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Several phenol derivatives are suspected endocrine disruptors and have received attention in risk assessment studies for several decades owing to the structural similarity between estrogens and phenolic compounds. We assessed the endocrine disrupting effect of the phenolic compound para-phenylphenol (PPP) through acute tests and evaluating chronic endpoints in an invertebrate model, Daphnia magna. Exposure of D. magna to PPP induced substantial adverse effects, namely, reduced fecundity, slowed growth rate, delayed first brood, and a reduction in neonate size. Furthermore, we investigated the mRNA expression of relevant genes to elucidate the mechanism of endocrine disruption by PPP. Exposure of D. magna to PPP induced the substantial downregulation of genes and markers related to reproduction and development, such as EcR-A, EcR-B, Jhe, and Vtg. Consequently, we demonstrated that PPP has an endocrine disrupting effect on reproduction and development in D. magna.
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Ecotoxicology and Environmental Safety 243 (2022) 113965
Available online 19 August 2022
0147-6513/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Endocrine-disrupting potential and toxicological effect of
para-phenylphenol on Daphnia magna
Hyunki Cho
a
, Chang Seon Ryu
a
, Sang-Ah Lee
a
, Zahra Adeli
a
, Brenda Tenou Meupea
a
,
Youngsam Kim
a
,
b
,
*
, Young Jun Kim
a
,
b
a
Environmental Safety Group, KIST Europe Forschungsgesellschaft mbH, 66123 Saarbrücken, Germany
b
Division of Energy & Environment Technology, University of Science & Technology, Daejeon 34113, South Korea
ARTICLE INFO
Edited by Dr. Caterina Faggio
Keywords:
Ecotoxicology
mRNA expression
Fecundity
Ecdysteroids
Invertebrate
ABSTRACT
Several phenol derivatives are suspected endocrine disruptors and have received attention in risk assessment
studies for several decades owing to the structural similarity between estrogens and phenolic compounds. We
assessed the endocrine disrupting effect of the phenolic compound para-phenylphenol (PPP) through acute tests
and evaluating chronic endpoints in an invertebrate model, Daphnia magna. Exposure of D. magna to PPP induced
substantial adverse effects, namely, reduced fecundity, slowed growth rate, delayed rst brood, and a reduction
in neonate size. Furthermore, we investigated the mRNA expression of relevant genes to elucidate the mechanism
of endocrine disruption by PPP. Exposure of D. magna to PPP induced the substantial downregulation of genes
and markers related to reproduction and development, such as EcR-A, EcR-B, Jhe, and Vtg. Consequently, we
demonstrated that PPP has an endocrine disrupting effect on reproduction and development in D. magna.
1. Introduction
In modern society, the use of chemicals has become inevitable in the
manufacturing and consumer industries. Approximately 1500 new
chemicals are estimated to be synthesized every year (Alla et al., 2021),
and the registered number of chemicals/mixtures was more than 350,
000 in 2020 (Wang et al., 2020). Among these substances, more than
1000 are potential endocrine disrupting chemicals (EDCs) (Street et al.,
2018). Recently, the European Union mandated the registration of
chemicals that are veried or potential EDCs (on the basis of Sdata) in its
Registration, Evaluation, Authorization, and restriction of Chemicals
policy (http://echa.europa.eu/substances-restricted-under-reach).
Currently, 100 compounds are included in List 1 of these regulations
(http://edlists.org); of these, 72 are phenolic compounds. Because of
their structural similarities to natural estrogens, phenol derivatives such
as alkyl-, hydroxyl-, chlorinated-, and bis-phenols can affect the estro-
genic activity of the endocrine system (Omar et al., 2016). Because of
their variety and frequent usage, phenolic compounds have been a focus
of toxicological research over the past few decades. However, para--
phenylphenol (PPP) has received comparatively lacking attention,
despite its involvement in the synthesis of pesticides, dyes, and resins
(Larra˜
naga et al., 2016). Alarmingly, studies have reported PPP con-
centrations of 50265 µg/mL in lake water (Wick and Gschwend, 1998)
and 17 µg/mL in urine collected from pregnant women (Tefre de
Renzy-Martin et al., 2014). Therefore, it is important to understand the
endocrine disrupting potential of PPP to evaluate the risk it poses to the
environment.
