Environ Geochem Health (2019) 41:1847–1860

https://doi.org/10.1007/s10653-018-0159-z (0123456789().,-volV)
(0123456789().,-volV)

ORIGINAL PAPER

Combined effects of ocean acidification and crude oil

pollution on tissue damage and lipid metabolism in embryo–
larval development of marine medaka (Oryzias melastigma)
Lingbin Sun . Jinpeng Ruan . Mengchao Lu . Meng Chen . Zhongliang Dai .
Zhenghong Zuo

Received: 27 December 2017 / Accepted: 20 July 2018 / Published online: 31 July 2018
Ó Springer Nature B.V. 2018

Abstract Ocean acidification (OA) and crude oil
pollution have been highlighted as some of the most
pervasive anthropogenic influences on the ocean. In
marine teleosts, early life-history stages are particu-
larly vulnerable to disturbance by CO2-driven acidi-
fication as they lack pH-mediated intracellular
regulation. Embryos exposed to trace levels of crude
oil constituents dissolved in water exhibit a common
syndrome of developmental abnormalities. So far,
little is known about the combined effects of OA and
crude oil on the early life history of marine fish. Eggs
and larvae of the marine medaka (Oryzias melastigma)
were treated with CO2 (1080 latm atmospheric CO2),
Electronic supplementary material The online version of
this article (https://doi.org/10.1007/s10653-018-0159-z) con-
tains supplementary material, which is available to authorized
users.
the water-soluble fraction (WSF) of crude oil (500 lg/
L) and a CO2 (1080 latm atmospheric CO2)/WSF
(500 lg/L) mixture within 4 h after oviposition.
Isolated and combined OA/WSF had no detectable ef-
fect on embryonic duration, egg survival rate and size
at hatching. Histopathological anomalies of tissue and
lipid metabolic disorder were significant when CO2 or
WSF was given alone at 30 days of age. Combination
of CO2 and WSF enhanced their toxicity compared to
their separate administration. Since the early life-
history stage of marine fish is thought to be impacted
more heavily by increasing CO2 partial pressure
(pCO2) levels and crude oil pollution, OA and crude
oil pollution have the potential to act as an additional
source of natural mortality.
Keywords Ocean acidification
Crude oil
pollution
Tissue damage
Lipid metabolism

L. Sun
J. Ruan
M. Lu
M. Chen
Z. Zuo (&)

State Key Laboratory of Cellular Stress Biology, School
of Life Sciences, Xiamen University, Xiang’an South
Road, Xiamen 361102, China Introduction
e-mail: zuozhenghong@xmu.edu.cn
Despite the vast expanse of the oceans, there is no area
L. Sun
Z. Dai
that remains unaffected by acidification, pollution,
Department of Anesthesiology, Second Clinical Medical
College (Shenzhen People’s Hospital), Jinan University, global warming, and overharvesting fisheries (Cole
Shenzhen, China et al. 2016; Keshavarzifard et al. 2017). Recently,
more and more attention has focused on ocean
M. Chen
acidification (OA), with an increasing concern about
State Key Laboratory of Marine Environmental Science,
Xiamen University, Xiang’an South Road, the adaptive capacity of species responding to an
Xiamen 361102, China acidified ocean and with predictions being made

