Opening a new gateway towards the applications of chitosan nanoparticles stabilized Pickering emulsion in the realm of aquaculture

Carbohydrate Polymers 265 (2021) 118096

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Opening a new gateway towards the applications of chitosan nanoparticles
stabilized Pickering emulsion in the realm of aquaculture
´k, Josef Velíˇsek, Jan Mra
´z
Bakht Ramin Shah *, Petr Dvoˇra
ˇ e Budˇejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of
University of South Bohemia in Cesk´
Hydrocenoses, Czech Republic

A R T I C L E I N F O

A B S T R A C T

Keywords:
Pickering emulsions
Eugenol
Characterization
Fish anesthesia
Common carp

In the current study, we synthesized eugenol (EU) based Pickering emulsion (PE) stabilized by food grade in­
gredients such as chitosan (CS) and tripolyphosphate (TPP) not only to enhance water miscibility of EU but also
to decrease stress and damage to the immune system of fish due to anesthetic procedures. The formulated EUPEs
were characterized in terms of droplet size, size distribution and the effects of environmental conditions e.g. pH

and temperature on the behavior of the EUPEs. The results showed that EU PEs with 5% EU had smaller size with
uniform distribution and were stable in the range of pH 5–7.5 and temperature 30–80 ◦ C. The anesthetic effect of
the EUPE was investigated by taking Common carp as a sample species. Interestingly, it was found that the
induction time to anesthesia and recovery for the fish that received the PE was significantly shorter than that
received EU at the same eugenol concentration (50 ppm). Most importantly, the improved hematological and
bio-chemical parameters in the PE group further confirmed the immuno-protective and stress control efficacy of
the PE. The results of this study propose a novel useful and potential application of PE in fishery where sedation
is needed.

1. Introduction
Emulsions are regarded as thermodynamically unstable systems of
two immiscible or partly miscible phases e.g. oil and water (BertonCarabin & Schroăen, 2015). The interfaces between these phases are
required to be stabilized by some type of amphiphilic or surface active
compounds (Matos et al., 2018). The surfactants used to stabilize
conventual emulsions such as nano or micro (McClements, 2012), can
exert adverse effects on health as well as environment (Perrin et al.,
2020). On the other hand, Pickering emulsion (PE) is an emulsion which
is stabilized by solid particles instead of surfactants (Pickering, 1907;
Ramsden, 1904). The particles are partly wetted by both the phases and
are irreversibly adsorbed at their interfaces thereby conferring these
emulsions with stability for months or even years. In contrast with
conventional emulsions (stabilized by small molecular surfactants), PEs
have garnered tremendous attention in scientific community due to its
non-toxic safe and long-term stable nature and therefore, are known as
much more advantageous and efficient option for the encapsulation and
delivery of a wide range of bioactive compounds (Shah & Mraz, 2020).
So far a wide range of particles have been implied to stabilize PEs

including inorganic or synthetic silica, latex, clay and so on (Yang et al.,
2017) or natural food grade particles derived from protein such as lac­

toferrin, soy protein isolate (SPI), pea protein isolate (PPI) etc. (Shao &
Tang, 2016); from lipids in the form of fat crystal which have been
documented as potential stabilizers for water in oil (W/O) emulsions
(Rousseau, 2013). However, carbohydrates-based particles in the form
of modified starches comprise a large group of emulsifiers for PEs. This
predominantly includes cellulose, starch and chitosan (CS) etc. (Chen
et al., 2020; Soltani & Madadlou, 2016). CS is generally obtained by the
alkaline deacetylation of chitin (the second most abundant polymer in
nature after cellulose) present in the exoskeletons of crustaceans as well
as the cell walls of some algae and fungi (Mohammed et al., 2013). Being
inexpensive, commercially available, biocompatible, biodegradable due
to the free amino and hydroxyl groups along its backbone, CS has a wide
range of useful applications in biomedicine, pharmaceutics and food
industries (Mwangi, Ho, Ooi, et al., 2016). During the last few decades
there has been a considerable research interest in using CS nanoparticles
(NPs) as drug delivery carriers for sustained and controlled release of the
encapsulated drugs and as stabilizers for stabilizing the oil water in­
terfaces of PEs. To synthesize these NPs, different methods have been

* Corresponding author at: University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Centre of
ˇ e Budˇ
Aquaculture and Biodiversity of Hydrocenoses, Institute of Aquaculture and Protection of Waters, Na S´
adk´
ach 1780, 370 05 Cesk´
ejovice, Czech Republic.
E-mail address: (B.R. Shah).
/>Received 4 November 2020; Received in revised form 8 March 2021; Accepted 22 April 2021
Available online 24 April 2021
0144-8617/© 2021 The Author(s).
Published by Elsevier Ltd.