The identication of EDCs is conducted in terms of their interference
with estrogen, androgen, and thyroid hormone or steroidogenesis
pathways. Screening assays have focused on in vitro bioassays of
mammalian cells to assess the effect of EDCs on vertebrate species
(Robitaille et al., 2022). However, an assessment of invertebrate species
should be undertaken to comprehensively elucidate the adverse effects
of EDCs in the environment because invertebrates comprise more than
90 % of animal species in the ecosystem (Brennan et al., 2006). Contrary
to the vertebrate endocrine system, invertebrates possess ecdyster-
oidogenesis. Consequently, various in vivo bioassays have been devel-
oped for invertebrates like chironomids, earthworms, mites, and
collembolans. Among invertebrate models, Daphnia magna, a freshwater
crustacean, has been well-documented and exploited as an efcient test
Abbreviations: EDC, Endocrine disrupting chemical; PPP, para-Phenylphenol; OECD, Organization for Economic Co-operation and Development; YCT, yeast,
cerophyll, and trout chow; EP, Eppendorf; PBS, phosphate-buffered saline; ROS, reactive oxygen species; qRT-PCR, real-time quantitative reverse transcription PCR.
* Corresponding author at: Environmental Safety Group, KIST Europe Forschungsgesellschaft mbH, 66123 Saarbrücken, Germany.
E-mail address: youngsam.kim@kist-europe.de (Y. Kim).
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety
journal homepage: www.elsevier.com/locate/ecoenv
https://doi.org/10.1016/j.ecoenv.2022.113965
Received 19 May 2022; Received in revised form 3 August 2022; Accepted 9 August 2022
Ecotoxicology and Environmental Safety 243 (2022) 113965
2
subject for identifying and monitoring the toxicity of EDCs. This species
possesses several traits advantageous for in vivo assays: high biological
sensitivity to environmental uctuations, a short lifespan and repro-
ductive cycle, and cost-effective maintenance (Kang et al., 2014).
Several studies have conrmed that D. magna: (i) is representative of
freshwater invertebrates, (ii) responds to vertebrate hormones, (iii) has
end-point indicators suggesting cross-reactivity, and (iv) can provide
relevant comparisons between ecdysteroid and steroid systems (Alla
et al., 2021; Colbourne et al., 2011; Kashian and Dodson, 2004; Miya-
kawa et al., 2018).
In this study, we hypothesize that exposing D. magna to sublethal
concentrations of PPP would disrupt hormonal activities with resultant
adverse growth and reproductive outcomes. End-point parameters were
mortality, growth, time to rst reproduction, neonate size, and number
of offspring.
2. Materials and methods
2.1. Test solution preparation
PPP was purchased from Sigma-Aldrich (D4540; St. Louis, MO, USA).
To prepare the stock solution, 50 mg PPP was dissolved in 10 mL DMSO
to render a concentration of 5000 mg/L. To obtain test concentrations,
deionized water was used to dilute the stock solution 10 times for acute
toxicity tests and 20 times for chronic toxicity tests. The stock solution
was renewed every week. For the test solution, ISO and Elendt M4 media
were prepared according to the OECD guideline 202 (OECD, 2018).
2.2. D. magna culture
D. magna ephippia was purchased from Micro Biotests (Gent,
Belgium) and hatched under a 16 h light/8 h dark cycle using a 7000 lux
light for 72 h in a controlled climate incubator. Culture conditions
conformed to OECD guidelines 202 and 211 (OECD, 2018). The popu-
lation consisted of 15 D. magna individuals reared in a 2 L glass beaker
containing 1.5 L Elendt M4 medium, maintained at 20.0 ±1.0 C under
the same light cycle. D. magna were fed daily with Chlorella vulgaris
(~1.5 ×10
8
cells/mL at a carbon concentration of 0.1 mg
C/D. magna/day) purchased from the Culture Collection of Algae at
Cologne University (Essen, Germany). Additionally, 0.5
μ
L/mL of yeast,
cerophyll, and trout chow (YCT) were supplied thrice a week. New ne-
onates were removed daily, and the beakers and cultural media were
renewed thrice a week to maintain good water quality. The pH and
dissolved oxygen content were checked before replacing equipment or
conducting tests. To ensure the reliability of test conditions, inter-
laboratory tests were applied according to ISO 6341 and using potas-
sium dichromate (Sigma-Aldrich, St. Louis, MO, USA) as reference
toxicant.
2.3. Immobilization test
An acute toxicity test was carried out according to OECD guideline
202. The third brood of 20 neonates (<24 h old) from our daphnid
culture was exposed to concentrations of 5.00, 4.00, 3.00, 2.75, 2.50,
2.25, 2.00, 1.00, 0.50, and 0.10 mg/L PPP, as well as to the control
series. Groups were divided into four replicates with ve daphnids for
each concentration in ISO medium. Each group was placed in six-well
culture plates lled with 10 mL solution and incubated for 48 h.