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regarding future ecosystem responses (Davis and
Caldeira 2010; Lewis et al. 2013; Pespeni et al.
2013; Schunter et al. 2016). Owing to human activ-
ities, the atmospheric concentration of CO2 increased
by nearly 40%, from preindustrial levels of approxi-
mately 280 latm to nearly 400 latm in 2013. Mea-
surements already demonstrate a 0.1 unit drop in the
pH of the surface oceans (Widdicombe et al. 2013).
Increasing anthropogenic emissions of CO2 are
claimed to cause a 0.4 units drop in pH by 2100
(Denman et al. 2011). OA also affects the diversity of
marine fish and is of particular concern for the
vulnerable larval stages critical to populations (Sti-
asny et al. 2016). There are studies concerning the
acute effects of elevated CO2 on fishes including the
alteration of acid–base regulation, respiration status,
blood circulation and nervous system functions, and
the long-term effects including affected larval growth,
lipid accumulation, impaired olfactory function, hom-
ing ability and transgenerational response of marine
fish to ocean acidification (Frommel et al. 2012;
Kurihara and Ishimatsu 2008; Michaelidis et al. 2007;
Murray et al. 2014; Mu et al. 2015; Schunter et al.
2016). If acidification was to continue, acidification
impacts will continue to deteriorate many marine
species for decades.
While the physiological impacts of OA for many
marine biota have been widely studied, the potential
for OA to interact with additional environmental
stressors remains poorly understood. Of particular
concern for environmental monitoring purposes is the
potential for the predicted changes in ocean pH to alter
the behavior and bioavailability of chronic environ-
mental pollution, such as oil pollution. Oil pollution
has been highlighted as one of the most pervasive
human impacts on the ocean. Accidents such as the
1989 Exxon Valdez spill and the 2010 Deepwater
Horizon disaster pose the difficult challenge of
assessing the ecological impacts of the spill. Fish
exposed to trace levels of crude oil constituents
dissolved in water exhibit a common syndrome of
developmental abnormalities. These disorders include
pericardial and yolk sac edema, small jaw and body
axis defects (Carls et al. 1999, 2008), and in addition,
growth rate depression, behavior and brain damage are
also noted in juveniles several months after exposure
ceased (Arukwe et al. 2008; Heintz et al. 2000;
Whitehead et al. 2012). Although short-term embry-
onic exposure to sublethal concentrations of oil causes
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Environ Geochem Health (2019) 41:1847–1860
toxicity that is delayed and not counteracted by the
protective effects of cytochrome P450-dependent
metabolism, after embryonic oil exposure for nearly
1 year, adult zebrafish show subtle abnormalities in
heart shape, indicative of reduced cardiac output.
These delayed physiological impacts on cardiovascu-
lar system at later life stages provide a potential
explanation linking reduced individual survival to
population-level ecosystem responses of fish species
to chronic, low-level oil pollution (Hicken et al. 2011).
There is growing evidence showing that many
environmental stressors can act in a combined manner,
thereby synergistically, antagonistically or additively
affecting many physiological processes of marine
organisms (Barron et al. 2003; Darling and Cote 2008;
Gooding et al. 2009; Lewis et al. 2016). Although
scarce, some recent studies show that Alaska North
Slope crude oil is phototoxic and that ultraviolet
radiation can be a significant and causative factor in
the mortality of early life stages of herring exposed to
oil and chemically dispersed oil (Barron et al. 2003);
increasing temperature and CO2 have positive and
additive effects on the growth rate of the echinoderm
Pisaster ochraceus (Gooding et al. 2009); ocean
acidification also significantly increases copper toxi-
city in mussels (Mytilus edulis) and purple sea urchins
(Paracentrotus lividus) with distinct acid–base
responses (Lewis et al. 2016), and the interactive
effects of temperature and CO2 affect shell dissolution
in the Mytilus chilensis (Duarte et al. 2014). However,
little is known about the combined effects of OA and
crude oil on the early life-history stages of marine
organism (Arnberg 2016). Early life-history stages of
fish with very high rates of oxygen consumption are
likely to be among the most susceptible to changes in
ambient CO2 because their high rates of gas exchange
can be expected to result in particularly low blood
pCO2 approaching ambient pCO2 (Nilsson et al. 2007;
Ferrari et al. 2011). Thus, the magnitude of expected
changes in pCO2 indicates that coastal systems may be
more sensitive to other stressors than previously
thought. These studies show that OA and oil pollution
are occurring concomitantly with human activities, but
no research has yet clearly evaluated the combined
effects. Changes in the speciation of crude oil with
water pH could affect the bioavailability and toxicity.
Meanwhile, oil pollution could also influence the rates
of metabolism and respiration, causing changes in the
levels of lipid metabolism and growth. Crude oil is
Environ Geochem Health (2019) 41:1847–1860
composed by complex hydrocarbon mixtures, with
varying solubilities depending on their octanol–water
partition coefficients. Among them, polycyclic aro-
matic hydrocarbons (PAH) rank as relatively soluble,
more soluble than alkanes having an equal number of
