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Organ-specific toxicity evaluation of stearamidopropyl
dimethylamine (SAPDMA) surfactant using zebrafish embryos
Ola Al-Jamal, Hadeel Al-Jighefee, Nadin Younes, Roba Abdin,
Maha A. Al-Asmakh, A. Bahgat Radwan, Mostafa H. Sliem,
Amin F. Majdalawieh, Gianfranco Pintus, Hadi M. Yassine,
Aboubakr M. Abdullah, Sahar I. Da'as, Gheyath K. Nasrallah
PII: S0048-9697(20)33972-3
DOI: https://doi.org/10.1016/j.scitotenv.2020.140450
Reference: STOTEN 140450
To appear in: Science of the Total Environment
Received date: 2 May 2020
Revised date: 20 June 2020
Accepted date: 21 June 2020
Please cite this article as: O. Al-Jamal, H. Al-Jighefee, N. Younes, et al., Organ-specific
toxicity evaluation of stearamidopropyl dimethylamine (SAPDMA) surfactant using
zebrafish embryos, Science of the Total Environment (2020), https://doi.org/10.1016/
j.scitotenv.2020.140450
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Organ-specific Toxicity Evaluation of Stearamidopropyl Dimethylamine
(SAPDMA) Surfactant Using Zebrafish Embryos
Ola Al-Jamal1†, Hadeel Al-Jighefee1,2†, Nadin Younes1†, Roba Abdin2, Maha A. Al-Asmakh1,2,
A. Bahgat Radwan3, Mostafa H. Sliem3, Amin F. Majdalawieh4, Gianfranco Pintus5, Hadi M.
Yassine1, 2, Aboubakr M. Abdullah3, 6, Sahar I. Da'as7, Gheyath K. Nasrallah1, 2*
1Biomedical Research Center, Member of QU Health, Qatar University, P.O. Box 2713 Doha,
Qatar.
2Department of Biomedical Science, College of Health Sciences, Member of QU Health, Qatar
University, P.O. Box 2713 Doha, Qatar.
3Center for Advanced Materials, Qatar University, P.O. Box 2713 Doha, Qatar.
4 Department of Biology, Chemistry, and Environmental Sciences, College of Arts and Sciences,
American University of Sharjah, P.O. Box 26666 Sharjah, United Arab Emirates.
5Department of Medical Laboratory Sciences, University of Sharjah P.O. Box 27272 Sharjah,
United Arab Emirates.
6Department of Chemical Engineering, College of Engineering, Qatar University, P.O. Box 2713
Doha, Qatar.
7Department of Human Genetics, Sidra Medicine, P.O. Box 26999 Doha, Qatar.
† Equal Contributions
* Correspond to: Gheyath K. Nasrallah
Department of Biomedical Science, College of Health Sciences, Member of QU Health, Qatar
University, Doha, Qatar
Women’s Science building, C01, Tel: +974 4403 4817, Fax: +974-4403-1351, P.O Box: 2713,
email: gheyath.nasrallah@qu.edu.qa
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Abstract
Surfactants are widely used in the industry of detergents, household products, and
cosmetics. SAPDMA is a cationic surfactant that is used mostly in cosmetics, conditioning
agents and has recently gained attention as a corrosion inhibitor in the sea pipelines industry. In
this regard, literature concerning the ecotoxicological classification of SAPDMA on aquatic
animals is lacking. This study aims to evaluate the potential ecotoxicity of SAPDMA using the
aquatic zebrafish embryo model. The potential toxic effects of SAPDMA was assessed by
different assays. This includes (i) mortality/survival assay to assess the median lethal
concentration (LC50); (ii) teratogenicity assay to assess the no observed effect concentration
(NOEC); (iii) organ-specific toxicity assays including cardiotoxicity, neurotoxicity (using
locomotion assay), hematopoietic toxicity (hemoglobin synthesis using o-dianisidine staining),
hepatotoxicity (liver steatosis and yolk retention using Oil Red O (ORO) stain); (iv) cellular
cytotoxicity (mitochondrial membrane potential ) by measuring the accumulation of JC-1 dye
into mitochondria. Exposure of embryos to SAPDMA caused mortality in a dose-dependent
manner with a calculated LC50 of 2.3 mg/L. Thus, based on the LC50 value and according to the
Fish and Wildlife Service (FWS) Acute Toxicity Rating Scale, SAPDMA is classified as
“moderately toxic”. The No Observed Effect Concentration (NOEC) concerning a set of
parameters including scoliosis, changes in body length, yolk, and eye sizes was 0.1 mg/L. At the
same NOEC concentration (0.1 mg/L), no organ-specific toxicity was detected in fish treated
with SAPDMA, except hepatomegaly with no associated liver dysfunctions. However, higher
SAPDMA concentrations (0.8 mg/L) have dramatic effects on zebrafish organ development (eye,
heart, and liver development). Our data recommend a re-evaluation of the SAPDMA
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employment in the industry setting and its strictly monitoring by environmental and public health
agencies.
Keywords: Stearamidopropyl Dimethylamine (SAPDMA) Surfactant; zebrafish; organ-specific
toxicity; LC50; NOEC.
1. Introduction
In comparison with conventional solvents, surfactants have attracted considerable
attention in various applications due to their unique physico-chemical properties and abilities to
be tailor-made to suit various applications (Al-Kandari et al., 2019; Radwan et al., 2017; OECD,
2013). In this regard, a very strong impulse on surfactants research is coming from their growing
employment in many important practical and fundamental industries like petroleum oil recovery,
water, environmental pollutions, and most importantly corrosion inhibition (Radwan et al.,
2017). Corrosion inhibitors have always been considered to be the first line of defense against
corrosion in the oil extraction and processing industries (Chilingar et al., 2008; Migahed and Al-
Sabagh, 2009; Popov, 2015). Therefore, current and future challenges in this field are the
findings of environmentally friendly “green”, cost-effective, organic corrosion inhibitors, which
are of great interest to oil and gas operating companies associated with deep-sea excavation
(Darling and Rakshpal, 1998). Recent studies have shown that surfactants employment is one of
the best-known “green” methods of corrosion protection (El-Lateef, 2014; Malik et al., 2011;
Radwan et al., 2017; Sliem et al., 2019; Zhu et al., 2017).