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B.R. Shah et al.

Carbohydrate Polymers 265 (2021) 118096

implied so far including pH induce CSNPs (Liu et al., 2012) or by cross
linking through ionotropic gelation method using different compounds
such as glutaraldehyde (GLA), glyoxal (GO), epichlorohydrin (ECH),
ethylene glycol diglycidyl ether (EGDE) and tripolyphosphate (TPP)
(Babakhani & Sartaj, 2020; Shah, Li, et al., 2016). However, TPP is
regarded as low cost and environmentally safe in comparison to the rest
of these cross-linking agents which are known to be toxic and exert

adverse effects on the environment (Vakili et al., 2014). In our previous
studies we successfully synthesized CS cross linked TPP NPs which
proved to be efficient stabilizers for a long-term stable PEs (Shah, Li,
et al., 2016; Shah, Zhang, et al., 2016). Available literature supports the
claim that till date PEs have a broad spectrum of useful applications in
food, cosmetic and pharmaceutical industries including delivery of
bioactive compounds and in the synthesis of porous scaffold,
environment-responsive material and thermo-responsive material so on
(Jiang et al., 2020; Yang et al., 2017). In addition, most recently, a
research group fabricated zein particle stabilized clove oil (CO) PE and
found that the addition of the synthesized COPE boosted up the anti­
microbial efficacy of the chitosan-based edible films (Xu et al., 2020).
Eugenol (EU) an essential oil present in CO (70–90% by weight) is
obtained from the buds, leaves, flowers and stem of the clove tree
(Syzygium aromaticum) (Soltanian et al., 2018). It contains active com­
pounds isoeugenol and methyleugenol and is considered a potential
anesthetic in fish (Hur et al., 2019). However, due to the issue of its poor
dissolution in water, researchers employ alcohols or detergents as dis­
solving agents which can exert unfavorable effects on the fish and other
living organisms (Gholipourkanani et al., 2015). Fish anesthesia is often
required during different experimental procedures or during their
transportation, grading or vaccination to make them sedated for
extended period of time. And applying higher doses of anesthetics can
put a huge amount of stress on fish, that can result in anomalous
metabolic rates, oxygen utilization, blood pressure, and blood physio­
logical responses, which can even last for several hours after recovery
from anesthesia and can ultimately affect the nutritional quality of fish
for human consumption (Herrera et al., 2019; Park et al., 2009). Now in
order to avoid these unfavorable conditions, it is desired to reduce the
stress to the fish up to the maximum level during anesthetic procedures.

And hence ecofriendly, non-toxic and cost-effective formulations such as
PEs stabilized by food grade ingredients will be the best choice to serve
the role. So far no such study has ever been conducted to use EUPEs
based formulations as anesthetizing agents in fishery. Therefore, the
main aim of the current study was to fabricate and characterize EUPEs
stabilized by CSTPP NPs and evaluate their anesthetizing role in com­
mon carp as a sample specie.

room temperature (≈25 ◦ C). After overnight stirring the solution was
filtered to remove the large dust or undissolved particles. A solution of
TPP at the concentration of 1.0 mg/ml (0.1 wt%) was prepared by dis­
solving TPP powder in ultrapure water and stirred to ensure its complete
dissolution. The NPs were then prepared by dropwise addition of TTP
solution onto the CS solution at a ratio of 1:1 (w/w) under continuous
stirring.
2.3. Preparation of the required EUPEs
Using the synthesized CSTPP NPs as stabilizer, the desired PEs were
prepared with different EU contents (5, 10, 20, 30, 50 and 70 wt%). To
do so, aqueous phase containing NPs (95, 90, 80, 70, 50 and 30 v%
respectively) was taken in a glass vial and was mixed with the oil phase
(EU) at the specified amounts. This mixture was then vigorously ho­
mogenized using UltraTurrax® T25 device equipped with a S25N-18G
shaft (IKA, Germany) rotating at a speed of 10,000 rpm for 3 min. The
emulsions so prepared were immediately transferred to tightly covered
glass bottles and stored at room temperature (≈25 ◦ C) for further
analysis.
2.4. Emulsions characterizations
2.4.1. Mean droplet size & droplet size distribution
Particle size (mean diameter) and size distributions of the PEs sam­
ples were determined using a Malvern Mastersizer 3000 instrument

(Malvern Instruments Ltd., Worcestershire, UK). The samples were
dispersed in deionized water (≈1000 ml) and stirred at a rate of 2200
rpm to avoid multiple scattering effects. The refractive indices of 1.53
and 1.33 were used for clove oil droplets and water (the dispersant),
respectively.
2.4.2. Effect of pH
The effect of pH was assessed by adjusting pH of the samples to
different values i.e. 5, 6, 7 and 8 using either 0.1 M NaOH or 0.1 M HCl
solution. Thereafter, mean particle diameter and size distributions of
these samples were measured in the same way as described above in
Section 2.4.1.
2.4.3. Effect of temperature
In order to evaluate the effect of temperature on the behavior of the
emulsions, emulsion samples were incubated in a water bath at 30, 50,
60, 80 and 90 ◦ C for 30 min. The visual changes of the emulsions were
observed. Then the mean particle diameter and size distributions of the
samples were determined after the samples cooled down to room tem­
perature following the same procedure mentioned in Section 2.4.1.