carbon atoms. PAH solubility is an important feature,
considering that many PAH rank among the most toxic
components of crude oil (Zeng et al. 2015). Conse-
quently, our study is necessary to better understand
how the interaction between OA and oil pollution
affects the individual organisms. We examined a suite
of physiological and toxicity responses, previously
described as relevant for other marine organism
(Frommel et al. 2012; Arnberg 2016), to combined
OA and crude oil exposures in the marine medaka
(Oryzias melastigma). The marine medaka (O. me-
lastigma) is a potential marine model fish that is used
in diverse fields of fish biology and ecotoxicology. To
evaluate the combined effects of OA and crude oil on
the early life history of marine fish, the eggs and larvae
of the marine medaka (O. melastigma) were treated
with CO2, the water-soluble fraction (WSF) of crude
oil and CO2/WSF. The expression of genes involved in
lipid metabolism was also analyzed to better under-
stand the toxicities.
Methods and materials
Ethics statement
Animal experiments were carried out in strict accor-
dance with the recommendations in the Guide for the
Care and Use of Laboratory Animals. The protocol
was approved by Xiamen University Institutional
Animal Care and Use Committee (XMUEA-0080).
All surgery was performed under anesthesia using
MS-222 (tricaine methanesulfonate), and all efforts
were made to minimize suffering.
Preparation of the water-soluble fraction
The crude oil used in this study was obtained from
Santa Cruz, Argentina. It was stirred in a 50-L
stainless-steel mixing vessel equipped with a mechan-
ical stirrer and a bottom drain and kept in a cold room.
The WSF was prepared from crude oil and seawater in
a volumetric ratio of 1:100 as previously described
(Ali et al. 1995). An 8-h mixing period was used with
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no settling period. After mixing, the oil and water
phases were allowed to separate for 6 h before the
water phase was siphoned off and utilized immedi-
ately in experiments. Hydrocarbon concentrations
were determined by fluorospectrophotometer (Varian
Australia Pty Ltd, Melbourne, Victoria, Australia)
with excitation wave length of 310 nm and emission
wave length of 365 nm (Wang et al. 2003). The
standard used was a mixed petroleum hydrocarbon
standard provided by the Third Institute of Oceanog-
raphy, State Oceanic Administration (Xiamen, China).
The final hydrocarbon concentration was 500 lg/L.
To maintain WSF equilibrium, procedure was set in
an air-conditioned room (21 °C) and in the darkness.
During exposure duration (96 h), WSF stock was
stored at 4 °C in the dark to avoid compound
degradation or photo-activation.
Medaka culture and exposure
Marine medaka (O. melastigma) were obtained from
City University of Hong Kong and has been reared in
our laboratory for over 10 generations. Standard
operating procedures (SOPs) for large-scale culturing
of O. melastigma were established. Marine medaka
were maintained under optimal growth and breeding
conditions (5.8 mg O2 L-1, 28 ± 2 °C, pH 7.2 in a
14-h light: 10-h dark cycle) (Kong et al. 2008). Eggs
from the female medaka were collected each day a few
hours after spawning and gently separated by rolling
clusters between the fingertips to break the connective
filaments (Marty et al. 1990; Hamm et al. 2001).
To examine the long-term effects of future ocean
acidification and oil pollution scenarios, cohorts of
marine medaka (O. melastigma) were exposed for to
either the ambient CO2 level (380 latm; control) or to
one of three different exposure model scenarios:
1080 latm CO2 (a pessimistic year 2100 scenario),
500 lg/L WSF of crude oil (the WSF concentrations
were chosen based on reported levels following the oil
spill) (McNutt et al. 2012; Nahrgang et al. 2010), and
combined OA and crude oil.
Embryos used in the exposures were all between the
four- and eight-cell stages at the start of the exposure.
The pCO2 levels were obtained by a CO2 enricher
(CE-100B, Wuhan Ruihua Instrument and Equipment,
China), and the target pCO2 levels were checked with
a carbon dioxide sensor (GM70, Vaisala, Finland).
Each replicate consisted of 100 viable embryos, and
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the embryos of each replicate were distributed in
90-mm glass dishes containing 40 mL carbon-treated
water with four treatments: control, 500 lg/L WSF of
crude oil, 1080 latm CO2 and a combined OA/WSF
exposure, and half of the culture seawater was
renewed every 24 h and aerated (0.5 L min-1) with
ambient or CO2-enriched air. There were three
replicates for each treatment. During exposure, car-
diac edema was commonly observed by light micro-
scope in the embryos.
After hatching (between 7 and 12 days), larvae
were transferred to aquaria inflated with a small
aquarium pump. The CO2 enrichment system was
used to regulate the CO2/pH conditions in a 30-L
reservoir tank. Experimental seawater CO2 was
manipulated using a carbon dioxide sensor that
continuously measured pH and injected CO2. Aquaria
were vigorously aerated to maintain appropriate air
saturation and to promote mixing. The aquaria were
aerated with ambient (380 latm CO2) or CO2-en-
riched (1080 latm CO2) air at 0.5 L min-1. Half of
the culture seawater was exchanged every day. Each
population at each CO2 level was maintained in jars at
28 °C. The experiment was carried out in three
replicates, and fish were sampled after exposure for
19 and 30 days post-fertilization (dpf).
From the beginning of the experiment, pH and