Nevertheless, whether these compounds are truly eco-friendly and free of environmental
toxic effects especially towards aquatic organisms is a question that remains to be answered yet
(Belanger et al., 2006; Olkowska et al., 2014; Rhein, 2007). Even though surfactants are usually
labeled as “green”, a recent in vivo study explored the toxicity of three commonly employed
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surfactants (sodium dodecyl sulfate, dodecyl dimethyl benzyl ammonium chloride, and fatty
alcohol polyoxyethylene ether) revealing that two of them are very toxic to zebrafish embryos
even at very low concentrations (1 μg/mL) (Wang et al., 2015). Therefore, the eco-friendly
“fame” of many of these widely used compound need to be revisited and further in vivo studied
need to be performed to better investigate the potential toxicity of both new and marketed
compound to ultimately withdraw highly toxic and non-biodegradable compounds from
commercial use and replace them with environment-friendly ones.
Stearamidopropyl dimethylamine (SAPDMA) is a widely used cationic surfactant and the
most common surfactant used in hair conditioners and personal care products, usually at
concentrations below 5% (Minguet et al., 2010). Recently, our collaborators reported that
SAPDMA is a very efficient corrosion inhibitor of API X120 steel under extremely aggressive
conditions (Radwan et al., 2017), a discovery that may prompt the wide employment of
SAPDMA in the industry field of steel pipes-using companies. Given the potential environment
implication associated with the massive employment of this surfactant, especially by sea oil
companies, conducting a comprehensive toxicity and safety evaluation of SAPDMA in an
aquatic animal model remains an essential aspect to be answered. Indeed, to the best of our
knowledge, no studies are present in the literature reporting on this specific aspect of SAPDMA.
According to the Environment Canada Domestic Substance List, SAPDMA is classified as a low
hazardous ingredient and not expected to be potentially toxic or harmful (Canada.ca., 2014).
However, according to PubChem and European Chemical Agency, SAPDMA is very toxic to the
aquatic environment with long-lasting effects and can cause serious eye and skin damage
(National Center for Biotechnology Information., 2020). Thus, the safety employment of
SAPDMA in aquatic environments is still uncertain and needs to be addressed urgently. We
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believe that answering this question may provide new insights concerning the mechanisms
associated with surfactants toxicity in aquatic animals and provide useful information to local
and international public health and environmental agencies concerning the actual SAPDMA
toxicity.
The present study was undertaken to assess the general in vivo and organ-specific toxicity
(cardiac, hepatic, and neuromuscular) to comprehensively evaluate any potential toxicity of
SAPDMA using zebrafish (Danio rerio) embryo as a model for marine fauna. Zebrafish is
recognized by the National Institute of Environmental Health Science (NIEHS, USA) and the
Institute for Environment and Sustainability (IES, Europe) as an excellent system to study
environmental toxicity and is accepted by the National environmental toxicity and is accepted by
the National Institutes of Health (NIH, USA) as an alternative model for exploring human
diseases (Bar-Ilan et al., 2009; Dooley and Zon, 2000; National Institutes of Health., 2016;
Parng, 2005). Since no toxicity studies have been performed on SAPDMA, we investigated a
wider range of concentrations (0.1, 1.0, 5.0, 10, 50, 100, 500, and 1000 mg/L) to find both the no
observed effect concentration (NOEC) and the median lethal concentration (LC50). In this regard,
the selected concentrations were consistent with previously published work using surfactants
(Wang et al., 2015) and within the toxicity rating scale provided by the U.S. Fish and Wildlife
Service (USFWS)(El-Harbawi, 2014).
2. Materials and Methods
2.1 Chemicals
Zinc oxide (ZnO, catalog #721077-100G) dispersion (nanoparticles), of diameter <100
nm, was purchased from Sigma-Aldrich (St. Louis, MO, USA). ZnO nanoparticles, previously
characterized by (Bai et al., 2010), is known to cause mortality and morphological deformities to
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zebrafish embryos and was previously used as a positive control in toxicology studies (Bai et al.,
2010; Choi et al., 2016; Kteeba et al., 2017). Thus, Zinc oxide was used as a positive control
(PC) in all performed experiments. N-phenylthiourea (PTU) (Sigma, Germany) in egg water (E3
media) was used as a media to raise zebrafish embryos in vitro. In addition, it is used to inhibit
pigment formation in the developing zebrafish embryos to facilitate their visualization under the
microscope. All E3 media constituents including 5.0 mM sodium chloride (NaCl), 0.17 mM
potassium chloride (KCl), 0.33 mM calcium chloride dihydrate (CaCl2·2H2O) and 0.33 mM
magnesium sulfate heptahydrate (MgSO4·7H2O) were obtained from Sigma, USA. For the
toxicity experiments, stock solutions such as ZnO, PTU, and E3 media were prepared as
described in (Al-Kandari et al., 2019).
2.2 Preparation of SAPDMA
SAPDMA was prepared through amidation of stearic acid with dimethylamino propyl
amine with a molar ratio of 1.2:1 to 0.8:1 (Maisonneuve et al., 2000). Most preferably, less than
5% excess of amine should be employed. The reactants were added to a reaction vessel in a
nitrogen atmosphere under 90-105 psi pressure range at 40-210 °C. The temperature was held at
140 °C for almost 2 hr before additional ramping and then gradually increased to 180 °C (5
°C/30 min). The reaction’s overall duration was about 10-24 hr to produce in the final SAPDMA
product according to the following reaction:
Dimethylamino propyl amine
+
Stearic acid
Stearamidopropyl dimethylamine
(SAPDMA)
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The main stock was prepared by dissolving SAPDMA gel in distilled water using the
ratio of 0.286 g/100 mL to obtain an aqueous solution of 2860 mg/L concentration. To prepare
the desired concentrations, an intermediate stock was prepared by adding 1048.95 µL of the
main stock to 1951.05 µL of PTU, and then it was vortexed to achieve homogenous suspension.
