0.05 (an adjusted p-value < 0.05) was designated as significantly
enriched GO terms and pathways in DEGs.

3
X. Hong et al. Science of the Total Environment 832 (2022) 155017

2.8. Quantitative real-time PCR (RT-qPCR)

Transcriptions of DEGs were confirmed by using the RT–qPCR. Briefly,

ovaries from three fish were pooled as one replicate sample, and each expo-
sure group was prepared in triplicate. Total RNA extraction and RT–qPCR
analysis were performed as described in the Supporting Information, Text
S4. The relative gene expression was normalized to that of β-actin via the
method of 2-ΔΔCt. The primers used for all relevant genes were listed in
Table S4.

2.9. Statistical analysis

The assumptions of normality and equality of variance were evaluated

by Kolmogorov-Smirnov and Levene's tests, respectively. Statistically sig-
nificant differences between each treatment and control group were deter-
mined by one-way ANOVA followed by Tukey's multiple range test using
SPSS 25.0 software. Significant differences from control treatments were
assessed using two-sample t-tests (Tukey's adjusted probabilities with sepa-
rate variances). The OriginPro 2020 was used for data plotting. (*) P-value
< 0.05 were considered statistically significant.

3. Results
3.1. Exposures
The concentrations of the two antidepressants were below the limit of
detection in the control group. The measured concentrations (mean ± stan-
dard deviation, % T24/T0 of the exposure solution) of venlafaxine in the ex-
posure tanks (0.1, 1, 10 and 100 μg/L) were 0.09 ± 0.02 μg/L (91.1%),
0.94 ± 0.06 μg/L (94.5%), 9.10 ± 0.45 μg/L (91.9%) and 93.23 ± 3.03
μg/L (93.4%), and those of citalopram were 0.08 ± 0.02 μg/L (80.7%),
0.82 ± 0.04 μg/L (83.6%), 7.84 ± 0.56 μg/L (78.9%) and 75.10 ± 4.12
μg/L (76.7%), respectively. Therefore, the nominal antidepressant concen-
trations were maintained in the exposure system, and the nominal values
are used in this study.
3.2. Survival and growth
Fig. 1. Reproductive inhibition of zebrafish after exposure to venlafaxine and
citalopram. Comparative (a) average egg production and (b) fertilization rate in
different groups. The results are expressed as mean ± SD. Each group contains
nine replicates (n = 9). The asterisk indicates significant differences.
3.5. Reproductive behaviour
Given the effects of venlafaxine and citalopram exposure on egg produc-
tion, we next asked whether reproductive behaviour was particularly sensi-
tive to venlafaxine and citalopram's effects and, if so, which reproductive
behavioral parameters were affected. After exposure, no significant varia-
tion in mating rate was observed in the exposure group (Fig. 4a). For the
mating duration, only 100 μg/L venlafaxine and citalopram exposure
groups were significantly decreased when compared with the controls

Lifecycle exposures to venlafaxine and citalopram did not affect the sur-
vival or sex ratio of fish (Table. S5). There were no effects on adult zebrafish
growth parameters of body length and body weight in citalopram treat-
ments. However, fish exposed to 100 μg/L venlafaxine had significantly
higher growth promotion than control fish (Table. S6; P < 0.05).

3.3. Reproductive inhibition

After exposure for 180 days, the mean number of egg production of the
female zebrafish from the exposure groups manifested a statistically signif-
icant decline compared with the controls (Fig. 1a; P < 0.05). Specifically,
the mean number of egg production that were exposed to 10 and 100
μg/L venlafaxine displayed a 24.0% and 41.7% reduction relative to basal
levels (184.5 ± 40.6) in controls, respectively; treatment with 1 and 100
μg/L citalopram resulted in 28.6% and 34.1% reduction, and more than
50% reduction of the mean number of eggs (56.8%) was found in the 10
μg/L citalopram treatment group. Of reproductive success, no significant
variation in fertilization rate was found in all exposure groups (Fig. 1b).

3.4. Sperm traits

Both venlafaxine and citalopram, irrespective of exposure level, did not

affect either concentration (106/μL) or viability (%) of sperm (Fig. 2). Fur- Fig. 2. Effects of venlafaxine and citalopram on sperm traits of male zebrafish.
thermore, the vigor and pattern of sperm motion (e.g., MOT, VCL, VAP, (a) Sperm concentration and (b) sperm viability of male fish from different
LIN, VSL, and WOB) appeared to be unaffected in all venlafaxine and groups. The results are expressed as mean ± SD. Each group contains nine
citalopram exposed groups compared to their controls (Fig. 3a and b). replicates (n = 9). The asterisk indicates significant differences.