dissolved oxygen were measured at 08:00 and 20:00 h
each day. The pH and dissolved oxygen concentration
of the experimental seawater were measured using an
autoclavable pH probe (Mettler Toledo) and oxygen
electrode (Mettler Toledo). A microscope was used to
observe the embryonic and larval development.
Embryos were incubated under static conditions at
28 °C and remained in the exposure solution during
heart rate. The heart rates were obtained from 20-s
video segments collected from individual embryos
(Incardona et al. 2009). Embryos were observed each
day till hatch or death (maximum exposure period of
12 days). We recorded mortality (hatch ability) and
time to hatching (from the start of exposure). And
larvae were observed from hatching to 19 and 30 days
post-fertilization. The larval malformations were
sorted and recorded, and their occurrence frequencies
were determined in all the groups (Zhang et al. 2008).
Larval length was observed by light microscopy every
day. The seawater and tissues were measured for
polycyclic aromatic hydrocarbons (PAHs).
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Environ Geochem Health (2019) 41:1847–1860
Lipid analysis
Total lipids were extracted from dry fish by using a
chloroform/methanol (2:1) solvent mixture similar to
that used by Folch et al. (1957). Because of a small
size, each sample was a pool of fish, and six replicates
were used. Dried samples (200 mg) were sonicated
using a Scientz-IID sonicator (Scientz Biotechnology
Co., Ltd, Ningbo, China) in 5 mL of chloroform/
methanol (2:1) for approximately 30 s. The biomass
was then collected at the bottom of the test tube by
centrifugation, and the solvent was removed to a
weighed vial (EP scientific P/N 340-40C, Miami, OK).
Extraction was repeated twice as described above. The
organic extractions, pooled into a weighed vial, were
dried by blowing a stream of argon gas for 12 h. The
total lipids were then quantified using the sulfo-
phospho-vanillin reaction. Absorbance was measured
at 540 nm with a spectrophotometer after supple-
menting the vanillin and phosphoric acid with heated
lipid and sulfuric acid.
The triglyceride and free fatty acid levels in the fish
were determined using a commercial kit (Triacylglyc-
erol Kit and NEFA Kit, Jiancheng, Nanjing, China).
The total cholesterol content was measured using a
total cholesterol kit (Shanghai Kehua Dongling Ltd.)
following the manufacturer’s instructions.
Real-time PCR analysis
Identification of the sequence was carried out using the
basic nucleotide analysis against National Centre for
Biotechnology Information (NCBI) data (http://www.
ncbi.nlm.nih.gov/BLAST/). The mRNA expression of
peroxisome proliferator-activated receptors (PPARs)
and retinoid X receptors (RXRs) in fish was detected
following the method used in our laboratory (Sun et al.
2011). In this study, five candidate reference genes, the
hypoxanthine guanine phosphoribosyltransferase 1
(HPRT1), glyceraldehyde-3-phosphate dehydroge-
nase (gapdh), succinate dehydrogenase complex sub-
unit A (SDHA), tubulin (TUB) and b-actin (ACTB)
were evaluated for their suitability in normalizing
gene expression under experimental conditions.
According to transcriptome sequencing using next-
generation technologies, gapdh is the suitable choice
to normalize the gene expression for the test condi-
tions and for this research (Table S1). Each sample
was a pool of six fish, and 9 replicates were used. Each
Environ Geochem Health (2019) 41:1847–1860
mRNA level was normalized to gapdh. The Relative
Expression Software Tool (REST-MCSÓ-version 2)
was used to calculate the relative expression of target
gene mRNA (Pfaffl et al. 2002). The real-time quan-
titative PCR primers and the PCR efficiencies are
shown in Table S2.
Histology
The larvae were killed at 30 dpf and selected for
assessment of histopathological changes. Any external
abnormalities (e.g., frayed fins, cloudy eyes, ulcers,
skin discolorations, parasites) were assessed using a
stereomicroscope (Olympus SZ61). Twenty-five lar-
val fish from each treatment were preserved in Bouin’s
fixative solutions for histological analysis. To ensure
uniform and complete fixation, larvae were fixed for
48 h, and then, larvae stored in 70% ethanol were
paraffin-embedded, sectioned into 5-lm sections, and
mounted on slides. Five-micrometer sections of gills,
liver, eye, pancreas and kidney were routinely stained
with hematoxylin and eosin. Sections were then
screened using light microscopy for proper visualiza-
tion and imaged on an Olympus BH2 microscope with
an Olympus digital camera DP26, and cellSens Entry
1.6 software (Olympus Corporation) was used for
image processing. Images of exposure-related tissue
changes were subsequently examined to see whether
they met the standard criteria for normal tissue
distribution based on the method described (Frommel
et al. 2012) and compared with control sections. A
total of controls, crude oil-exposed fish, acidification-
exposed fish and the combined treatment exposed fish
were successfully sectioned at 30 dpf, and most of the
histological damage found was classified as regressive
changes that terminate in functional impairment or
loss of the organ and vacuolation, structural alter-
ations, degeneration, atrophy and necrosis. The stan-
dard for evaluation degree of tissue damage in various
organs (Fig. 1) was adopted and the degree of tissue
damage calculated.
Analytical chemistry
The WSFs and larval pooled samples were analyzed
for the 16 US Environmental Protection Agency
priority PAHs (16 USEPA PAHs). Larval homoge-