Following gentle agitation, immobilization was checked within 15 s
2.4. Reproduction test
Reproduction tests followed the procedure described in OECD
guideline 211 and were carried out using a semi-static design in Elendt
M4 medium. The third brood of D. magna were randomly pooled from
Daphnia culture. Sixteen daphnids were individually held at 0.2, 0.4, and
0.8 mg/L PPP, as well as in the control series for 21 d. The 100 mL glass
beakers were lled with 60 mL prepared solution. D. magna were fed
daily with C. vulgaris (~1.5 ×10
8
cells/mL, 0.1 mg C/D. magna/day)
and thrice a week with YCT (0.5
μ
L/mL). New neonates were counted
and removed daily. The beakers and solution were replaced thrice a
week to maintain good water quality.
2.5. Growth test
D. magna growth was measured three times per week using an optical
microscope (Model CKX41, Olympus Inc., Tokyo, Japan). The magni-
cation, which ranged from 2 ×to 4 ×, was selected based on the size of
observed individuals. Daphnids were transferred onto glass slides along
with a few drops of their originating medium. Body length was
measured from the center of the eye to the base of the apical spine using
ImageJ software. Additionally, forty neonates from the third brood
cultured at each PPP concentration were randomly pooled to measure
their size by following the same procedure.
2.6. Reactive oxygen species (ROS) detection assay
Each sample was prepared with 20 neonates or 2 adults (17 days
old). After desired exposure time, daphnids were transferred to Eppen-
dorf (EP) tubes and gently rinsed with 1 mM phosphate-buffered saline
(PBS). Samples were then homogenized thoroughly in 200
μ
L PBS and
placed in an ice bath prior to being centrifuged at 3000 ×g (4 C, 10
min). Protein content was determined using a bicinchoninic acid kit
(Thermo Fisher Scientic, Waltham, MA, USA). ROS level was deter-
mined using a cellular ROS assay kit (Abcam, Cambridge, UK), which is
based on a 2 ´,7 ´-dichlorouorescein diacetate reaction. Assays were
performed according to the manufacturers instructions using 20
μ
L of
supernatant from the homogenized samples. The obtained uorescence
intensities were normalized with readings from control samples, which
consisted of untreated daphnids.
2.7. RNA isolation and real-time quantitative reverse transcription PCR
(qRT-PCR)
Five adult D. magna (17 days old) were exposed to PPP according to
the desired time. They were then transferred to EP tubes and rinsed with
distilled water three times before being homogenized in TRIzol reagent.
A column-based kit (Qiagen, Valencia, CA, USA) was used to isolate total
RNA, of which approximately 1000 ng was reverse-transcribed using a
high-capacity RNA-to-cDNA kit (Applied Biosystems, Forster, CA, USA).
qRT-PCR assays were performed using Fast SYBR Green Master Mix
(Applied Biosystems) on a 7500 FAST real-time PCR system (Applied
Biosystems). Relative gene expression levels were calculated using the
2
ΔΔCt
method, and D. magna actin was used as an endogenous control
for data normalization. The primer sequences and references are pre-
sented in Table S1.
2.8. Heatmap and hierarchical clustering analysis of mRNA expressions
Relative mRNA gene expression was represented on a heatmap
constructed using the gplots package (version 3.13) in R (version 4.1.2).
Gene expressions were clustered with hierarchical clustering using the
Euclidean distance metric and visualized in color at the center of the
heatmap. The columns and rows represent D. magna gene expressions
and PPP inoculation concentrations, respectively.
2.9. Statistical analysis
Statistical analysis was performed using Originpro 9.65 (OriginLab
Corporation, Northampton, MA, USA). EC
50
value was determined by
LevenbergMarquardt non-linear tting (doseresponse curve with
variable Hill slope) based on immobilization data. The normality of qRT-
H. Cho et al.
Ecotoxicology and Environmental Safety 243 (2022) 113965
3
PCR data was conrmed using the ShapiroWilk test. Statistical differ-
ences between control and exposed groups were analyzed using one-way
analysis of variance followed by Tukeys multiple comparison tests.
Asterisks indicate the signicance of statistical differences (*p <0.05,
**p <0.01, and ***p <0.001).
3. Results
3.1. Acute effects of PPP on D. magna mortality and ROS production
Acute immobilization tests and ROS production assays were per-
formed to investigate acute PPP toxicity and to determine the concen-
trations in a long-term exposure test at sublethal concentrations. Using a
wide range of test concentrations for PPP (0.150.0 mg/L), we calcu-
lated the EC
50
to be 2.71 mg/L by tting a doseresponse curve (Fig. S1).
For the determination of the concentrations in chronic exposure, a
preliminary mortality test in long-term exposure was performed in three
concentrations (0.5, 1.0, and 1.5 mg/L). Daphnids in two concentrations
(1.0 and 1.5 mg/L) showed a mortality rate over than 20 % in 10 d.