2. Experimental section
2.1. Materials and methods

2.4.4. Optical images
Optical micrographs of the ZNCSPs based PEs were taken by an
Optical microscope OLYMPUS (U-TVO.63XC, Tokyo, Japan) fitted with
a digital camera (Olympus, DP 50). Emulsions samples were placed
directly onto a microscope slide and softly covered with a cover slip. The
images were recorded at 25 ◦ C under the magnification from 10 to 40×.

CS (3 × 105–7 × 105 Da), ≥99.5% acetic acid (AA), TPP, NaOH were

purchased from Sigma-Aldrich, Sokolovska, Prague, Czech Republic. EU
(100%) was purchased from Mach chemical spol. s.r.o. Ostrava, Czech
Republic. NaCl and 36–38% HCl were purchased from PENTA phar­
maceuticals Radiova, Prague, Czech Republic. All of the materials
guaranteed analytical grade and were used without further purification.
Water used in the preparations of all solutions was purified by deion­
ization and filtration with a IWA 30 iol, WATEK apparatus (Czech Re­
public) to a resistivity higher than 18.0 MΩ⋅cm.

2.5. Fish trials
2.5.1. Experimental fish
A total of 28 common carps with average body weight 206 ± 41, total
body length 223 ± 21 and specific length of 182 ± 12 were selected from
the already acclimatized common carp in the aquarium of the Institute
of Aquaculture and Protection of Waters, Faculty of Fishery and Pro­
ˇ ´e Budˇejovice. The
tection of Waters, University of South Bohemia in Cesk
fish were individually stocked in plastic tanks (10 L) equipped with
aeration. Parameters such as water dissolved oxygen, pH and tempera­
ture of the water were measured to be 90%, 7.3 and 19 ◦ C respectively.
In order to test the anesthetizing efficacy of the designed EUPEs, the

2.2. Preparation of chitosan tripolyphosphate nanoparticles (CSTPP NPs)
CSTPP NPs were synthesized using the same method as described in
our previous work (Shah, Li, et al., 2016). Briefly, 0.5 wt% CS solution
was prepared by dissolving CS powder in AA solution maintaining CS to
AA ratio of 2:3 and was diluted with deionized water to obtain the
required volume. The resultant mixture was then stirred for overnight at
2

B.R. Shah et al.

Carbohydrate Polymers 265 (2021) 118096

selected fish were divided into seven groups (six experimental and one
control with no EU) with four fish in each group. EU at three different
concentrations i.e. 12.5, 25 and 50 ppm either in the form of PE (0.25
ml, 0.5 ml & 1 ml of 5% PE/l respectively) or free (original form) were
applied to the respective tanks with the individual fish.

3. Statistics
All experiments were done at least in triplicates. The data were
presented as mean ± SD. Statistical evaluation of the anesthesia in­
duction and recovery times as well as hematological and biochemical
parameters were carried out by analysis of variance (ANOVA) procedure
using the Statistix 8.1 software. Least significant difference (LSD) at 5%
level was used for multiple comparison tests among the treatments.

2.5.2. Onset and recovery from anesthesia
The observations of the stages 5 & 6-anesthesia onset and recovery
(according to Table 1) were made after using either EUPE or EU under
completely similar experimental conditions. The duration to achieve
stages 5 & 6 anesthesia (A5 & A6) for each replicate were noted using a
stopwatch. Once an individual fish would reach the onset of stage 6
anesthesia, a dip net was used to immediately remove it from the
experimental tank. The fish was then washed off in clean water tank and
transferred to another 10 L, well-oxygenated tank termed as ‘Recovery’
tank (i.e., no anesthesia present) maintained at 19 ◦ C and observed until
it fully recovered. During this recovery period, the fish behavior was

observed and times to observe stages 5 & 6 recovery (R5 & R6) were
noted using a stopwatch.