nates were used for the analysis of PAH accumulation.
Internal standards were added to the supernatant, and
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the sample was extracted twice with acetone/n-hexane
(both HPLC grade) mixture (3/7; v/v) for 4 h. After
solvent partitioning, the extract was concentrated (N2,
25 °C). Purification of the extract was performed
using liquid chromatography after dilution with an
acetone/n-hexane mixture (3/7; v/v) on an alumina
microcolumn. Finally, a total of 16 USEPA PAHs
were quantified using the Varian CP-3800 gas chro-
matograph linked with a mass spectrometer (Varian
1200 Quadrupole MS/MS, Varian Inc., USA) based on
the reported method (Helaleh et al. 2005). Data
acquisition and processing were performed using
Varian MS Workstation, version 6.8. Quantification
of the analytes was undertaken by an external
calibration of sixteen EPA priority QTM PAH mix,
which contained naphthalene (NAP), acenaphthylene
(ACY), benzo[k]fluoranthene (BKF), acenaphthene
(ACE), fluorene (FLU), phenanthrene (PHE), anthra-
cene (ANT), benzo(a)anthracene (BAA), chrysene
(CHR), pyrene (PYR), benzo(a)pyrene (BAP), fluo-
ranthene (FLT), dibenzo(a,h)anthracene (DBA),
benzo(b)fluoranthene (BBF), indeno(1,2,3-cd)pyrene
(IND) and benzo(g,h,i)perylene (BGP). The recover-
ies of individual PAHs were estimated using a certified
standard compound solution (recoveries [ 90%) and
the samples (recoveries = 80 ± 10%). The detection
limits of the PAHs were 0.07–1.43 lg/L. The limit of
quantification limits ranged from 0.23 to 4.76 lg/L
(Table S3). The relative standard deviation (RSD) of
individual PAH identified in sample extracts was less
than 33%.
Data processing
Results are reported as mean ± standard error (SE)
except for histological comparisons. Statistical anal-
ysis was performed using SPSS software (SPSS Inc.,
Chicago, IL, USA). Two-way ANOVAs were con-
ducted to assess combined effects of pCO2 and crude
oil in embryo–larval development of marine medaka
(O. melastigma). A value of P \ 0.05 was used to
indicate significant difference.
Results and discussion
OA and oil pollution are of concern studying marine
ecosystems because of the high potential as stressors
to teleost fish and their demonstrated toxicity. We
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Fig. 1 Larval length over
1 week post-hatch on a
logarithmic scale. The data
are expressed as mean ± SE
(n = 25). Asterisks (*)
indicate that the values are
significantly different from
that of the control at
P \ 0.05 versus the control
as assessed using two-way
ANOVA
showed that exposure to low-level WSF did not affect
larval development and growth (Fig. 1). The results of
the present study confirm and extend previous work
showing identified effects of CO2-enriched conditions
on fishes, including growth rate, tissue damage and
lipid accumulation (Checkley et al. 2009; Frommel
et al. 2012). This is the first study to investigate the
combined effects of OA and crude oil pollution on the
growth of marine fish, and the degree of histological
damage observed in the eye, kidney, pancreas and
liver were significantly increasing. Our data showed
that combination of near-future OA and crude oil
pollution significantly increases the degree of histo-
logical damage compared to their separate adminis-
tration. In addition, the large amounts of lipid droplets
accumulated around the liver were significantly ele-
vated on group exposed to the pCO2/WSF compared
with isolated pCO2 or WSF (Figs. 2, 3).
Water chemistry
Water chemistry was analyzed to determine the alkanes
and 16 USEPA PAHs in the WSF dilutions used for the
definitive exposure experiment. For 16 EPA priority
PAHs, total concentration was 9.24 ± 1.26 lg/L
(Table S3) and the individual compound with the
highest concentration was phenanthrene. The 16
USEPA PAHs for measurements in the larval pooled
samples were below detection limit. We tested the
experimental seawater exposed to CO2-induced
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Environ Geochem Health (2019) 41:1847–1860
acidification by - 0.39 pH units (here * 1080 latm
atmospheric CO2: see Table S4) on the embryos and
larvae of medaka.
Because of widely diverse group of teleosts in the
marine environment, a wide range of biological
responses to oil spill is reported. Oil and its degradation
products have acute and chronic toxicity to marine fishes
and include death from direct oiling, as well as impaired
feeding, growth and development (Heintz et al. 2000;
Arukwe et al. 2008; Whitehead et al. 2012). As the
major contributors of oil spills, PAHs is well recognized
as toxic organic compounds responsible for genotoxi-
city, metabolism, carcinogenicity and mutagenicity.
Given the persistence, toxicity of carcinogenic PAHs,
many studies and health risk assessments have been
conducted on their accumulation in various marine
species. Carcinogenic PAHs, such as benzo[a]pyrene,
exert their carcinogenic effects only after metabolic
activation, resulting in highly reactive diol-epoxides
capable of forming covalent DNA adducts that can lead
to mutations through errors in DNA replication (Arlt
et al. 2015). Even if some of them (DBA, IND and
BGP) are not detected, it should be noted (Table S3).
Observations on development, larval growth
Embryos between 0.5 and 4 h post-fertilization were
exposed to CO2 (1080 latm atmospheric CO2), WSF
of crude oil (500 lg/L) and CO2 (1080 latm atmo-
spheric CO2) plus WSF (500 lg/L), mimicking
Environ Geochem Health (2019) 41:1847–1860 1853