4
X. Hong et al.
Fig. 3. Time series plot of sperm motility (MOT), straight line velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), path linearity (LIN), and wobble (WOB)
measures in the control group, (a) venlafaxine and (b) citalopram treatments (n = 9). The results are expressed as mean ± SD.
Fig. 4. Effects of venlafaxine and citalopram on the reproductive behaviour of
zebrafish. (a) The successful mating rate in different groups; (b) Mating duration
and (c) mating interval of fish from different groups. Box plots show 25th, 50th
(mean), and 75th percentiles. Each group contains nine replicates (n = 9). The
asterisk indicates significant differences. Note: Successful mating rate, percentage
of the total time fish spent in mating; Mating duration, cumulative time fish spent
in mating; Mating interval, the average time interval between each mating
behaviour.
5
Science of the Total Environment 832 (2022) 155017
(Fig. 4b; P < 0.05). Regarding mating intervals, we observed a significant
increase in 10 and 100 μg/L venlafaxine and citalopram exposure groups,
and a notable increase showed in 1 μg/L venlafaxine (Fig. 4c; P < 0.05).
3.6. Transcriptomic profiling
HiSeq RNA sequencing platform was applied to profile the DEGs of ova-
ries. The DEGs in response to the 10 μg/L venlafaxine and citalopram expo-
sure were visualized with the volcano plot (Fig. S1). In total, 944 and 1181
significant DEGs were identified, respectively. Among these DEGs, the ex-
pression of 361 genes was commonly altered in venlafaxine and citalopram
exposure groups compared to controls, and more than 60% of them showed
up-regulation.
3.7. Gene ontology analysis
To further understand the underlying functions and pathways of these
DEGs, we carried out GO function annotation and enrichment analysis
(Fig. 5) as well as pathway analysis. This GO analysis revealed differences
in altered GO terms between venlafaxine and citalopram exposure groups.
Zebrafish exposed to venlafaxine (10 μg/L), exhibited most DEGs involved
in the biological processes of muscle and skeletal muscle contraction. The
top five enriched GO terms in cellular components were “supramolecular
fiber,” “supramolecular polymer,” “sarcomere,” “myofibril,” and “contrac-
tile fiber” (Fig. 5a). As in the case of the citalopram exposure group, the top
two enriched GO terms in biological processes were “defense response” and
“chemical homeostasis,” and six additional GO terms in cellular component
were “extracellular matrix,” “collagen trimer,” “intermediate filament,”
“intermediate filament cytoskeleton,” “hemoglobin complex,” and
X. Hong et al.
muscle contraction
muscle system process
regulation of body fluid levels
blood coagulation
hemostasis
coagulation
striated muscle contraction
skeletal muscle contraction
multicellular organismal movement
musculoskeletal movement
supramolecular fiber
supramolecular polymer
sarcomere
myofibril
contractile fiber
Z disc
I band
protein-lipid complex
plasma lipoprotein particle
lipoprotein particle
endopeptidase activity
enzyme inhibitor activity
peptidase inhibitor activity
peptidase regulator activity
endopeptidase inhibitor activity
endopeptidase regulator activity
serine-type endopeptidase activity
serine-type peptidase activity
serine hydrolase activity
serine-type endopeptidase inhibitor activity
0.02 0.03 0.04 0.05 0.06
GeneRatio
defense response
chemical homeostasis
interspecies interaction between organisms
defense response to other organism
sodium ion transport
response to bacterium
sodium ion transmembrane transport
defense response to bacterium
gas transport
oxygen transport
extracellular matrix
collagen trimer
intermediate filament
intermediate filament cytoskeleton
hemoglobin complex
haptoglobin-hemoglobin complex
secondary active transmembrane transporter activity
endopeptidase inhibitor activity
endopeptidase regulator activity
peptidase inhibitor activity
peptidase regulator activity
symporter activity
solute:sodium symporter activity
solute:cation symporter activity
serine-type endopeptidase inhibitor activity
neurotransmitter:sodium
symporter activity
0.02 0.04
GeneRatio
Fig. 5. Gene ontology (GO) analysis on DEGs of zebrafish upon exposure to
(a) venlafaxine and (b) citalopram at the concentration of 10 μg/L. Significance
criteria: |log2(Fold change)| ≥ 1 and adjusted p ≤ 0.05 (corrected by Benjamini-
Hochberg method). Note: BP, biological processes; CC, cellular component; MF,
molecular function.
“haptoglobin-hemoglobin complex” (Fig. 5b). Most notably, this GO analy-
sis also revealed similarities between venlafaxine and citalopram exposure
groups such as DEGs involved in the molecular function, which mainly