4. Results and discussion
4.1. Fabrication and characterization of EUPEs
4.1.1. Optimization and droplet size of the EUPEs
In order to prepare stable PE systems, the oil to water ratio should be
optimized as it will affect particles concentration which is the vital
factor in controlling the droplet size of PEs. With this consciousness,
initially we prepared the required EUPEs with different oil (EU) frac­
tions of 5, 10, 20, 30, 50 and 70 wt% shown in Fig. 1a. As can be seen,
the formulations with higher oil contents of 30, 50 and 70 wt% didn’t
turn into well homogenized emulsions systems and the oil fractions were
clearly seen separated soon after the homogenization. On the other
hand, formulations with lower oil contents i.e. 5, 10 and 20 wt% gave
well homogenized and stable emulsions systems at the same homoge­
nizing speed and time. The reason for this emulsification failure at
higher oil contents could be due to the poor coverage of the oil water
interface by the stabilizing CS-TPP NPs. Based on these outcomes, we
therefore, didn’t further characterize the formulations with higher oil
contents but rather proceeded with the ones having lower oil contents.
Optical images in Fig. 1b, c and d for emulsions with oil contents 5, 10
and 20 wt% respectively, exhibited an increase in size with increasing
oil contents. This trend in increase with increasing oil contents was
further confirmed by measuring mean droplet diameter through laser
diffraction instrument (Malvern Mastersizer 3000, UK).
It is evident from Fig. 2 that although the three emulsions had uni­
form particle distributions, the droplet sizes in terms of the Sauter mean
diameter (d32) and volumetric diameter (d43) increased significantly
with increasing the oil contents from 5 to 20 wt%. These results are in

agreement with the previous study where increase in sizes with increase
in oil contents of olive oil based PE was observed at all tested pH values
(i.e. 2, 4 and 7) (Jutakridsada et al., 2020). As a matter of fact, this can
be attributed to the alteration in the oil to particles ratio caused by
increasing the oil contents in the system. This phenomenon has been
well explained in previous work by Arditty and colleagues who
described the phenomenon of droplet size variation with the amount of
particles by a so-called limited coalescence. The emulsion droplets tend
to coalesce as a result of the decrease in the total particles concentration
required to completely cover the oil-water interfaces (Arditty et al.,
2003). Similar results of the link between particles concentration and
droplet size were also presented by Tzoumaki et al., who obtained
emulsions with smaller droplets, enhanced stability and distinct elastic
responses by increasing the concentrations of chitin nanocrystals (ChNs)
used to stabilize oil-in-water emulsion (Tzoumaki et al., 2011).

2.5.3. Blood sample collection, hematological and biochemical assay
Blood samples were collected from all the anesthetized fish included
in six experimental groups and one control group (without EUPE and EU
administration). The samples were drawn from vena caudalis using a
syringe with heparin as anticoagulant (Heparin inj., Leciva, Czech Re­
public) at a concentration of 5000 IU heparin sodium salt in 1 ml.
Plasma samples obtained by centrifugation of blood at 3000g for 15 min
were stored at − 80 ◦ C until analysis. The hematological parameters
were measured that included red blood cell count (RBC), haemoglobin
concentration (Hb), hematocrit (PCV), white blood cell count (WBC)
mean erythrocyte volume (MCV), mean corpuscular haemoglobin con­
centration (MCHC), mean corpuscular haemoglobin (MCH), differential
% WBCs (lymphocyte, monocyte, neutrophile granulocytes segment,
neutrophile granulocytes bands, developmental phases – myeloid

sequence). The indices used to evaluate hematological profile were
based on unified methods for hematological examination of fish (Svo­
bodova et al., 1991). The bichemical parameters were measured that
included glucose (GLU), total protein (TP), albumin (ALB), globulin
(GLOB), triacylglycerols (TAG), aspartate aminotransferase (AST),
lactate (LACT), inorganic phosphate (PHOS), cholesterol (CHOL).
Globulin content was calculated by subtracting albumin values from
serum total protein. Measurement of biochemical plasma parameters
was conducted using the Catalys One analyser (IDEXX Laboratories Inc.,
Maine, USA).

Table 1
Detailed description of different stages of anesthesia induction and recovery in
EU based tests performed in common carp (Park et al., 2009; Yousefi et al.,
2018).
Stage

Characteristic behavior

Anesthesia
A1
Normal swimming; opercular movement and normal general movement
A2
Swimming speed slowed; rolling from side to side
A3
Partial loss of equilibrium; swimming erratic
A4
Complete loss of equilibrium; swimming perfectly inside out; pectoral fin,
pelvic fin and dorsal fin movement stop
A5

Little sedation; anal fin and tail fin movement stop
A6
Perfect sedation; only opercular movement
Recovery
R1
R2
R3
R4
R5
R6