Fig. 2 Total lipid (a), free

fatty acid (b), triglyceride
(c) and total cholesterol
(d) content of larval medaka
for 19 and 30 dpf. Total lipid
content of dry weight with
standard deviation for each
of the four treatments
(control (380 latm
atmospheric CO2), 500 lg/
L WSF, 1080 latm
atmospheric CO2 and CO2
[1080 latm atmospheric
CO2/WSF (500 lg/L)] at 19
and 30 dph. Data are
presented as mean ± SE
(n = 6). Means of treatments
not sharing a common letter
are significantly different at
P \ 0.05 as assessed using
two-way ANOVA

changes in pH and oil spill pollution. No significant Exposure to WSF did not affect larval growth, but
differences were detected in heart beats, egg survival the groups treated with isolated pCO2 or pCO2 plus
rate or embryonic duration (Fig. S1). WSF showed negative effect on larval growth at 19

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Fig. 3 Histological
changes of the livers stained
with Oil Red O and
counterstained with
hematoxylin in
O. melastigma exposed to
500 lg/L WSF and CO2 for
30 d. a Control; b 500 lg/L
WSF; c 1080 latm
atmospheric CO2; d CO2
(1080 latm atmospheric
CO2/WSF (500 lg/L). Bar:
100 lm. Lipid drops judged
by red staining and indicated
by black arrow
and 22 dpf (Fig. 1). Interestingly, at 30 dpf larvae from
the association of pCO2/WSF seemed to be positively
affected and attained 4.7% more length relative to the
control (Fig. 1). The larvae under increased pCO2 and
crude oil pollution, however, resulted in allocating
resources to continued growth, at the cost of organ
development, and may have outgrown the critical
surface-to-volume ratio necessary for effective cuta-
neous acid–base regulation.
Both OA and crude oil pollution can impact
animal growth and the biotransformation processes
(Arukwe et al. 2008; Frommel et al. 2012;
Ohiozebau et al. 2016) and thus might act severely
or synergistically. Although adult fish tolerate
short-term exposures to pCO2 that exceed those
predicted for the next 300 years (2000 latm),
potential effects of increased CO2 on growth and
survival during the early life stages of fish remain
poorly understood (Ishimatsu et al. 2008). In order
to maintain outward CO2 diffusion gradients,
larval fish are challenged by severe acid–base
disturbances due to proportionally higher extracel-
lular pCO2 values (Melzner et al. 2013). These
acid–base disturbances may then significantly
increase the cost for cellular homeostasis, which
would impact energy storage and the reallocation
of energy needed for survival under other
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Environ Geochem Health (2019) 41:1847–1860
conditions. Mu et al. (2015) observed that CO2-
driven seawater acidification (pH 7.6 and pH 7.2)
had no detectable effect on hatching time, hatching
rate or heart rate of embryos on early life stages of
marine medaka (O. melastigma). However, the
deformity rate of larvae in the pH 7.2 treatment
was significantly higher than that in the control
treatment (Mu et al. 2015). Exposure to pCO2 in
the early life stages of Menidia beryllina causes
severely reduced survival and growth rate (Bau-
mann et al. 2012). WSF is known to contain high
concentrations of alkylated PAHs, low molecular
weight PAHs and related compounds (Kim et al.
2013). Tissues can easily and rapidly accumulate
soluble PAHs (Collier et al. 1995). A potential oil
spill in the vicinity of capelin spawning sites may
affect egg/larval survival and further recruitment
of the exposed population if the oil contaminates
the spawning substrate (Paine et al. 1992). Obvi-
ously, our research showed that the groups treated
with isolated pCO2 or pCO2/WSF between 19 and
22 dpf showed negative effects on larval growth.
However, at 30 dpf, larvae from the association of
pCO2/WSF seemed to be positively affected. It is
possible that the earliest life stages of fish are most
susceptible to acidification.
Environ Geochem Health (2019) 41:1847–1860
Lipid analysis
At 19 dpf, medaka larvae from the pCO2/WSF
treatment had a significantly elevated free fatty acid
content compared with the control (Fig. 2). The
contents of total lipid, triglyceride and total choles-
terol of the four groups were not significantly differ-
ent. In the same way, the total lipid, free fatty acid and
total cholesterol in 30 dpf medaka larvae treated with
isolated pCO2 or WSF had no difference compared