From the intermediate stock, various dilutions (5 mL total volume) were prepared, in which PTU
was used as the diluent to obtain final concentrations of 0.1, 0.5, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,
4.0, and 5.0 mg/L.
2.3 Zebrafish embryos culture
Two types of zebrafish embryos were used in the toxicity experiments; wild-type AB strain
and the naturally transparent Casper strain. Zebrafish embryos were maintained in an
environmentally controlled lab (photoperiod: 14 hr light/10 hr dark cycle with a water
temperature of 28 °C) (Korenbrot et al., 2013) using the Aquaneering system (San Diego,
California, USA) in zebrafish laboratory in the Biomedical Research Center (BRC) at Qatar
University, Doha, Qatar. Zebrafish were prepared for mating by placing two pairs of adult male
and female fish in a single mating tank separated by a divider and left in the dark overnight. The
Next morning, spawning was triggered by removing the divider and the embryos were left to
mate for 5 hr. After that, fertilized eggs were collected, and the healthy ones were selected and
washed with PTU-E3 media in a new petri dish before conducting the experiments. All
performed experiments on zebrafish were carried out following the animal protocol guidelines
mandated by Qatar University Institutional Animal Care and Use Committee (IACUC) and
policy on zebrafish research established by the Department of Research in the Ministry of Public
Health, Doha, Qatar.
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2.4 Acute toxicity (acutoxicity) assays
Mortality and developmental toxicity of SAPDMA were investigated with an acute
toxicity assay adopted by the Organization of Economic Co-operation and Development (OECD)
guideline for testing chemical toxicity (Nº 203 and 236) (OECD, 2013, 2019). As described in
section 2.2, different concentrations of SAPDMA were prepared from the intermediate stock
(0.1, 0.5, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 mg/L). Then, zebrafish embryos were
dechorionated 24 hr post-fertilization (hpf) using 450 µL of 1.0 mg/mL pronase (St. Louis, MO,
USA) as described in (Rieger, 2013). Afterward, healthy dechorionated embryos were incubated
in 5 mL of fresh PTU-E3 media containing (i) eleven different concentrations (0.1, 0.5, 0.8, 1.0,
1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 mg/L) of SAPDMA; (ii) positive control (PC) ZnO (1.5
mg/L); and (iii) negative control (NC) PTU-E3 media. The cumulative mortality and
morphological deformities were observed and recorded for 3 consecutive days (48, 72, and 96
hpf) under a standard stereomicroscope microscope. The number of dead embryos and
deformities were scored by gross microscopic examination of each embryo. The embryos that
showed coagulated fertilized eggs, no somite formation, undetectable heartbeat, and undetached
tail-bud from the yolk sac were considered dead. Embryos that showed defects or variations in
body length, eye, heart and yolk size, and scoliosis were considered deformed (teratogenic
phenotypes). LC50 values were calculated using GraphPad Prism 7 software (version 7.01, San
Diego, USA) by fitting a sigmoidal curve to mortality data at 95% confidence interval as
described elsewhere (Nasrallah et al., 2018a; Nasrallah et al., 2018b) . Variations in the body
length, eye, and yolk sac size were captured using HCImage software at 21x magnification and
then measured using ImageJ software version 1.52a (NIH, Washington DC, USA) bundled with
Java 1.8.0_172 (Nasrallah et al., 2019). Both mortality and teratogenicity percentage were used
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to calculate the no observed effect concentration (NOEC) of SAPDMA. The NOEC is the
highest concentration of SAPDMA that does not show a significant (p<0.05) morality or
teratogenic defects compared to the negative control (PTU-E3 media) during the exposure
period. Twenty-five embryos were used for each tested concentration of SAPDMA and both
controls. The experiment was done 3 times.
2.5 Cardiotoxicity assay
The blood flow of two major blood vessels in zebrafish embryos was measured to assess
the effect of SAPDMA on cardiac function. The posterior cardinal vein (PCV) and dorsal aorta
(DA) were imaged at 96 hpf, which is the time where the heart usually fully develops (Denvir et
al., 2008). At 96 hpf, embryos were placed on a depression slide with 1-2 drops of 3%
methylcellulose. Embryos were positioned on their side with the same orientation for
measurement of the DA and PCV. Videos of the tail were recorded for all embryos at the same
site where the two major blood vessels were visible (Figure S1) using Zeiss SteREO Discovery
V8 Microscope equipped with Hamamatsu Orca Flash high-speed camera and a workstation
equipped with HCImage software (Hamamatsu Photonics, USA). This camera can record image
sequences with 100 frames per second (fps) speed.
By tracking RBC movements in the two major blood vessels using image analysis
algorithms of MicroZebraLab Blood Flow software from ViewPoint (version 3.4.4, Lyon,
France, three parameters were measured; blood flow velocity, vessel diameter, and heartbeat.
After that, wall shear stress was calculated using the following formula:
Where µ is the blood viscosity (dynes/cm2), V is the average blood velocity (µm/s) and D is
the vessel diameter (µm) (Eisa-Beygi et al., 2018).
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2.6 Hemoglobin staining
To evaluate the effect of SAPDMA on hemoglobin synthesis, a hematopoietic assay was
performed by staining the embryos with o-dianisidine stain (Catalog #D9143-5G, Sigma, USA)
based on a protocol previously described in (Leet et al., 2014). This assay is based on the
oxidation of o-dianisidine by hemoglobin, which produces a dark red stain in hemoglobin
containing cells. Casper strain was used for the staining. At 24 hpf, the embryos were
dechorionated and incubated with three different concentrations of the surfactant (0.1, 0.5, and
0.8 mg/L) and with PTU-E3 media only as of the negative control. At 72 hpf, the stain was
prepared by mixing 0.6 mg/mL of o-dianisidine, 0.65% hydrogen peroxide, 0.01 M sodium
acetate at 4.5 pH, and 40% (v/v) ethanol solution (Leet et al., 2014). Following staining, embryos
were post-fixed in 4% paraformaldehyde at 4 °C for at least 1 hr. 3% (w/v) methylcellulose was
used to fix the embryos on the slide for imaging under a bright field microscope (Stemi 508
Zeiss, Oberkochen, Germany) at 50x magnification. Zeiss AxioCam ERc 5s professional digital
camera was used for imaging. ImageJ software was used to quantify the intensity and size of the
red-stained areas in the embryos’ yolk sac.