4.1.2. Effects of temperature and pH on the behavior of the EUPEs
Droplet size of an emulsion has a crucial role in its stability. The
smaller the emulsion droplets, the more stable the emulsions are and
vice versa. This higher stability of an emulsion with smaller droplets can
be due to the strong resistance of smaller droplets against different destabilization phenomena like aggregation, coalescence, and floccula­
tion because of the Brownian motion that overcomes gravitational forces
(Hidajat et al., 2020). Thus a stable emulsion is the one capable of
resistance to the instability mechanisms such as Ostwald ripening
(development of the larger droplets by the expense of the smaller ones)
or very much similar to that coalescence as well as the flocculation,
where the droplets collide and bunch together to make flocs (larger

Resume opercular movement
Preferential movement of pectoral fin and tail fin
Dorsal fin, pelvic fin and anal fin movement
Swimming perfectly inside out
Swimming erratic; redress the balance
Normal swimming; responsiveness to visual stimuli

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Carbohydrate Polymers 265 (2021) 118096

Fig. 1. Digital photographs of the PEs prepared with different EU contents (wt%) a) and optical micrographs of the ones prepared with 5 b) 10 c) and 20 d) wt%
of EU.

Fig. 2. Mean droplet sizes and size distributions of the EUPEs prepared with 5, 10 and 20 wt% EU.

droplets) which then finally lead to sedimentation or creaming of the
emulsion due to gravitational separation (Low et al., 2020).
Based on these facts, the emulsion prepared with 5 wt% EU was
selected to evaluate the effects of temperature and pH on its stability in
terms of visual observations and droplet sizes, as it had smaller sizes
compared to the ones prepared with 10 and 20 wt% (Fig. 2).
In the first instance, the effect of temperature was evaluated by
incubating emulsion samples (original temperature 20 ◦ C) in a water
bath at 30, 50, 60, 80 and 90 ◦ C for 30 min. Visual observations of the
samples showed that there was no cream formation or even sedimen­
tation of the particles on the bottom of the sample tubes till 80 ◦ C. And
hence, although the mean droplet diameter slightly increased at tem­
perature 30 ◦ C, no significant changes were observed in the sizes
thereafter till 80 ◦ C (Fig. 3a). Also, all the samples showed uniform size

distributions (Fig. 3b) which further confirmed the capability of the PE
to withstand the influence of temperature. However, when the tem­
perature was raised to 90 ◦ C, the emulsion apparently turned into cream

and therefore at this temperature no mean droplet size of the emulsion
was measured. This observation can be attributed to the enhanced en­
counters among the particles due to the rise in temperature and ulti­
mately their average kinetic energy, thereby leading to enhancing their
motion and rate of collision finally making them to agglomerate
(Mwangi, Ho, Tey, & Chan, 2016). Similarly, the effect of pH on the
emulsion stability was monitored by adjusting pH of the samples
(original pH 4.3) to 5, 6, 7, 7.5 and 8. As no destabilization occurred
during changing pH from 5 to 7.5, it can be stated that emulsion was
highly stable against droplet coalescence and creaming till pH 7.5. The
droplet sized increased linearly with increasing pH values (Fig. 3c) but
4


B.R. Shah et al.

Carbohydrate Polymers 265 (2021) 118096

Fig. 3. Influence of temperature on the droplet size a) size distribution b) and influence of pH on the droplet size c) and size distribution d) of the EUPE prepared
with EU contents of 5 wt%.

exhibited uniform distribution (Fig. 3d).
This finding can be well explained by the fact of relatively low net
droplet charge at high pH with lowered electrostatic repulsion between
the droplets (Shah, Li, et al., 2016) which could even be reason for the
complete destabilization of the emulsion at pH 8 (shown in the inset of
Fig. 3c). These results can be regarded to present an ideal scenario of
releasing the encapsulated EU as main anesthetizing agent into the
water (with pH ≈ 7.3) in which the experimental fish are stocked.
Furthermore, to assess the influence of storage time on its stability,

the emulsion was stored at room temperature for three months and was
observed visually with time. It was found that no visual change appeared
in the behavior of the emulsion during this time which clearly indicated
its long-term stable nature.