with the control (Fig. 2). However, the group treated
with the combination of pCO2/WSF was more affected
than the group treated with isolated pCO2 or WSF
(Fig. 2). In the liver, we further confirmed the
presence of lipid droplets, whose bulk increased in
the group treated with the pCO2/WSF (Fig. 3). Thus,
we examined the mRNA expression of genes which is
associated with the lipid metabolism such as perox-
isome proliferator-activated receptors (PPARs) and
retinoic acid receptors (RXRs). At 19 dpf, the
expression levels of PPARs and RXRb transcripts in
the group treated with the combination of pCO2/WSF
were up-regulated compared with the control, while
exposure to pCO2 increased the expression of RXRa
and RXRc transcripts (Fig. 4). However, at 30 dpf, no
significant change was observed in the gene expres-
sion of any of the other groups except for the group
treated with isolated pCO2, which showed down-
regulated PPARb mRNA expression (Fig. 4).
Accumulation of fat in the nonadipose tissue of
marine fish exposed to three levels of pCO2 is shown
in liver and gut (Frommel et al. 2012), but little is
known about fat accumulation in relation to pCO2/
WSF. Steatosis is associated with persistently low
urinary pH, low urinary NH4? and high titratable acid-
ity (Bobulescu et al. 2008). With respect to acid
equivalents, proton (H?) excretion in fish is generated
by apical vacuolar-type (H?)-ATPase and/or a
sodium/proton exchanger, the latter dependent on
sodium/potassium-ATPase (Claiborne et al. 2002).
Exposure of the marine teleost, Opsanus beta, at levels
of 750, 1000 and 1900 latm CO2 results in signifi-
cantly increased plasma HCO3- (Esbaugh et al. 2012).
We inferred that the explanations for fat accumulation
seem to center on the increases in pCO2 and HCO3-
that occur in the body during pH compensation for
acid–base balance. In vivo results indicate that WSF of
petroleum increased the activity of microsomal
enzymes that are critical in lipid metabolism,
1855
suggesting a synthesis induction, or an enzyme-
regulatory mechanism (Lavarı́as et al. 2007). Thus,
the effect of pCO2 and pCO2/WSF on liver steatosis is
likely attributable to PPARs and RXRs activation in
the nonadipose tissue (Fig. 4), which in turn decreases
protein biosynthesis and disrupts metabolic pathways
by redistributing lipids to the tissues, leading to
intracellular accumulation of free fatty acids, triglyc-
erides and lipid metabolites (Fig. 2). Increased lipid
content, although an energy store, may not necessarily
be beneficial to the teleosts when it presents as
accumulated droplets in specific organs. These data
suggested that OA and WSF may act synergistically to
increase lipid content.
Histological examination
Moreover, our determination of the sensitive phase of
growth, development, and reproduction coincided
with the major histological damage observed in the
larvae under pCO2/WSF treatment. Visible tissue
damage was found in the eye, kidney, pancreas and the
liver of larvae 30 dpf (Fig. 5), and the degree of
damage significantly increased with pCO2/WSF expo-
sure (Fig. 6). The retinal layer of larval eyes had an
irregular profile 30 dpf when treated with isolated
pCO2 or pCO2/WSF (Fig. 5a). Compared with those in
the pCO2 and WSF groups, the lesions of combined
exposure were more severe. There was some evidence
of vacuolation in the epithelial cell cytoplasm of the
kidney with changes in the staining characteristics.
Furthermore, loss of the neck cells of the pronephric
tubules and atrophy was observed in the group treated
with the pCO2/WSF (Fig. 5b). There was an increase
in the incidence of hepatic lesions (including necrotic
cells) in individuals exposed to pCO2 (Fig. 5c).
Together with the large amounts of lipid droplets
accumulated around the liver (Fig. 3), we also
observed lipid droplets accumulated and the decrease
in Islet size around the pancreas in the group treated
with the combination of pCO2/WSF (Fig. 5d). No
significant effects were found in the gills and ovary
(Fig. S2).
Treatment of Atlantic cod larvae with 4200 latm
CO2 results in severe to lethal tissue damage in many
internal organs (Frommel et al. 2012). However, even
if present CO2 emission trends continue, it would be
difficult to reach extra high levels of acidification in
ocean habitats of the future excerpt for hypoxic
123
1856 Environ Geochem Health (2019) 41:1847–1860