2.7 Locomotion (neuromuscular toxicity) assay
For the locomotion assay, fertilized zebrafish embryos were collected in a petri dish
containing E3 media. Abnormal and unfertilized embryos were discarded, and healthy embryos
were incubated at 28.5 °C. At 96 hpf, healthy embryos were transferred to 12-well plates with E3
media containing SAPDMA solution at 0.1, 0.5, and 0.8 mg/L concentrations in addition to the
positive (1.5 mg/L ZnO) and negative (E3 media) controls. Then, the embryos were additionally
incubated for 24 hr at 28.5 °C. At 120 hpf, the embryos were transferred to a 96-well plate by
placing one embryo per well to get a total of 12 embryos for each treated group. Neurotoxicity
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was analyzed by locomotion assessment using ViewPoint ZebraLab technology as described
previously (Nasrallah et al., 2018a; Younes et al., 2020). The 96-well plates containing the
treated embryos were placed in a system chamber at 28.5 °C and irradiated for 20 min with white
light for an acclimation period to allow the embryos to adapt to the environment. Then, the
movement of the embryos was measured under the following conditions: an initial 10 min period
of darkness accompanied by two repeated bright light cycles for 10 min, which was separated by
10 min of darkness. The neurotoxicity was determined through measurement of the average total
distance moved after a cycle of 60 min and by assessing the response of the embryos by the dark-
light cycles. The results were compared to the negative and positive controls using E3 media and
1.5 mg/L ZnO, respectively. Each embryo was analyzed individually in the 96-well plate.
2.8 Hepatotoxicity evaluation
The hepatotoxicity assays were performed using the Tg[cmlc: GRP] transgenic AB strain
of zebrafish. This strain expresses the RFP in the hepatocytes thus allowing a good quality of
liver imaging. To evaluate the toxic effect of SAPDMA on zebrafish liver, the following
parameters were assessed: liver size (to measure necrosis and hepatomegaly) and yolk retention
(to measure liver lipid metabolism) as previously described in (Abou-Saleh et al., 2019; Younes
et al., 2020). At 96 hpf, embryos were incubated for additional 24 hr at 28 ºC with the following
treatments: (i) E3 media (NC) (ii) 1% EtOH (PC), and (iii) 0.1 mg/L SAPDMA.
2.8.1 Liver area analysis
For the liver size measurement, the fluorescent liver of the embryos was imaged with a
fluorescence stereomicroscope (Olympus MVX10) using a digital camera (Olympus DP71). RFP
filtered images of the liver were taken and their areas were analyzed using DanioScope software
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(Noldus, Wageningen, Netherlands) for hepatomegaly and necrosis detection (Abou-Saleh et al.,
2019; Younes et al., 2020).
2.8.2 Detection of yolk retention
Yolk retention was evaluated using Oil Red O (ORO) staining (Catalog #1320-06-5,
Sigma-Aldrich, USA), which is a lysochrome, fat-soluble dye used to stain neutral triglycerides
and lipids. At 24 hpf, healthy embryos were selected and allowed to develop normally until 96
hpf. At 96 hpf, zebrafish embryos were incubated in different SAPDMA concentrations along
with the positive (1% ethanol) and negative control (PTU-E3 media). After 24 hr of incubation
(120 hpf), which is the time at which the liver fully develops, embryos were stained as described
in (Yoganantharjah et al., 2017). Briefly, 0.035 grams of ORO powder added to 7 ml of 100%
isopropanol and stirred overnight on a magnetic stirrer at room temperature. Then, working ORO
stain was prepared by mixing 1 part of ORO stock to 1 part 10% isopropanol (in MilliQ water).
The treated embryos were washed from PTU-E3 media with 60% isopropanol and then replaced
with 1 mL of ORO working solution for 75 min. ORO stain was discarded, and embryos were
washed quickly (30 sec) with 60% isopropanol and then rinsed again for 3 min in 60%
isopropanol, followed by a 30 sec wash in 1% PBS. After that, the ORO stain was extracted from
the embryos for quantification. Sample sizes of 5 embryos were pooled together per eppendorf
tube to extract an adequate volume of ORO stain. Replicates for each treatment group ranged
from 5-6 eppendorf tubes (5 embryos each). PBS was removed from each eppendorf and then
250 mL of 4% ethanol made up in 100% isopropanol was added to each eppendorf tube. Samples
were briefly vortexed and incubated overnight at room temperature to make sure that the ORO
stain was completely extracted. Then, 200 mL of the solution that contained the extracted ORO
stain was pipetted into respective wells of a 96-well plate and OD (absorbance) was read on a
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Tecan GENios Pro 200 at 495nm (Yoganantharjah et al., 2017). Moreover, multiple reads per
well (filled circle, 6×6) were performed.
2.9 Mitochondrial membrane potential assay (ΔΨm)
The mitochondrial membrane potential is one of the key parameters to look for when
studying mechanisms related to cell health and when testing compounds. ΔΨm is usually
measured using cationic fluorescent dyes that accumulates in the anionic mitochondrial matrix.
Such dyes can be used for qualitative measurement in fluorescence microscopy or quantitative
measurement in flow cytometry or microplate spectrophotometry. Several mitochondrial
membrane potential probes can be used such as TMRE, JC-1, and JC-10 probe (Sakamuru et al.,
2016).