PE groups, whereas no change in the behavior of fish was observed even
after 30 min of applying EU in their respective water tanks. This dif­
ference was also reflected in the analysis of different blood parameters of
both the groups. For example, it can be seen in Table 2, that hematocrit
(PCV) of 0.3925 in the PE group with 25 ppm of EU was significantly
higher (P < 0.05) than EU group (0.3425). These results comply with the
previous studies where significant increase in PCV levels were observed
after administrating EU as an anesthetic in O. tshawytscha (Cho & Heath,
2000) and olive flounder (Paralichthys olivaceus) (Hur et al., 2019).
Interestingly, when the EU at a concentration of 50 ppm in the form
of EUPE or EU was administrated, the induction and recovery times from
anesthesia were significantly lower (P < 0.05) for the EUPE group than
EU. As shown in Fig. 4a, the induction time of stage 5 and 6 anesthesia
(A5 and A6) with ≈3.3 min, was significantly lower (P < 0.05) than the
EU group with A5 and A6 of 12.4 and 14.2 min respectively. Similarly,
the recovery time for stage 5 and 6 recovery (R5 and R6) of 11.9 and 15
min respectively for EUPE group was significantly lower (P < 0.05) than
R5 and R6 of 15 and 19.9 min respectively for the EU group (Fig. 4b).
These findings of EUPE are closely coincided with the claims of previous
studies mentioning optimum anesthetic concentrations to induce anes­
thesia around 3 min and recovery around 10 min (Park et al., 2009).
This promising capability of EUPE was further hallmarked by
different hematological and biochemical plasma parameters as given in
Tables 2 and 3 respectively. It is evident from Table 2 that neutrophil
granulocytes segment (%) of 1.8000 ± 1.1106 for EUPE group was

significantly higher (P < 0.05) than EU group with 0.5000 ± 0.7572, but
was non-significant with the control group (1.4750 ± 0.4717) where no
EU (neither EUPE nor EU) was administered. Similarly, another
important indicator developmental phases – myeloid sequence (%) with
1.8000 ± 1.2410 for EUPE group was significantly higher (P < 0.05)

4.2. Evaluating anesthetizing efficacy of the synthesized EUPE in fish
model (common carp)
With the aim to introduce EU based PE an unprecedented non-toxic
formulation composed of biocompatible and biodegradable materials as
anesthetizing agent in fish, we evaluated the anesthetizing role of the
prepared EUPE by choosing common carp as a sample species. The
selected fish were divided into different groups, and three different
concentrations of EU i.e. 12.5, 25 and 50 ppm in the form of EUPE or free
EU were tested to evaluate their anesthetizing efficacy. It was found that
the EUPE ensured complete dissolution of the loaded EU as no oil
droplets were visualized on the water surface after the administration.
Conversely, the oil droplets were seen on the water surface even after
powerful agitation when free EU was applied. And therefore, it could be
the reason that even at lower concentration of EU i.e. 12.5 and 25 ppm,
anesthesia up-to stage 4 (A3 & A4 shown in Table 1) was achieved in the
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Carbohydrate Polymers 265 (2021) 118096

Table 2
Hematological parameters of common carp following treatment with different concentrations of EU in the form of EUPE or free EU.

Hematological parameters

Control

PCV

0.3525
0.0150
79.048
2.3276
1.9875
0.0981
10.525
1.3672
39.859
2.5597
177.80
13.556
224.37
4.6701

±
bc
±
a
±
a
±
a
±

a
±
a
±
a

96.000
1.2083
0.8250
0.3403
1.4750
0.4717
0.1000
0.2000
1.6000
0.8446

±
bc
±
ab
±
ab
±
a
±
ab

Hb (g/l)
RBC (T/l)

WBC (G/l)
MCH (pg)
MCV (fl)
MCHC (g/l)
Differential WBC count
Lymphocyte (%)
Monocytes (%)
Neutrophil granulocytes segment
(%)
Neutrophil granulocytes bands (%)
Developmental phases – myeloid
sequence (%)

Group I (12.5 ppm EU)

Group II (25 ppm EU)

Group III (50 ppm EU)

PE

EU

PE

EU

PE

EU

0.3238 ± 0.0075
c
80.481 ± 5.4021
a
1.7975 ± 0.3715
a
13.275 ± 4.9466
a
45.896 ± 7.8457
a
185.37 ± 33.917
a
248.68 ± 17.346
a

0.3250 ±
0.0220 c
78.153 ±
11.806 a
1.7525 ±
0.2323 a
7.8750 ±
2.4281 a
45.272 ±
10.132 a
187.05 ±
19.772 a
240.10 ±
28.368 a

0.3925 ± 0.0419
a
86.658 ± 15.477
a
1.8900 ± 0.3153
a
6.7500 ± 3.3789
a
47.721 ± 16.919
a
210.04 ± 22.324
a
223.40 ± 53.929
a

0.3425 ±
0.0328 bc
88.627 ±
6.5280a
1.9725 ±
0.2478 a
11.525 ±
4.5353 a
45.780 ±
9.1281 a
176.46 ±
32.849 a
260.24 ±
27.369 a

0.3725 ±
0.0260 ab
88.806 ±
6.7754 a
2.0050 ±
0.2478 a
9.7500 ±
2.5331 a
44.837 ±
5.7419 a
187.97 ±
23.121 a
239.60 ±
28.651 a