Fig. 4 The mRNA

expression of PPARs and
RXRs genes in the larval
medaka exposed to 500 lg/
L WSF and CO2 for 19
(a) and 30 days (b). Values
were normalized against
gadph. The data are
expressed as mean ± SE
(n = 9). Asterisks (*)
indicate that the values are
significantly different from
that of the control at
P \ 0.05 (*). PPAR:
peroxisome proliferator-
activated receptor; RXR:
retinoid X receptor

regions. In the present study, we confirmed the
hypothesis that low-dosage seawater pH under other
environmental stressors, such as crude oil, threaten
marine life easily through synergic effects. Based on
the quantitative histological study of the damage
parameters, there was no statistically significant deficit
related to 500 lg/L WSF at 30 dpf, although we
detected some impairment related to 1080 latm CO2
exposure. However, larvae exposed to pCO2 under
crude oil conditions presented a more sensitive
measure of morphological damage. Our results
demonstrated widespread tissue damage as a result
of low-level pCO2 and WSF exposure during embryo/
larval development of marine medaka (O. me-
lastigma). Without the modulation for ionic exchange
mechanisms, embryos and larvae of fish are thought to
be relatively susceptible to disturbance in ambient pH
(Larsen et al. 1997; Gilmour and Perry 2009; Perry and
Gilmour 2009). Compared with our present data, most
of the histological evaluation of tissue damage found
was classified as regressive change involving loss of
organ integration and characteristic vacuolation,
degeneration and necrosis. Regarding the incidence
of liver damage in larvae, the relatively high lipid
content alongside the presence of lipid vacuoles in the
liver might be related to the acidification-induced lipid
accumulation found in these larvae, as previously
mentioned (Frommel et al. 2012), suggesting that such
disturbances probably reflected hepatic dysfunction
possibly related to disorders in lipid metabolism. In
the present study, fatty tissue accumulation around the
pancreas of combined treatment fish was observed. In
addition, we observed the decrease in Islet size, which
indicates a loss in pancreatic exocrine function
(Snoeck et al. 1990; Nghiem et al. 2004).
Conclusions
Our results indicate that in a future ocean scenario,
ocean acidification will affect growth rates and
development in marine fish, with oil pollution partially
adding some observed effects of increased pCO2.
Larval development from pCO2 exposure had

123
Environ Geochem Health (2019) 41:1847–1860
Fig. 5 Tissue damage in histological sections of larvae under
increased pCO2 and WSF of crude oil at 30 dpf. Histological
sections of eye, kidney, liver and pancreas from O. melastigma
at four different treatments. Control (380 latm atmospheric
CO2), 500 lg/L WSF, 1080 latm atmospheric CO2 and CO2
Fig. 6 Quantification of degree of damage in various organs
with increasing pCO2 and WSF of crude oil. Mean percentage of
larvae at 30 dpf showing the degree of damage (white bars are
normal, with shading increasing with increasing severity of
positively affected and attained more length relative to
the control, and the interaction of pCO2 and oil spill
pollution synergistically affected larval length.
1857
(1080 latm atmospheric CO2/WSF (500 lg/L). The structures
identified in these sections are: eye: the irregular profile of
retinal layer; kidney: pronephric tubules (t), vacuoles (V), loss of
neck cells (NC), atrophy (AT); liver: enlarged lipid vacuoles
(LV); pancreas: vacuoles (V). Bar: 100 lm
damage) at four different treatment levels (control, 500 lg/L
WSF, 1080 latm atmospheric CO2 and CO2 [1080 latm
atmospheric CO2/WSF (500 lg/L)] for each different type of
tissue. N = 25–27 larvae per replicate
Additionally, the decrease in pH created a microen-
vironment that was both hostile to lipid metabolism,
particularly during the early stages of the experiment,
123
1858
and there was a significant additive interaction effect
of both parameters. In summary, marine fish will
likely be affected more strongly by acidifying oceans
than by WSF of crude oil, but oil pollution effects can
modify acidification impacts on marine fish.
We suggest that enhanced developmental rates at
high pCO2 concentration and oil spill pollution
increase the need of essential lipid for building
reproductive tissue. However, our data complemented
other studies which found tissue damage in response to
increased lipid accumulation in marine fish, leading to
disease with characteristic histological features. Our
results further proved that OA and crude oil pollution
have the potential to act as an additional role of natural
mortality, and affected fish habitat and a population of
already exploited fish resources. Our study clearly
highlighted the importance of understanding the CO2-
induced OA, together with WSF of crude oil, to assess
the combined toxicity and detect the sensitivity of
medaka larvae.
Acknowledgements This work was supported by the Natural
Science Foundation of Fujian Province of China (No.
2018J01067), the Program for Xiamen Southern
Oceanographic Center (14GST69NF33), and the Ocean Public
Welfare Scientific Research Special Appropriation Project
(201405017); Professor John Hodgkiss of The University of
Hong Kong is thanked for his help with English.
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