Based on this, three concentrations (0.2, 0.4, and 0.8 mg/L) were
selected for the chronic toxicity test. These concentrations were used for
further ROS detection assays to rule out any substantial effect from
oxidative stress on the results of (sub-) chronic endpoints. Neonate and
mother daphnia were exposed to these three PPP concentrations for 6
and 24 h. ROS levels increased with dosage in 6 h exposure samples,
showing statistically signicant differences for the 0.8 mg/L groups (p <
0.05) (Fig. S2). However, there was no signicant change in ROS levels
with 24 h exposure.
3.2. Chronic effects of PPP on D. magna mortality, reproduction, and
time to rst brood
Next, the mortality of a control daphnia series (7.25 %) was vali-
dated according to OECD guideline 211, which states that the mortality
of mother daphnids in a control group should not exceed 20 % at the end
of a chronic test (OECD, 2018). In compliance, our mother daphnids
showed a mortality rate less than 20 % across all groups at 21 d; the
mortalities ingroups exposed to PPP were 12.5 % (at 0.2 and 0.4 mg/L)
and 18.75 % (at 0.8 mg/L), respectively. The mean number of neonates
in the control series over 21 d was 61.6 ±2.8, conforming to OECD
guideline 211 that states that the average number of offspring per ani-
mal should exceed 60 by the end of the test (OECD, 2018). The repro-
ductive output of the 0.2 mg/L exposure group did not show signicant
reduction compared to that of the control series, with the mean number
of neonates being 52.8 ±2.4. However, groups exposed to 0.4 and 0.8
mg/L PPP contained signicantly less neonates (p <0.001) at 44.6 ±2.1
and 38.5 ±3.2, respectively (Fig. 1a). The decrease in the number of
neonates was dose-dependent. Exposure to PPP affected not only
reproductive success but also the timing of the rst brood, which was
signicantly longer (p <0.001) in all exposure groups compared with
that in the control series (Fig. 1b). The average time of the rst brood in
the control group was 9.8 ±0.2 d; timings of the rst brood in PPP
exposure groups were 10.4 ±0.2 d (0.2 mg/L PPP), 10.8 ±0.3 d (0.4
mg/L), and 11.3 ±0.3 d (0.8 mg/L).
3.3. Chronic effects of PPP on size of D. magna adults and neonates
Considering the endocrine disrupting effect of phenol compounds,
we hypothesized that sublethal concentrations of PPP could inhibit not
only reproductive output but also development. Although the average
size of mothers changed in a dose-dependent manner, statistical signif-
icance was rst observed after 15 d, when daphnids in the highest test
concentration group (0.8 mg/L PPP) were shorter than their counter-
parts in the control group (p<0.01). There was no statistically signi-
cant difference in growth between the control and other exposure
groups until 18 d. At this point, the average length of daphnids in the
control series was 3.4 ±0.2 mm, whereas length of daphnids in the
exposed groups were 3.1 ±0.4 mm (0.4 mg/L, p<0.05) and 3.0
±0.4 mm (0.8 mg/L, p<0.01). All PPP exposure groups showed sta-
tistically signicant and dose-dependent growth reduction at 21 d, with
average daphnid lengths of 3.3 mm ±0.2 (0.2 mg/L, p<0.05), 3.2
±0.4 mm (0.4 mg/L, p<0.001), and 3.2 ±0.4 mm (0.8 mg/L,
p<0.001) (Fig. 2a). Furthermore, the size of neonates from third broods
were examined. Similarly, all groups exposed to PPP showed statistically
signicant dose-dependent body length reduction compared with the
control series. The average daphnid length in the control series was 1.69
±0.1 mm, whereas those in experimental groups were 1.63 ±0.07
(0.2 mg/L), 1.53 ±0.07 (0.4 mg/L), and 1.45 ±0.07 (0.8 mg/L) mm
(Fig. 2b).
3.4. Transcriptional change of reproduction and stress response genes and
hierarchical clustering analysis
For understanding the genomic response of D. magna to PPP expo-
sure, we examined the expression of mRNA genes related to develop-
ment and reproduction in the ecdysteroid signaling pathway. This
included: ecdysone receptor alpha (EcR-A), ecdysone receptor beta (EcR-
B), vitellogenin (Vtg1), vitellogenin 2 (Vtg2), hormone receptor 3 (HR3),
Fig. 1. (a) Number of total reproduction of the
surviving D. magna after 21 d of exposure to
para-phenylphenol (PPP). Boxes indicate the
standard derivation; central line indicates the
mean value. (b) Average time to rst brood
upon exposure to PPP. Bars represent the mean
value. Error bars represent the standard error of
the mean. Statistical differences were analyzed
by one-way analysis of variance (ANOVA) and
Tukeys multiple comparison tests. Asterisks
indicate statistically signicant differences
(***p<0.001).