0.3650 ± 0.0297
ab
85.315 ± 4.6171
a
1.8325 ± 0.1047
a
9.5000 ± 3.9370
a
46.753 ± 4.9958
a
200.01 ± 23.743
a
235.19 ± 25.914
a

97.950 ± 0.8583
ab
0.6000 ± 0.6928
b
1.0250 ± 0.2754
abc
0.0000 ± 0.0000
a
0.4250 ± 0.6131
cd

98.975 ±
0.6076 a
0.3250 ±
0.3948 b
0.5750 ±
0.4272 c
0.1250 ±
0.2500 a
0.0000 ±
0.0000 d

95.350 ± 2.2368
c
2.4500 ± 1.7823
a
0.8250 ± 0.2217
bc
0.2375 ± 0.2750

a
1.1375 ± 0.5764
abc

98.100 ±
1.3928 ab
0.9500 ±
1.2557 ab
0.5000 ±
0.4082 c
0.0000 ±
0.0000 a
0.4500 ±
0.5196 cd

94.425 ±
2.2232 c
1.7250 ±
1.0468 ab
1.8000 ±
1.1106 a
0.2500 ±
0.5000 a
1.8000 ±
1.2410 a

96.625 ± 2.2750
abc
1.8500 ± 1.8448
ab

0.5000 ± 0.7572
c
0.3250 ± 0.2217
a
0.7000 ± 0.5944
bcd

Similarly, significant difference (P < 0.05) in lymphocyte (%) i.e. 95.350 ± 2.2368 and 98.100 ± 1.3928 for EUPE and EU group respectively was found between the
two groups. As lymphocytes have fundamental importance in body’s immune system, these findings highlight significant influence of anesthesia on the immunity of the
fish.

Fig. 4. Comparison between the times elapsed during the stages of anesthesia induction and recovery in the EUPE and EU groups of the experimental common carp.

than 0.7000 ± 0.5944 for the EU group.
Furthermore, as given in Table 3, we also analyzed different
biochemical plasma parameters. It is important to mention that being
one of the most common stress indicators in fish, glucose cannot be
ignored while speaking about stress. Because under stress conditions,
the release of stress hormones is altered which cause changes in the
blood as well as tissue chemistry including increasing blood glucose
levels, in an attempt to prepare the fish for emergency situations
(Makaras et al., 2020). Referring to the fascinating results of EUPE group
for the glucose levels in Table 3, it can be seen that the glucose values of
4.7950 ± 0.7735 mmol/l for EUPE was significantly lower (P < 0.05)
than for the EU with 7.1800 ± 0.7963 mmol/l. What’s more, the glucose
levels of the EUPE group (4.7950 ± 0.7735 mmol/l) was almost similar

with the control group (4.0775 ± 0.9859 mmol/l) having no significant
difference (P < 0.05). Looking at these findings it can postulated that the
EUPE reduced the stress level of fish by ≈50% than free EU. These re­

sults clearly favor the immuno-protective as well as stress reducing
potentials of the synthesized EUPE. The fast induction of anesthesia and
recovery of fish in the EUPE group due to short time of exposure would
favor less stress in these fish compare to the ones in the EU group
exposed for longer time. This can be the key immuno-protective factor as
there is a well-established link between stress and immune system
modulation of fish (Tort, 2011).

6


B.R. Shah et al.

Carbohydrate Polymers 265 (2021) 118096

Table 3
Biochemical plasma parameters of common carp following treatment with different concentrations of EU in the form of EUPE or free EU.
Biochemical plasma
parameters

Control

ALB (g/l)

11.750
a
24.500
a
176.00
a

1.3125
a
36.000
ab
4.0775
c
2.2850
ab
1.3725
c
3.0875
abc

GLOB (g/l)
AST (U/l)
PHOS (mmol/l)
TP (g/l)
GLU (mmol/l)
TAG (mmol/l)
LAC (mmol/l)
CHOL (mmol/l)

Group I (12.5 ppm EU)
PE
± 0.9574
± 2.3805
± 118.74
± 0.1204
± 2.7080
± 0.9859

± 0.2694
± 0.1335
± 0.2708

10.750
b
23.250
ab
141.25
a
1.3650
a
33.500
bc
5.4650
bc
2.2325
ab
4.5675
a
2.8025
c

Group II (25 ppm EU)

EU
± 0.5000
± 1.5000
± 23.796
± 0.0311

± 1.7321
± 1.3986
± 0.1056
± 2.8222
± 0.2702

11.750
ab
21.750
bc
176.25
a
1.2800
a
33.750
bc
6.9825
a
1.9500
cd
5.4675
a
2.8300
bc