enriched in regulation of peptidase and endopeptidase activity.
3.8. Pathway analysis
Enriched pathways affected by 10 μg/L venlafaxine and citalopram
were obtained based on the PANTHER, Reactome, and KEGG database
(Table 2; Table S4). The top three enriched pathways across all databases
were complement cascade, striated muscle contraction, and activation of
matrix metalloproteinases when female zebrafish were exposed to
venlafaxine (Table S7). In contrast, citalopram exposed females showed
high significance (P < 0.00001) for the enriched pathways of Na+/Cl−
dependent neurotransmitter transporters, transport of bile salts and
organic acids, metal ions, and amine compounds, and SLC-mediated
Science of the Total Environment 832 (2022) 155017
transmembrane transport compared to controls. We also identified a set
of DEGs involved in the regulation of Na+/Cl− dependent neurotransmit-
ter transporters (venlafaxine, 6 hits, 35% of total genes in this pathway;
BP
citalopram, 10 hits, 59%) and creatine metabolism (venlafaxine, 6 hits,
35%; citalopram, 7 hits, 41%) pathways that were altered both at
Count
venlafaxine and citalopram exposed females in comparison with controls
10 (Table S8).
15
20
25
3.9. Validation of gene transcriptional levels by quantitative real-time PCR
CC
p.adjust The gene expressions of Na+/Cl- dependent neurotransmitter trans-
0.0005
porters signaling (slc22a2, slc6a18, slc6a14, and slc6a19b), immune system
0.0010
(arpc1b, wasb, itgb2, cxcr1, ahsg2, gyg1b, and cul1a), and creatine metabo-
0.0015 lism (ckma, ckmb, and ckbb) were determined by RT–qPCR (Fig. 6a). Gener-
ally, the gene transcription levels determined by RT–qPCR were equivalent
to the changing trend and amplitude observed in the transcriptome results
(Fig. 6b). Based on the transcriptional levels of selected 14 DEGs, the data of
MF
RT–qPCR and RNA-Seq were in linear correlation (R2 for 10 μg/L
venlafaxine and citalopram were 0.87 and 0.85, respectively) (Fig. S2).
4. Discussion
The central focus of the present study was to evaluate the reproductive
effects of venlafaxine and citalopram on fish after a full lifecycle exposure.
Data on fecundity and fertilization rate associated with reproductive out-
comes provide direct insight into reproductive capability. In this study,
BP
we examined the mean number of egg production and fertilization rate
after zebrafish exposure from the early embryonic stage to adults (180
Count
10
dpf). We found that in addition to the previously reported decline of cumu-
20 lative spawning in zebrafish following 6 weeks of venlafaxine exposure at
30 5–6 μg/L (measured concentration) (Galus et al., 2013), fish exposed to en-
vironmentally relevant citalopram also displayed a 28.6% reduction of
CC
p.adjust
spawning across all treatments. To our knowledge, the effects of lifecycle
0.01 exposure to waterborne citalopram on fecundity in fish have not been
0.02 well studied. Other SSRIs such as fluoxetine have been shown to reduce
0.03 the fecundity of zebrafish (32 μg/L for 7 days) (Lister et al., 2009). Interest-
ingly, unlike citalopram, Parrott and Metcalfe (2017) reported a 46% cu-
mulative increase in the spawning of fathead minnow after exposure to
MF
venlafaxine (88 μg/L for 167–168 days), and such increases have not
been reported elsewhere. This is likely attributable to increased sperm
counts reported as a result of exposure to other antidepressants (Bertram
0.06 et al., 2018) or species-specific sensitivities (Martin et al., 2019). Another
possible explanation for the increased reproductive output in fathead min-
nows is the combination of a reproductive strategy of relatively low fecun-
dity coupled with intensive protection (guard the eggs against potential
predators) and its high variability of egg production (Holzman, 2014;
Parrott and Metcalfe, 2017). Therefore, we hypothesize that when exposed
to the highest concentrations of venlafaxine, this species may increase its fe-
cundity to ensure that there are enough surviving offspring to maintain
population stability.
At the applied long-term venlafaxine and citalopram exposed concen-
trations between 0.1 and 100 μg/L, we observed no effect of all exposed
levels on fertilization rates in zebrafish F1 generation relative to controls
cons with the findings of Parrott and Metcalfe (2017) in fathead minnow.
Sperm traits (e.g., sperm concentration, sperm viability, and sperm motion)
are important predictors of fertilization success (Snook, 2005) and have
been widely used to characterize a male-specific reproductive effect. In
this study, neither venlafaxine nor citalopram affected any measured
sperm traits. To date, only two studies investigating the effects of