H. Cho et al.
Ecotoxicology and Environmental Safety 243 (2022) 113965
4
nuclear hormone receptor gene (HR96), juvenile hormone esterase
(Jhe), chitinase, and cytochrome P45018a1 (cyp18a1). Two post-
treatment time points (6 and 24 h) were selected for a comparative
analysis of gene expression change, because the levels of gene expression
may recover to basal levels over time (Bang et al., 2015; Imhof et al.,
2017). The expression levels of EcR-A and EcR-B varied across experi-
mental groups at 6 h; however, expression levels were signicantly
downregulated at 24 h after exposure to all concentrations of PPP
(Fig. 3a). Jhe expression showed upregulation after 6 h of exposure to
0.2 and 0.4 mg/L PPP, but downregulation at any PPP concentration
after 24 h (Fig. 3b). The expression of Vtg1 and Vtg2 was downregulated
at all PPP concentrations after both 6 and 24 h (Fig. 3b); and that of
HR96 was upregulated after 24 h (Fig. 3c). The other genes related to
development, chitinase and cyp18a1, did not show a substantial change
with any PPP dosage.
We also measured the expressions of the following stress and
oxidative response genes: two heat shock response genes (Hsp70 and
Hsp90); genes expressing four antioxidant enzymes, namely, superoxide
dismutase (sod), catalase (cat), glutathione peroxidase (gpx), and
glutathione S transferase (gst); and three metallothionein genes (Mt-a,
Mt-b, and Mt-c) (Fig. 4). The expression of Hsp70 and Hsp90 was
downregulated upon exposure to 0.4 mg/L and to 0.2 and 0.4 mg/L PPP,
respectively, after 24 h. The expression of cat increased signicantly
with exposure to 0.8 mg/L PPP for 6 h but substantially decreased with
exposure to 0.2 and 0.8 mg/L PPP for 24 h. The expression of gpx sub-
stantially decreased with exposure to 0.2 and 0.4 mg/L PPP for 6 h, and
the expression of gst substantially decreased with exposure to 0.2 and
0.8 mg/L PPP for 24 h. No signicant changes were observed in the
expression of sod or the three metallothionein genes.
The relative gene expressions affected by PPP exposure were classi-
ed as either down-regulated or up-regulated compared with their
expression in the control group. The down-regulated genes were gst,
EcR-A/B, sod, gpx, cat, Jhe, and Vtg1/2, and the up-regulated genes were
cyp18a1, chitinase, Mt-a/b/c, Hsp70/90, HR96, and HR3 (Fig. 5). Hier-
archical clustering analysis determined high positive correlations
(r >0.95) between gst and EcR-A, gpx and cat, Vtg1 and EcR-B, and
Hsp70 and Hsp90. Additionally, a high correlation coefcient (r >0.95)
was observed except for the clustering. The correlation coefcients and
their p-values are provided in Supplementary information (Fig. S3).
4. Discussion
Toxicological studies that explore phenolic compounds as suspected
EDCs and toxicants have been conducted for several decades. Numerous
phenolic compounds, such as bisphenol A (BPA), 4-nonylphenol, and
2,4-dichlorophenol, are used as precursors for important industrial
commodities. These chemicals have been evaluated in terms of their
effect as water pollutants in freshwater environments (Alla et al., 2021;
Terasaka et al., 2006). The reproductive capacity of D. magna was
signicantly reduced upon exposure to 0.08 mg/L 4-nonylphenol (48 h,
EC
50
0.13 mg/L) (Brennan et al., 2006). In the same study, the effects of
BPA and diethylstilbestrol were also assessed. Fecundity was signi-
cantly inhibited at BPA concentrations higher than 1.0 mg/L (48 h, EC
50
7.75 mg/L) and diethylstilbestrol concentrations over 0.2 mg/L (48 h,
EC
50
1.55 mg/L). At 46.7 mg/L, 2,4-dichlorophenol induced a statistical
difference in daphnid reproductive capacity (Gersich and Milazzo,
1988). Among phenyl-substituted phenols, the toxicity of
ortho-phenylphenol has been reported at 48 h immobilization; its EC
50
was determined to be 1.5 mg/L by Kühn et al. (1989) and 2.7 mg/L
according to Ramos et al. (1998).