In this study, the JC1- mitochondrial membrane potential assay kit (Catalog #T3168,
Sigma, USA) was used to measure the ΔΨm. This kit contains JC-1 fluorescent dye
(tetraethylbenzimidazolocarbocyanine iodide) that accumulates in the mitochondria based on its
membrane potential. This dye is suitable for labeling the mitochondria in live cells of zebrafish
embryos to study the effect of SAPDMA on mitochondrial health and apoptosis (Sakamuru et al.,
2016). The procedure used for the preparation and treatment of the embryos was the same as that
in the acute toxicity assay. At 24 hpf, 30 embryos per concentration were transferred to a 12-well
plate with PTU-E3 media containing SAPDMA solution at 0.1, 0.5, and 0.8 mg/L concentrations
in addition to the positive (1.5 mg/L ZnO) and negative (PTU-E3 media) controls. At 96 hpf,
embryos were incubated with 5 µM of JC-1 dye for 30 min in the dark at 28 °C and then washed
3 times with PTU-E3 media. Then, embryos were transferred to a 96-well black plate, 5 embryos
were added per well with 100 µL of PTU-E3 media. The green and red JC-1 signals were read
using a microplate reader, Tecan GENios Pro. The working principle of the fluorescence plate
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reader is similar to fluorescence microscopy, although the plate reader will only record the total
fluorescence. The green and red JC-1 signals were measured at Ex 485 nm /Em 535 nm and Ex
535 nm /Em 590 nm respectively. Moreover, multiple reads per well (filled circle, 6×6) were
performed. Subsequently, the ratio of green to red fluorescence was measured.
2.10 Statistical analysis
For the acute toxicity assessment, cumulative mortality was expressed as a percentage of
dead embryos at 96 hpf. Descriptive statistics (DS) such as mean (m) and standard deviation
(SD) were calculated for the cardiotoxicity assay, locomotion assay, cytotoxicity, hepatotoxicity,
and o-dianisidine staining. Data were presented as mean ± SD. Statistical analysis was performed
using one-way analysis of variance (ANOVA) followed by the Dunnet test as compared to the
negative control group. GraphPad Prism 7 software was used to remove all significant outliers.
Significance (*) = p < 0.05; (**) = p < 0.01; (***) = p < 0.001.
3. Results and Discussion
3.1 SAPDMA can be classified as moderately toxic to the environment
At first, the potential adverse effect and the morality score of SAPDMA were examined at
24-96 hpf, which is the period where zebrafish embryos are most sensitive to external
compounds and drugs (Olkowska et al., 2014). Measurement of the cumulative mortality
percentage was done at 96 hpf, which is the recommended observation time (Cornet et al., 2017).
According to the mortality data of ZnO nanoparticles (Figure 1A) that feed the mortality curve,
the calculated LC50 value for ZnO was 3.6 mg/L (Figure 1B). For SAPDMA, the mortality score
of the treated embryos increased in a dose-dependent manner starting from 0.1 mg/L
concentration reaching 100% at 3.5 mg/L (Figure 1A). In line with the mortality curve (Figure
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1A), the calculated LC50 for SAPDMA was 2.3 mg/L (Figure 1B). According to the Fish and
Wildlife Service Acute Toxicity Rating Scale that classify compounds toxicity based on the LC50
(Rasool et al., 2018), SAPDMA surfactant can be classified as “moderately toxic”
Figure 1. Embryos viability at different concentrations of SAPDMA and ZnO with the
calculations of LC50. (A) Mortality rate of zebrafish embryos exposed to different concentrations
of SAPDMA compared to the negative and positive control groups. (B) Inhibitor dose-response
curve for the calculation of LC50for ZnO and SAPDMA. *p < 0.05, **p < 0.01 and ***p < 0.001
n = 75.
(A)
(B)
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3.2 SAPDMA cause significant teratogenic phenotypes at very low concentrations
After assessing the survival rate, the morphological and developmental teratogenic effects of
SAPDMA on zebrafish embryos were evaluated and recorded at 96 hpf of treatment to determine
the NOEC, which is the concentration that shows no statistically significant mortality and
teratogenicity (p < 0.05), within a stated exposure period compared to control (Nasrallah et al.,
2018a; OECD, 2013; Suter et al., 1993). Thus, according to the morality score of SAPDMA
(Figure 1B), three concentrations of SAPDMA were selected to assess the teratogenic
phenotypes; heart edema, scoliosis, body length, yolk and eye size (Figure 2B-E). As shown in
Figure 2A-E, only 0.1 mg/L of SAPDMA, did not cause any teratogenicity. Thus, the NOEC of
SAPDMA was chosen to be 0.1 mg/L were all treated embryos showed normal morphology
compared to the negative control.
Yolk sac edema was the most significant and commonly observed teratogenic effect in
embryos treated with 0.5 and 0.8 mg/L SAPDMA (Figure 2B, E). The significant increase in the
yolk sac size strongly indicates that SAPDMA may have an impact on the normal metabolism
and nutrients uptake of these embryos, leading to nutrients accumulation and fluid retention in
the yolk sac. Therefore, in concordance with the acute toxicity findings (section 3.1), these
results provide another line of evidence that SAPDMA surfactant can have a potentially toxic
effect on the embryonic development of zebrafish embryos when tested at higher concentrations.
SAPDMA did not induce any significant effect on the body length and eye size compared to
the negative control except at 0.8 mg/L concentration (Figure 2C, D). Hence, embryos treated
with 0.8 mg/L SAPDMA presented a wide range of developmental defects in all measured
parameters.
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All organ-specific toxicities, including cardio, neuro, hepato, and hematopoietic toxicity
were evaluated at the NOEC (0.1 mg/L). We also used 0.5 and 0.8 mg/L to test the dose-
dependent effect of SAPDMA.
Figure 2. Teratogenic effects of acute toxicity in zebrafish embryos following treatment with
SAPDMA. (A) Representative pictures (96 hpf) of acute toxicity experiments of ZnO
nanoparticles-exposed embryos (PC), PTU-E3 media (NC), and SAPDMA surfactant. Note the
deformed embryos at 1.5 mg/L ZnO (yolk (red arrow), scoliosis (green arrow), and cardiac
edema (blue arrow)). Images were captured using ZeissStemi2000-C stereomicroscope (21X).