venlafaxine and citalopram on sperm quality of fish, like fathead minnow
and zebrafish, showed similar results with no effect on spermatozoa abun-
dance and spermatogenesis (density of different stages of spermatogenic
cells) (Prasad et al., 2015a; Schultz et al., 2011). Likewise, an example
from Martin and co-authors (Martin et al., 2019) revealed that no obvious
disruptions of sperm quality were found in male mosquitofish (Gambusia
holbrooki) exposed other SSRIs fluoxetine as well as in Japanese medaka
6
X. Hong et al.
Fig. 6. Gene transcription in the ovaries of female zebrafish after chronic exposure to venlafaxine and citalopram at the concentration of 10 μg/L. (a) Heat map of selected
genes expression levels based on RNA-seq data. (b) RT-qPCR. Columns represent the fold change of RT-qPCR results and data were represented as the mean ± SD (n = 3).
(Oryzias latipes) (0–5 μg/L for four weeks) (Foran et al., 2004), which poten-
tially attributes to the fact of SSRIs sharing a similar mode of action in ver-
tebrates (Gould et al., 2021). By contrast, morphological analysis of the
effect of citalopram on spermatogenesis in the testis of zebrafish (40–100
μg/L for one month), displayed a significant inhibition of different stages
of spermatogenesis, including spermatogonium, secondary spermatocytes,
and spermatozoa (Prasad et al., 2015a). Such differences between studies
could be due to different species-specific sensitivities and/or exposure du-
rations. We suggest that further studies at multiple time points in a range
of fish species are warranted to elucidate the impacts of environmentally re-
alistic citalopram exposure on sperm traits.
As a key phenotypic trait, growing evidence across different fish species
suggests that reproductive behaviour is directly linked to the quantity and
quality of offspring, such as Japanese medaka, guppy, and mosquitofish
(Li et al., 2018; Salahinejad et al., 2022; Wang et al., 2018). To date, no
studies have attempted to investigate the potential impacts of lifecycle ex-
posure to environmentally realistic venlafaxine and citalopram on repro-
ductive behaviour in fish. We found that both of the two antidepressants'
exposure at environmentally relevant concentrations (0.1 and 1 μg/L) for
180 days did not alter the mating rate and mating duration; however, expo-
sure to 1 μg/L venlafaxine increased the time of mating interval by fish. In
line with our results, several short-term studies on fish demonstrated that
exposure to environmentally realistic venlafaxine and citalopram (0.31–1
μg/L) for less than one month likewise showed no alterations on reproduc-
tive behabviours (Holmberg et al., 2011; Olsén et al., 2014; Schultz et al.,
2011). Notably, bioactive metabolites of venlafaxine (N-desmethyl
venlafaxine) and citalopram (O-desmethyl citalopram) are found in the
aquatic environment at much higher concentrations than the parent com-
pounds. Therefore, the environmental significance of these results may be
enhanced (Patel et al., 2019). In risk assessment, the effects of short-term
studies are used to predict long-term environmental exposures and that
may underestimate the adverse impacts of compounds in question
(Legradi et al., 2018; Overturf et al., 2015), such as two investigations-
mainly concentrated on reproductive behaviours in Japanese medaka
after lifecycle exposure to other compounds, including triphenyl phosphate
(Li et al., 2018) and triclosan (Wang et al., 2018). Indeed, the present study
7
Science of the Total Environment 832 (2022) 155017
showed a lifecycle exposed fish being less likely to copulate than the short-
term studies (Holmberg et al., 2011; Olsén et al., 2014; Schultz et al., 2011).
To date, only the work by Parrott and Metcalfe (2018) provides direct evi-
dence that continuously exp. to a high-venlafaxine (88 μg/L for 5.5 months)
could substantially influence the nest-protecting behaviour, although man-
ifested in enhanced reproductive behaviour. Overall, our results indicate
that lifecycle exposure to environmentally realistic venlafaxine and
citalopram can dramatically weaken reproductive behaviour in zebrafish
adults, which is more pronounced in high concentrations.
Given the results for the reduced mean number of eggs in the females
exposed to venlafaxine and citalopram, the molecular mechanisms of repro-
ductive inhibition effects would be expected. In teleost fish, the role of sero-
tonin in reproduction has been well documented (Prasad et al., 2015b). It
has been reported that antidepressants (e.g., SNRIs and SSRIs) can influ-
ence the homeostasis of serotonin which was correlated with reproductive