Considering the toxicological results of other phenolic compounds,
and bearing in mind the limited results in other indicator species, PPP is
expected to display relatively high acute toxicity. The acute toxicity of
PPP considering 72 h LD
50
was 29.9 mg/L (175.4
μ
M) in the nematode
Caenorhabditis elegans (Jung et al., 2006) and 48 h immobilization EC
50
was 3.9 mg/L for D. magna as per the Ministry of the Environment in
Japan (http://env.go.jp). In our study, 48 h immobilization EC
50
for
D. magna was determined to be 2.71 mg/L PPP (Fig. S1). The 24 h EC
50
was obtained as 0.9 mg/L under ISO 6341 standards (Fig. S4). Daphnids
displayed relatively high sensitivity in the range of 0.62.1 mg/L PPP.
Our results are comparable to those reported for other indicator species
for exposure to isomer compounds (Kühn et al., 1989; Ramos et al.,
1998; Jung at el, 2006). Given that the 48 h immobilization EC
50
value
of other substituted phenols (such as phenyl-, n-chloro-, and
amino-phenol) on D. magna ranged from approximately 1.0 to 10 mg/L
(Dang et al., 2012), the EC
50
value of PPP in our study seems to be
remarkable (Fig. S1).
The overexpression of ROS and/or high mortality in D. magna can
occur because of exposure to toxicants and external stress caused by
pesticides (Horton et al., 2018; Nkoom et al., 2019), pharmaceutical
drugs (Duan et al., 2022; Guilhermino et al., 2021; Liu et al., 2019),
thermal stress (Bae et al., 2016; Im et al., 2019), and nanoparticles
Fig. 2. (a) Body length of each experimental group at 7, 9, 12, 15, 18, and 21 d. Boxes represent standard derivation; central line indicates the mean value. (b) Body
length of forty neonates from the third brood in each experimental group. Boxes represent the standard deviation; central line indicates the mean value. Statistical
differences were analyzed by one-way analysis of variance and Tukeys multiple comparison tests. Asterisks indicate statistically signicant differences (*p<0.05,
**p<0.01, and ***p<0.001).
H. Cho et al.
Ecotoxicology and Environmental Safety 243 (2022) 113965
5
(Puerari et al., 2021; V¨
olker et al., 2013). Although reduction in
fecundity was not observed in a few cases (Daniel et al., 2019; Kim et al.,
2010), the production of high amount of ROS may affect not only
immobilization/mortality related to acute toxicity but also the end-
points related to (sub-)chronic parameters. These results suggest that
external stress may result in an energetic investment in antioxidant ac-
tivities rather than development or reproduction. To rule out the sig-
nicant effect of ROS overexpression on endocrine disruption, ROS
production assays were performed prior to chronic toxicity tests. Our
results showed that the ROS expression level increased in a
dose-dependent manner at 6 h, but decreased again and stabilized at
24 h (Fig S2). Therefore, we hypothesized that the effect of ROS over-
expression could be minimized under the applied conditions.
In terms of a higher order effect at the individual level, reproductive
activity is a key endocrine-relevant endpoint for elucidating the effect of
chronic toxicity (Tkaczyk et al., 2021). The evaluation of the D. magna
reproduction test is widely performed as a classical standardized
endpoint in ecotoxicity tests (Dang et al., 2012). To the best of our
knowledge, the reproductive activity of D. magna exposed to PPP has not
been studied to date. As shown by reproduction test (Fig. 1a), PPP had a
remarkable impact on the fecundity of D. magna. As reproductive ac-
tivity, time to rst brood is a critical parameter related to reproduction
and development as well. Previous report demonstrated that delayed
time to rst brood showed strong correlation with a reduction in the
total number of offspring (Guilhermino et al., 1999). Exposure to the
herbicide molinate led to a 20% decrease in D. magna offspring, con-
current with a 19 % increase in the time to rst brood (S´
anchez et al.,
2004). In our study, upon exposure to PPP, the average time to rst
brood increased dose-dependently, with statistically signicant differ-
ences between the control series and exposure groups (Fig. 1b). The
proportional reduction in reproductive activity supports the identica-
tion of PPP as a suspected EDC.