(B) A graphical representation of the teratogenic effects observed in zebrafish embryos. Gross
Negative control (PTU-E3)
(A)
0.1 mg/L SAPDMA
Scoliosis
Yolk edema
Heart edema
Positive control (1.5 mg/L ZnO)
(B)
(C)
(D)
(E)
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microscopic examination was used (percentage of embryos with abnormalities was scored). (C-
E) Specific teratogenic effects detected following treatment with three different SAPDMA
concentrations compared to the NC and PC. (C) Average body length, (D) yolk, and (E) eye size
were captured using HCImage software and analyzed using ImageJ software version 1.52a. One-
way analysis of variance (ANOVA) followed by Dunnet test was used to compare the
differences between the average of the imaged areas between groups, **p < 0.01 and ***p <
0.001, n = 25.
3.3 SAPDMA does not affect cardiac parameters at the NOEC
Zebrafish is known to be an excellent model for studying the cardiotoxicity of drugs
(Zakaria et al., 2018). In this study, two of the earliest developing blood vessel in the tail were
examined; the posterior cardinal vein (PCV) and the dorsal aorta (DA) (Al-Kandari et al., 2019).
Four parameters were assessed in the PCV and the DA including; blood flow velocity, vessel
diameter, heart rate (pulse), and shear stress. Our findings showed that SAPDMA induces a
dose-dependent reduction in most of the measured cardiac parameters [(Figure S1, Figure 3A-D
(DA), and Figure 3E-H (PCV)]. The NOEC of SAPDMA (0.1 mg/L) did not affect any cardiac
parameters except for a reduction in the DA diameter (Figure 3B).
The reduction in the blood vessel diameter was more prominent in the DA than the PCV
in all tested concentrations. This could explain the significant induction of shear stress, caused by
SAPDMA, in the DA (Figure 3D), but not in the PCV (Figure 3H). Moreover, SAPDMA did not
induce a significant effect on the heart rate (pulse) in the PCV in all tested concentrations (Figure
3G). However, 0.8 mg/L of SAPDMA significantly decreased the heart rate in the DA (Figure
3C). We suggest that SAPDMA induces cardiotoxicity mainly by inducing vasoconstriction in
the DA and PCV, which lead to the interruption and dysfunction of other cardiac parameters.
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Figure 3. Effect of SAPDMA on the Dorsal Aorta (A) blood flow velocity, (B) diameter, (C)
heart rate (pulse), (D) shear stress, and the Posterior Cardinal Vein (E) blood flow velocity, (F)
diameter, (G) heart rate (pulse), (H) shear stress. Parameters were calculated from the DA and
PCV of the embryos following treatment with each indicated concentration. Twenty-five
embryos were used per concentration. One-way ANOVA was used to compare the differences
between the averages of the imaged areas between groups. *p < 0.05 and **p < 0.01, ***p <
0.001, n = 25.
(C)
(G)
(F)
(D)
(B)
(H)
(A)
(E)
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3.4 SAPDMA does not induce hematopoietic toxicity at the NOEC
The o-dianisidine stain was used to study the effect of SAPDMA on hemoglobin
synthesis in the red blood cells (RBCs) of zebrafish embryos. This is mainly done by detecting
the peroxidase activity in erythrocytes through direct measurement of hemoglobin synthesis or
indirect measurement of erythrocytes production by the bone marrow (erythropoiesis) (Al-
Kandari et al., 2019). The o-dianisidine stain is taken up only by hemoglobin-positive cells,
which means that the higher the hematopoietic activity, the larger the stained area in the
embryos. The amount of hemoglobin in RBCs or the number of hemoglobin-positive RBCs is
calculated by quantitative measurement of size and intensity of the red-stained areas in the
embryos’ yolk sac using ImageJ software. As shown in Figure 4A and 4B, no significant
difference in the number of stained cells was detected in embryos treated at the NOEC (0.1
mg/L) of SAPDMA compared to the negative control. However, embryos treated with 0.8 mg/L
SAPDMA were severely affected and showed a significant decrease in hemoglobin positive
cells. This could be due to the reduced blood flow and vessel diameter as shown in Figure S1 and
Figure 3A-F. Also, the reduced amount of hemoglobin could result from decreased RBCs
production by the bone marrow and reduced hemoglobin synthesis in erythrocytes due to a
blockage in the heme synthesis pathway (van der Vorm and Paw, 2017).
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Figure 4. Distribution of hemoglobin-positive cells using o-dianisidine stain at 72 hpf. (A)
Representative images of o-dianisidine stain concentrated in the yolk sac of the negative control,
0.1, 0.5 and 0.8 mg/L of SAPDMA. (B) The graph shows a quantification analysis difference in
the number of erythrocytes stained by o-dianisidine. Embryos treated with 0.8 mg/L SAPDMA
showed a significant reduction in the hematopoietic activity compared to the negative control.
One-way ANOVA was used followed by the Dunnet test to compare the difference between the
treated groups. ***p < 0.001, n = 30.
3.5 SAPDMA does not induce neuromuscular toxicity at the NOEC
Measurement of the locomotive behavior is very useful in assessing the effects of
SAPDMA on the nervous system and muscle development of zebrafish embryos. In this study,
locomotion and neurotoxicity were evaluated using ViewPoint ZebraLab technology by
measuring the average total distance moved after a 60-min cycle and by assessing the response
of the embryos to multiple dark/light phases. The results were compared to the negative and
positive controls using E3 media and 1.5 mg/L ZnO nanoparticles, respectively. Each embryo
was analyzed individually.
Consistently with the previous study (Chen et al., 2013), the neurotoxic positive ZnO
nanoparticles triggered a significant increase in locomotive behavior compared to the negative
control as shown in Figure 5. In addition, other studies reported an increase in locomotion
behavior associated with different nanoparticles such as chitosan (Sakamuru et al., 2016; Soffker
(B)
(A)
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et al., 2012). Most importantly, at the NOEC (0.1 mg/L), the locomotive behavior of SAPDMA-
treated embryos slightly decreased. However, at higher concentrations of SAPDMA, starting
from 0.8 mg/L, there was a significant reduction in the locomotive activity of the treated
embryos. The reduction in total distance move of the 0.8 mg/mL-treated embryos could be due to
different types of organ damage, in particular, the heart and muscle development and function.