function (Gould et al., 2021; Prasad et al., 2015a). To gain insights into the
canonical pathways disrupted by lifecycle venlafaxine or citalopram expo-
sure in zebrafish, transcriptome analysis was performed on the ovaries of
the control group and those treated with an antidepressant. A significantly
enriched pathway, Na+/Cl− dependent neurotransmitter transporters, was
identified in the ovaries across both the venlafaxine and citalopram-treated
groups. The transport function of serotonin reuptake transporters (SERT)
was reported to require through the cotransport of Na+/Cl− and the
countertransport of K+ (Chen et al., 2004). This result supports the hypoth-
esis that antidepressants may influence the homeostasis of serotonin by in-
terfering with SERT, and the underlying molecular regulatory mechanism is
the interference of the cotransport of Na+/Cl− rather than the
countertransport of K+. Our work also identifies two novel pathways by
which lifecycle exposure to venlafaxine or citalopram may alter reproduc-
tive function in zebrafish ovaries. For example, venlafaxine exposure
ovary displayed transcriptional regulation like with immune signaling
pathway. Moreover, both venlafaxine and citalopram showed significant
transcriptional enrichment in creatine metabolic signaling pathways. As
one of the most regenerative and highly energetic tissue, the female repro-
ductive organ has been undeniably linked to ovarian regeneration and en-
ergy metabolism (Wade and Schneider, 1992; Ye et al., 2016; Zou et al.,
X. Hong et al.
2021). On one hand, to our knowledge, one study has reviewed that the im-
mune system can modulate the secretion of hormones in the ovary and also
control the development of ovarian germline stem cells, including prolifer-
ation, differentiation, and further remodeling ovarian function, which will
eventually impact the reproduction (Ye et al., 2016). Therefore, disruption
of this pathway provides another new clue for the reduced ability of egg
production observed in the venlafaxine treatment. On the other hand, as
a key component of cellular energy homeostasis (e.g. oocytes), creatine me-
tabolism can maintain intracellular levels of adenosine triphosphate (ATP)
and some available li have linked perturbation in creatine metabolism to
declined fertility (Muccini et al., 2021). Taken together, perhaps both
venlafaxine and citalopram-induced disruption of these pathways could ex-
plain the reduced ability for offspring production; this hypothesis warrants
further examination.
5. Conclusion
In summary, we present a comprehensive study of the fish reproductive
effects of lifecycle exposure to low concentration antidepressants that in-
cludes sperm quality, reproductive behavioral, and molecular end points.
Instead of sperm quality but reproductive behaviour and spawning ability
are impacted when fish lifecycle exposure to environmentally relevant con-
centrations of antidepressants. These results likely alter the recognition that
people underestimate the long-term effects of antidepressants although at
concentrations that approach those commonly measured in environmental
compartments. One additional direction that may provide new perspectives
for the varied effects among different fish species is to focus on the sequence
variation of SERT (>30%, data source: http://asia.ensembl.org) that may
lead to SERT-independent pathways modulating fish reproduction follow-
ing SNRI or SSRI exposure. Our study provides novel mechanistic insights
into the established correlations between lifecycle antidepressants expo-
sure and reproductive toxicity in zebrafish.
Funding
This work was supported by the National Key Research and Develop-
ment Project of China [grant number 2019YFC1803402]; the National Nat-
ural Science Foundation of China [grant numbers 21976202, 42107291];
and the China Postdoctoral Science Foundation [grant numbers
2021M703405].
CRediT authorship contribution statement
Xiangsheng Hong: Funding acquisition, Investigation, Data curation,
Formal analysis, Writing – original draft, Writing – review & editing.
Rui Chen: Investigation, Data curation, Formal analysis, Writing – review
& editing. Le Zhang: Data curation, Formal analysis. Liang Yan: Data
curation, Formal analysis. Jiasu Li: Investigation, Data curation, Formal
analysis. Jinmiao Zha: Conceptualization, Writing – review & editing, Su-
pervision, Funding acquisition, Project administration.
Declaration of competing interest
The authors declare that they have no actual or potential competing fi-
nancial interests.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2022.155017.
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