One of the most common toxicological effects is developmental
modication, such as adverse changes in growth, molting, maturation,
and the size of neonates (Hamid et al., 2021). Previous studies have
indicated that limited concentrations of EDCs do not affect develop-
mental parameters as dramatically as they affect reproductive endpoints
(Oropesa et al., 2016; Svigruha et al., 2021). However, the effect of
Fig. 3. Relative expression of mRNAs following
6 and 24 h exposure of D. magna adults (17 days
old) to para-phenylphenol (PPP). (a) Ecdysone
receptors, (b) reproduction-related genes, and
(c) development-related genes. Bars represent
the mean value. Error bars represent the stan-
dard error of the mean (n 3, each sample
consisting of 5 individuals). Statistical differ-
ences were analyzed by one-way analysis of
variance and Tukeys multiple comparison
tests. Asterisks indicate statistically signicant
differences (*p<0.05, **p<0.01, and
***p<0.001).
H. Cho et al.
Ecotoxicology and Environmental Safety 243 (2022) 113965
6
endocrine disruption on ecdysteroidogenesis may cause abnormal
development, leading to a chronic effect on D. magna (Hutchinson,
2002; Mu and LeBlanc, 2002). Correlated with previous research on
EDCs (Haeba et al., 2008; Lambert et al., 2021), all our test groups
exposed to PPP showed statistically signicant differences in growth
parameters. Furthermore, the size of third generation neonates was
impacted negatively because mother maturity indirectly affects both the
size and number of neonates, as indicated by the correlation between
maternal size and brood chamber volume (Bartosiewicz et al., 2015;
Lampert, 1993).
To determine the molecular mechanism of PPP effects in D. magna,
we analyzed the expression of genes related to reproduction and stress.
Ecdysteroids and juvenile hormones are major regulatory factors of
reproduction and development, being associated with molting and
metamorphosis as well as vitellogenesis (Abe et al., 2015). Ecdysteroids
can be activated through their binding with the heterodimer of EcR and
RXR/USP (retinoid X receptor/untraspiracle) (Dai et al., 2016; Seyoum
et al., 2020). Juvenile hormone (JH), crucial for the molting process, is
reportedly metabolized into JH acid diol or phosphate by the catalytic
activity of Jhe, which regulates the concentration of JH (Hu et al., 2020;
LeBoeuf et al., 2018; Toyota et al., 2014). In addition, JH can suppress
Vtg expression through binding with the upstream JH-responsive ele-
ments and regulating chitinase expression (Merzendorfer and Zimoch,
2003; Tokishita et al., 2006). In this study, daphnid exposure to PPP
induced a signicant downregulation of their EcR-A, EcR-B, Jhe, and two
Vtg genes (Fig. 3). Adult daphnids have reciprocally synchronized cycles
of molting and reproduction. With EcR being responsible for the regu-
lation of molting, decreased expression of this gene may disrupt this
critical cycle (Miyakawa et al., 2018). The decreased level of Jhe may
increase the level of JH, which suppresses downstream vitellogenesis.
Indeed, the expression of vitellogenin genes (which are representative
reproduction markers in oviparous species) was signicantly down-
regulated, corresponding with the change in EcR and Jhe expression
levels (Fig. 3a and b). Combined with substantial downregulation of
ecdysone receptors and reproduction-related genes, it is suggested that
the adverse effects of the EcR and Jhe signaling pathways were
down-regulated by exposure to PPP. These results align well with our
observations of reduced fecundity and developmental retardancy, and
Fig. 4. Relative expression of mRNAs following
6 and 24 h exposure of D. magna adults (17 days
old) to para-phenylphenol (PPP). (a) Heat shock
response (Hsp) genes, (b) metallothionein (Mt)
genes, and (c) antioxidant-related genes. Bars
represent the mean value. Error bars represent
the standard error of the mean (n 3, each
sample consisting of 5 individuals). Statistical
differences were analyzed by one-way analysis
of variance and Tukeys multiple comparison
tests. Asterisks indicate statistically signicant
differences (*p<0.05 and **p<0.01).
H. Cho et al.
Ecotoxicology and Environmental Safety 243 (2022) 113965
7
indicates the negative chronic effects of PPP. Overall, our ndings re-
ected that exposing daphnids to PPP disrupted the expression of several
of their genes linked with ecdysteroid signaling and its hormone
receptor-mediated signaling pathways; this may be the result of
competitive receptor binding because of structural similarities between
PPP and ecdysteroids.
We furthermore analyzed the expression of cyp18a1, HR3, HR96, and
chitinase genes. In the endocrine-mediated pathways, cyp18a1, HR3,
and chitinase regulate the molting hormone 20-hydroxyecdysone
(20HE) (Giraudo et al., 2017; Sengupta et al., 2017). Cyp18a1 inhibits
the activity of 20HE through an ecdysteroid degradation pathway (Kim
et al., 2015; LeBoeuf et al., 2018; Park and Kwak, 2012; Toyota et al.,
2014). As a regulator of molting, EcR activates downstream genes
including HR3, which in turn acts as a positive regulator of the tran-
scription factor of cuticle genes (Asada et al., 2014; Charles, 2010).
HR96, on the other hand, regulates the lipid metabolism pathway as well
as detoxication genes in D. magna (Guittard et al., 2011). In the present
study, drastic gene modulation was not observed except for increased
HR3 and HR96 expression at 24 h post PPP exposure (Fig. 3c). Consid-
ering HR3 is regarded as an inducible gene via EcR signaling, the initial
downregulation of HR3 at 6 h may be explained by the compensatory
effect after EcR activation and vice versa (Hannas and LeBlanc, 2010).