For instance, at 0.8 mg/L exposure, most of the heart function parameters was affected (see
figure 3). There was also a delay/defect in muscle development. For example, as shown in Table
S1, the average length of three somites in the 0.8 mg/L-treated embryos was significantly (p<
0.001) less than the NC. Furthermore, the average total number of the somites per specific area
and also the distance between somites were significantly reduced (p < 0.001) in the 0.8 mg/L-
treated embryos compared to NC (Table S1). More importantly, exposure to 0.8 mg/L of
SAPDA caused severe mitochondrial damage (Figure 7), which is essential for ATP production
that is required for muscle and heart functions. Collectively, all of these abnormalities could lead
to a significant reduction in total distance move for the 0.8 mg/mL SAPMDA-treated embryos.
In conclusion, the findings of the neurotoxicity assay suggest that SAPDMA may have a toxic
effect on the neurons and muscle activity of zebrafish embryos at concentrations higher than the
NOEC.
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Figure 5. Locomotion and neurotoxicity assessment of zebrafish embryos after 24-hr exposure
to different concentrations of SAPDMA. (A) Shows the average total distance moved (measured
using ViewPoint, Micro lab system) every 5 min by the 120 hpf -old embryos treated with E3
media, 1.5 mg/L ZnO, 0.1, 0.5, and 0.8 mg/L of SAPDMA at 96 hpf. (B) Shows the average
distance moved (mm) per hour under dark/light cycles of the same embryos in (A). ***p <
0.001, n =12.
(A)
(B)
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3.6 SAPDMA induces hepatomegaly without affecting liver functions at the NOEC
Previous studies have shown the robustness of zebrafish for hepatotoxicity prediction
(Goessling and Sadler, 2015; Hill et al., 2005; Menke et al., 2011; Vliegenthart et al., 2014). This
robustness is supported by a high degree of genetic conservation for the enzymes and pathways
required in drug metabolisms, such as ARH receptors, CYP enzymes, or ADH isoenzymes,
which are present and functional from early developmental stages, including our experimental
window (Du et al., 2015; Klüver et al., 2014; Timme-Laragy et al., 2007). At 120 hpf, zebrafish
embryos consume the entire yolk and start to seek food from exogenous sources (Chu and
Sadler, 2009). At this point, the liver of the embryos should be fully functional to metabolize
external nutrients from the environment. The yolk consists of 70% lipids, which are mainly
metabolized by the liver (Jones et al., 2008). Therefore, an indirect indication of impaired liver
function is lipid accumulation in the yolk sac of the embryo. To further elaborate, if the liver
function is compromised, the metabolism and absorption of lipids will be delayed, which will
result in lipid retention in the yolk (Huang et al., 2013; Vliegenthart et al., 2014). Hence, we
performed two different hepatotoxicity assays. First, we assessed changes in the liver size in
response to different treatments. As expected, 1% ethanol showed a significant decrease in liver
size compared to the negative control, indicating liver necrosis (Abou-Saleh et al., 2019; Younes
et al., 2020; Zhang et al., 2014). Unexpectedly, embryos treated with the NOEC (0.1 mg/L) of
SAPDMA showed a significant increase in the liver size, indicating hepatomegaly, however, the
normal morphology of the liver (crescent shape) was maintained. Thus, we suggest that 0.1 mg/L
concentration of SAPDMA might cause hepatomegaly without significantly affecting the normal
function of the liver.
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Second, we assessed the neutral lipids accumulation and triglyceride content by staining
the embryos with ORO stain to ensure that the liver function was not affected. ORO stain was
extracted from zebrafish embryos and quantified via optical density (OD) analysis. Since ORO
stains neutral lipids and triglycerides, OD measurements can be taken as a direct and correlative
indicator for the present amount of these two components within zebrafish embryo compared to
the control. As shown in Figure 6, embryos treated with 1% ethanol (PC) showed a significant
increase in lipid accumulation by 47.6%, which is consistent with the previous studies (Abou-
Saleh et al., 2019). However, SAPDMA-treated embryos at the NOEC (0.1 mg/L) showed a
minor increase in lipid retention compared to the negative control, which was not significant.
This means that although the NOEC of SAPDMA (0.1 mg/L) induces hepatomegaly, it does not
affect the liver functions.
SAPDMA 0.1
Ethanol 1%
NC
(A)
PC NC
A) B)
PC NC
A) B)
NC
Ethanol 1%
(B
)
(C)
(D)
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Figure 6. (A) Representative images for liver size. (B) Representative images of yolk retention.
(C) Quantification and measurement of the RFP liver area (m2) for the controls and the tested
compounds. (D) Staining of neutral triglycerides and lipids in wild-type embryos and
quantification of ORO staining. Optical density analysis of ORO stained embryos using a
microplate spectrophotometer. Quantification took into account the intensity and amount of ORO
staining present within the whole zebrafish body. Paired two-tailed student t-test was performed
to compare the difference between the treated group and the negative control. *p < 0.05, n= 10
embryos for liver size measurement, n = 25-30 embryos, 5-6 replicates for ORO stain
measurement.
3.7 SAPDMA does not cause cytotoxicity at the NOEC
The mitochondrial membrane potential (ΔΨm) is a critical parameter of mitochondrial
function and it acts as an indicator of cell health. It is generated by proton pumps in the electron
transport chain and is an important component of energy storage processes during oxidative
phosphorylation (Vliegenthart et al., 2014). It regulates ATP synthesis, ROS generation, and
calcium influx into the mitochondria. Along with the proton gradient, ΔΨm generates a hydrogen
ion transmembrane potential, which is harnessed to make ATP. The level of ΔΨm and ATP are
usually kept stable, but sustained changes in ΔΨm and ATP may induce unwanted loss of cell
viability leading to many mitochondrial dysfunctions (Olkowska et al., 2014). Therefore, the
mitochondrial membrane potential is one of the key parameters to look for when studying
mechanisms related to cell health and when testing compounds’ toxicity.
JC-1 cationic dye was used to measure the ΔΨm in healthy and apoptotic cells. This dye
naturally exhibits green fluorescence and can cross the mitochondrial membrane and accumulate
inside forming reversible complexes known as J-aggregates (Yoganantharjah et al., 2017).