PE
± 0.5000
± 1.5000
± 44.192
± 0.0455

± 0.9574
± 0.6547
± 0.1013
± 0.9652
± 0.0648

10.750
b
19.250
d
102.50
a
1.1400
b
30.000
d
6.1050
ab
2.0250
bcd
2.3625
bc
2.4700
d

Group III (50 ppm EU)
EU

± 0.9574
± 0.9574

± 16.862
± 0.0906
± 0.8165
± 0.9425
± 0.1318
± 0.8982
± 0.1564

10.750
b
20.000
cd
122.00
a
1.2950
a
30.750
d
4.6825
bc
1.9200
d
4.0575
ab
2.3050
d

PE
± 0.9574
± 1.4142

± 33.872
± 0.1480
± 0.9574
± 1.1598
± 0.1454
± 0.9251
± 0.1666

10.500
b
21.250
bcd
165.50
a
1.3250
a
31.750
cd
4.7950
bc
2.2900
a
5.4725
a
3.1250
ab

EU
± 1.2910
± 0.5000

± 42.587
± 0.1439
± 1.7078
± 0.7735
± 0.1525
± 0.5861
± 0.1028

12.250
b
25.000
a
149.00
a
1.4250
a
37.750
a
7.1800
a
2.1900
abc
4.7850
a
3.1550
a

± 0.5000
± 1.1547
± 49.214

± 0.0954
± 2.3629
± 0.7963
± 0.2652
± 1.4121
± 0.2908

Glucose (GLU), total protein (TP), albumin (ALB), globulin (GLOB), triacylglycerols (TAG), aspartate aminotransferase (AST), lactate (LACT), inorganic phosphate
(PHOS), cholesterol (CHOL). The values are expressed as mean ± SD. Measurements were performed in triplicate (n = 4/group). Means in the rows with different letter
are significantly different (LSD test, P < 0.05).

5. Conclusion

concentrations of EUPE based on EU in common carp. Although no full
sedation was achieved at lower concentrations (i.e. 12.5 and 25 ppm
EU), however with higher concentration (50 ppm), the times for anes­
thesia induction and recovery was significantly lower for EUPE group
than EU groups. We also analyzed different hematological and
biochemical plasma parameters which showed that compared to EU,
EUPE significantly control the stress with no attenuating effects on the
immunity of the fish. Overall outcomes of the current study categorically
demonstrated EUPE to be a potential anesthetizing formulation in fish­
ery with immuno-protective and stress control capabilities.

In the current study we for the first time introduced EU based PE as
an anesthetizing formulation in fish. As drawn schematically in Fig. 5,
the EUPEs were successfully fabricated using CS-TPP NPs for stabilizing
the oil water interfaces. The preparation of the required EUPE was
initially optimized by its preparation with different oil contents. Droplet
size and size distribution of all the samples were measured and the one

with the smallest size was chosen for further analysis. Effects of influ­
encing environmental conditions such as temperature and pH on the
behavior of the EUPE were evaluated. The EUPE was found to be stable
against different tested temperature ranges up-to 80 ◦ C as no significant
change in droplet size was observed. On the other hand, the droplet size
was found to increase with increasing pH until pH 7.5 as at pH 8 the
emulsion was completely destabilized. Thereafter, the anesthetizing
efficacy of the formulated EUPE was evaluated by testing three different

CRediT authorship contribution statement
Dr. Bakht Ramin Shah: Conceptualization, Methodology, Formal
analysis, Data curation, Writing - original draft, Visualization, Ac­
tivities administration.

Fig. 5. Schematic representation of the NPs and EUPEs synthesis and the potential application of the EUPE as an anesthetizing formulation in comparison with free
EU in common carp.
7


B.R. Shah et al.

Carbohydrate Polymers 265 (2021) 118096

´k: Methodology, Investigation,
Dr. Peter Dvoˇra
Professor Josef Velíˇsek: Visualization, Investigation.
Associate Professor Jan Mraz: Funding acquisition, Writing - re­
view & editing.

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Declaration of competing interest
There is no conflict of interest in this manuscript.
Acknowledgement
The authors are grateful to the Ministry of Education, Youth and
Sports of the Czech Republic the CENAKVA project (LM2018099),
CENAKVA Center Development (CZ.1.05/2.1.00/19.0380) and Biodi­
versity (CZ.02.1.01/0.0/0.0/16_025/0007370) and National Agency for
Agricultural Research (QK1810296), for financial support. The authors
would like to express their sincere gratitude to the colleagues of labo­
ratory of Nutrition, Institute of Aquaculture and Protection of Waters
(IAPW), Faculty of Fisheries and Protection of Waters (FFPW), Univer­
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huge support in conducting fish trials.
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