Enhancement of HR96 activity in D. magna led to the altered expression
of genes related to lipid metabolism, resulting in delayed growth and
reduced fecundity (Sengupta et al., 2017). Our results suggest that the
development of D. magna is slowed down by PPP exposure as it disrupts
ecdysteroidogenesis.
Alteration of the ecdysteroid pathway is accompanied by ROS
overexpression upon exposure to xenobiotics. Hsp70 and Hsp90 are
prominent stress proteins expressed following cellular perturbation
(Chen et al., 2021). Metallothionein is a non-enzymatic protein
expressed under several stress conditions such as metal homeostasis
imbalance, oxidative stress, and abnormal metabolic regulation (Amiard
et al., 2006; Andrews, 2000; Asselman et al., 2012; Haq, 2003). In this
study, we observed that Hsp70/90 expression increased at 24 h, but the
expression of Mt-a/-b/-c was not regulated or slightly upregulated at
both time points (Fig. 4). The expression level of antioxidant enzymes
either uctuated or decreased. As such, antioxidant enzymes are
assumed to have a compensatory effect on the stress response to reduced
oxidative stress (Kim et al., 2018; Shields et al., 2021). The expression
levels of gst mRNA changed over time. Although Tukeys multiple
comparison test indicated no signicant difference with gst expression,
exposure to 0.8 mg/L PPP for 6 h induced the expression level at a fold
increase of 2.7 ±0.8. At 24 h, the level of gst mRNA decrease signi-
cantly. Previous studies reported that antioxidant activity aimed at
coping with oxidative stress may demand the synthesis of glutathione
synthesis, affecting indirectly gst mRNA level. However, the depletion of
antioxidant capacity under continuous oxidative stress can lead to a
decrease in the expression of antioxidant-related genes (Liu et al., 2019).
Other reports also reected a temporary increase in gst mRNA levels for
24 h, followed by a subsequent decrease at 48 h post treatment with
dibutyl phthalate (Wang et al., 2019). We hypothesize that the changes
in the level of gst expression is associated with antioxidant capacity.
Hierarchical clustering heatmaps showed distinct separation between
the downregulation of antioxidant enzymes/reproduction gene cluster
and upregulation of stress response genes (Fig. 5). These results indicate
that the downregulation of stress response genes may oppose the
compensation of antioxidant enzymes. However, taken together with
the ROS level (Fig. S3), these results may suggest that oxidative stress
may not have substantially adverse outcomes for (sub-)chronic toxicities
of PPP on D. magna.
5. Conclusion
PPP toxicity was evaluated using D. magna as an aquatic invertebrate
model. Combined with the transcriptional downregulation of repro-
ductive signaling pathways (evidenced by changes in EcR and Jhe
expression), the negative impact of PPP on D. magna development and
reproduction provided sufcient evidence that PPP displays endocrine
disrupting properties similar to those of other phenolic compounds.
However, our results of gene expression levels differed substantially
between the two time points at which they were measured, similar to
ndings by Street et al. (2019). These biological rhythms for the molting
and reproduction cycle require further investigation to clarify the cor-
relation of the signaling cascade during reproduction in D. magna.
Therefore, future studies need to include measurements at more time
intervals, using various concentrations of PPP, to clarify the signaling
modulation induced by this phenolic compound.
Funding
This work was supported by the Nanomaterial Technology Devel-
opment Program (NRF-2017M3A7B6052455) funded by the South
Korean Ministry of Science.
CRediT authorship contribution statement
HyunKi Cho: Validation, Methodology, Data curation, Formal
analysis, Writing review & editing. Chang Seon Ryu: Conceptuali-
zation, Validation, Methodology, Writing review & editing. Sang-Ah
Lee: Validation, Writing review & editing. Zahra Adeli: Validation,
Formal analysis. Brenda Tenou Meupea: Validation, Formal analysis.
Youngsam Kim: Conceptualization, Methodology, Validation, Formal
analysis, Investigation, Data curation, Visualization, Writing original
draft, Writing review & editing, Supervision. Young Jun Kim:
Conceptualization, Writing review & editing, Project administration,
Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
Fig. 5. Hierarchical clustering heatmap of mRNAs at each concentration of
para-phenylphenol (PPP). A graph was created with R using the heatmap. Rows
represent genes and biological replicates, and columns represent different
concentrations of PPP (control, 0.2 mg/L, 0.4 mg/L, and 0.8 mg/L) to which
daphnids were exposed.
H. Cho et al.
Ecotoxicology and Environmental Safety 243 (2022) 113965
8
the work reported in this paper.
Data availability
Data will be made available on request.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.ecoenv.2022.113965.
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