Unlike JC-1 molecules that exhibit green fluorescence, these aggregates show excitation and
emission in the red spectrum. Therefore, healthy cells with normal ΔΨm will form red
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fluorescent J-aggregates as the dye accumulates inside the active negatively charged
mitochondria (Yoganantharjah et al., 2017). On the other hand, the mitochondrial membrane in
unhealthy and apoptotic cells will be less negative due to the disruption of mitochondria and
changes in its membrane potential. Such alterations happen when the cell is undergoing early
stages of apoptosis which leads to the opening of permeability transition pore (MPTP) and
passage of ions and small molecules. The negativity of the membrane will decrease
(depolarization) until equilibrium is reached which in turn leads to the decoupling of the
respiratory chain and releasing of cytochrome C into the cytosol. This triggers the apoptotic
cascade (Rasool et al., 2018; Yoganantharjah et al., 2017). Therefore, JC-1 dye will not be able
to reach a concentration that is sufficient to enable the formation of J-aggregates and remains in
the monomeric form with its natural green fluorescence.
Based on these premises, the green/red fluorescence ratio of JC-1 dye can be used as a
direct indicator of the mitochondria’s polarization status. SPADMA-treated embryos were
stained according to the protocol mentioned in section 2.10. The intensity of green and red
signals was measured, and then the ratio of green/red fluorescence was determined. As shown in
Figure 7, there was no significant change in the green/red fluorescence ratio in ZnO-treated
embryos or those treated with 0.1 mg/L and 0.5 mg/L SAPDMA. This indicates a normal
polarization of the mitochondrial membrane, which means that the cells are healthy. In contrast,
embryos treated with 0.8 mg/L SAPDMA showed a significantly elevated green/red fluorescence
ratio indicating unhealthy or apoptotic cells. ZnO-treated embryos showed no significant change
in the green/red fluorescence ratio, which could be due to cell necrosis or cells undergoing late
stages of apoptosis (Wang et al., 2015), leading to the uptake of the JC-1 dye.
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Figure 7. The green/red fluorescence ratio measured in zebrafish embryos treated with different
SAPDMA concentrations. The ratio of J-monomers (green signal at 540 nm; dead cells) to J-
aggregates (red signal at 590 nm; live cells) was calculated using GraphPad Prism 7 software.
***p < 0.001, n = 25.
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4. Conclusions
Our study for the first time presents data that comprehensively investigates the organ-specific
toxicity of the cationic surfactant SAPDMA in vivo in the zebrafish embryo model. The
performed toxicity assays suggest that SAPDMA can cause adverse toxic effects in a dose-
dependent manner on the embryonic development of zebrafish. Indeed, the mortality score at 96
hpf following exposure steadily increased, starting with 9% in embryos treated with 0.1 mg/L
(NOEC) SAPDMA until it reached 100% in embryos treated with 3.5 mg/L SAPDMA. The LC50
and NOEC were found to be 2.3 and 0.1 mg/L, respectively. Thus, we concluded that SAPDMA
is classified as “moderately toxic” according to the U.S. Fish and Wildlife Service (USFWS)
acute toxicity rating scale (Table S2) (Rasool et al., 2018). Thus, we recommend that the use of
SAPDMA in the industry should be re-evaluated and monitored by different environmental and
public health agencies.
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Author Contributions:
Conceptualization: GKN; Methodology: ABR, MHS, OAJ, HAJ, RA and NY; Software: OAJ,
HAJ, RA and NY; Validation: GKN and GP; Formal Analysis: GKN, OAJ, HAJ, RA and NY;
Investigation: GKN, OAJ, HAJ, RA and NY; Resources: GKN, GP, HMY, and MA; Data
Curation: GKN; Writing – Original Draft Preparation: GKN, OAJ, HAJ, and RA; Writing –
Review and Editing: GKN, AFM, OAJ, HAJ, NY, MA, HMY, and GP; Visualization: GKN,
AFM, OAJ, HAJ, and NY; Supervision: GKN, GP and AMB; Project Administration, GKN and
MA; Funding Acquisition: GKN and MA.
Acknowledgment:
This work is supported by Qatar University's internal grant QUCP-CHS-17-18-2 Given to MAA
and Qatar University international research collaboration co-funds grant IRCC-2019-007 given
to GKN and FP. This work was made possible by UREP grant # (UREP26-097-3-040) from the
Qatar National Research Fund (a member of Qatar Foundation). The statements made herein are
solely the responsibility of the author(s). We would also like to thank Qatar National Library (a
member of Qatar Foundation) for sponsoring publication fees (gold open access) of this paper.
Conflict of interest:
Declarations of interest: none (all authors have no competing interests to declare).
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Credit Author Statement
Conceptualization: GKN; Methodology: ABR, MHS, OAJ, HAJ, RA and NY; Software: OAJ,
HAJ, RA and NY; Validation: GKN and GP; Formal Analysis: GKN, OAJ, HAJ, RA and NY;
Investigation: GKN, OAJ, HAJ, RA and NY; Resources: GKN, GP, HMY, and MA; Data
Curation: GKN; Writing – Original Draft Preparation: GKN, OAJ, HAJ, and RA; Writing –
Review and Editing: GKN, AFM, OAJ, HAJ, NY, MA, HMY, and GP; Visualization: GKN,
AFM, OAJ, HAJ, and NY; Supervision: GKN, GP and AMB; Project Administration, GKN and
MA; Funding Acquisition: GKN and MA.
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Declaration of competing interest
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
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Graphical abstract
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Highlights:
According to the U.S. Fish and Wildlife Service (USFWS) acute toxicity rating scale,
SAPDMA is classified as “moderately toxic” surfactant.
The exposure of the embryos to SAPDMA caused mortality in a dose-dependent manner
with calculated LC50 of 2.3 mg/L and NOEC at 0.1 mg/L.
SAPDMA induces cardiotoxicity by decreasing the Aortic pulse, stress sheer, and blood
flow velocity at concentration higher than the NOEC.
Starting from 0.8 mg/L, SAPDMA severely affects the hematopoiesis process and
locomotive activity.
SAPDMA at the NOEC (0.1 mg/L) might cause hepatomegaly without significantly
affecting the normal function of the liver.
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