BioRisk 172 117-126 (2022) Rg ak) Bre
doi: 10.3897/biorisk.17.77300 RESEARCH ARTICLE & B lO R IS k

https://biorisk. pensoft.net

Comparative study on the oxidative stress of
commercially important fish species from localities
with different ecological conditions along
the Bulgarian Black Sea coast

Albena Alexandrova', Jordan Raev’, Dimitar Dimitrov’, Nesho Chipev',
Elina Tsvetanova', Almira Georgieva', Violin Raykov?

1 Laboratory of Free Radical Processes, Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia, Bul-
garia 2 Marine Biology and Ecology Department, Institute of Oceanology, Bulgarian Academy of Sciences,
Varna, Bulgaria 3 Marine Geology and Archaeology Department, Institute of Oceanology, Bulgarian Academy
of Sciences, Varna, Bulgaria

Corresponding author: Albena Alexandrova (a_alexandrova_bas@yahoo.com)

Academic editor: Michaela Beltcheva | Received 29 October 2021 | Accepted 13 December 2021 | Published 21 April 2022

Citation: Alexandrova A, Raev Y, Dimitrov D, Chipev N, Tsvetanova E, Georgieva A, Raykov V (2022) Comparative
study on the oxidative stress of commercially important fish species from localities with different ecological conditions
along the Bulgarian Black Sea coast. In: Chankova S, Peneva V, Metcheva R, Beltcheva M, Vassilev K, Radeva G,
Danova K (Eds) Current trends of ecology. BioRisk 17: 117-126. https://doi-org/10.3897/biorisk.17.77300

Abstract

The aim of the present study was to perform a pilot assessment and analysis of the oxidative stress (OS) level
in four commercially important fish species (round goby, red mullet, sprat and horse mackerel) from different
localities of the Bulgarian Black Sea coast. The fish were sampled during trawl selectivity experiments. The OS
level in the fish was assessed by measuring lipid peroxidation (LPO), glutathione concentration (GSH), activi-
ties of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-trans-
ferase (GST), as well as acetylcholine esterase (AChE) in gills and liver. Round goby and red mullet caught in
the Nessebar Bay showed clear signs of OS with the highest levels of LPO and GST activities, accompanied
by the lowest AChE activities in both liver and gills. On the contrary, round goby caught near Maslen Nos (a
region with good ecological conditions) were least affected by OS with low LPO and high GSH concentra-
tions and SOD activity. There were no significant differences in the OS bioindicators of horse mackerel from
the different localities. Sprat caught in Nessebar Bay, compared to those caught from the other localities,
showed presence of OS indicated by lower GSH levels and relatively higher CAT, GPx and GST activities,
accompanied by low AChE activity in gills. It can be concluded that round goby and red mullet were more
vulnerable to OS induced by marine environmental factors than the horse mackerel and sprat. However, their
antioxidant defense system allows them to tolerate and adapt to the environment of their habitats. Further

studies are needed for the assessment of OS in important fish species in the Bulgarian part of the Black Sea.

Copyright Albena Alexandrova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY
4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

118 Albena Alexandrova et al. / BioRisk 17: 117—126 (2022)

Keywords

Bulgarian Black Sea, horse mackerel, oxidative stress, red mullet, round goby, sprat

Introduction

Fish are a major component of aquatic ecosystems and the good state and health of their
populations is crucial for ecosystem stability. Marine fish populations are subjected to in-
creasing environmental pollution and also to overfishing pressure (Daskalov 2003; Radu
et al. 2011; Totoiu et al. 2018). At present, Black Sea fish species with significant econom-
ic and ecological importance, also in the Bulgarian part, are sprat, turbot, red mullet, blue-
fish, horse mackerel and round goby (Raykov et al. 2019). ‘The assessment of the condi-
tion of the exploited fish species (Descriptor D3) in the Bulgarian Black Sea part showed
that there are no species in “Good condition’, and sprat, whiting, horse mackerel, mullet,
turbot and others are even classified in “Bad” condition (Panayotova and Todorova 2017).

The sustainable management of marine fish stocks requires not only regulation of
the fish populations, but also knowledge regarding the ability of the fish to respond
and adapt to multiple environmental stressors (Hagger et al. 2006, 2008). A general
reaction of marine organisms to environmental pressures (abiotic, biotic and anthro-
pogenic) involves the activation of oxidative processes in their cells where the gener-
ated reactive oxygen species (ROS) play the role of signaling molecules that trigger
the organisms’ adaptative responses to environmental changes (Birnie-Gauvin et al.
2017). However, under prolonged or acute exposure, excess ROS generation cannot
be compensated by the antioxidant defense system and provokes oxidative stress (OS)
(Villarreal et al. 2014; Chowdhury and Saikia 2020). The OS in marine fish can be
induced by multiple stress factors which were proved to adversely affect the growth, de-
velopment and reproduction of fish species (Stoliar and Lushchak 2012; Vinagre et al.
2012; Dolci et al. 2013; Mozhdeganloo and Heidarpour 2014; Florescu et al. 2021).
All these stressors (and related stresses) can result in community-scale effects (commu-
nity structures) and may ultimately drive species coexistence (Steinberg 2012).

The aim of the present study was to provide a pilot assessment and analysis of the
oxidative stress level of four commercially important fish species from representative
localities of the Bulgarian Black Sea coastal area.

Materials and methods
Sampling
The fish species selected for this study were: the demersal round goby (eogobius mela-

nostomus Pallas, 1814), two benthopelagic species — red mullet (Mullus barbatus Lin-
naeus, 1758) and horse mackerel (7rachurus mediterraneus Steindachner, 1868), and

Oxidative stress of fish from Bulgarian Black Sea 119

the pelagic species — sprat (Sprattus sprattus Linnaeus, 1758). The fish species were
identified in accordance with BSFishList (2020). The fish individuals were randomly
picked from several trawl selectivity experimental catches by pelagic Midwater otter
trawl (7 x 7 mm mesh size of the codend) from 3 localities of the Bulgarian Black Sea
aquatory (Table 1). On board, random samples of 9-12 fish individuals of every spe-
cies were shock frozen and transported to the laboratory.

In the laboratory, fish individuals were measured with a caliper and weighed on
scales with an accuracy of 0.01 g. The Fulton’s condition factor (K) of the fish individu-
als was computed according to Nash et al. (2006): K = 100*7W/TL?, where TW is the
total body wet weight in grams and TL is the total length in cm; the factor 100 is used
to bring K close to a value of one.

Tissue preparation

The fish were dissected and their liver and gills were extracted. The organs were ho-
mogenized in 0.1 M potassium phosphate buffer (pH 7.4) and centrifuged at 3000 g
for 10 min to obtain a post-nuclear fraction, used for determination of lipid peroxida-
tion and glutathione levels. A portion of post-nuclear fraction was re-centrifuged at
12000 g for 20 min at 4 °C for obtaining a post-mitochondrial supernatant, used for
measurement of the antioxidant enzymes activities.

Measurement of oxidative stress biomarkers

The OS biomarkers were measured spectrophotometrically using commercially avail-
able kits: Lipid Peroxidation (MDA) Assay Kit MAKO085, Glutathione Assay Kit
CS0260, SOD Assay Kit-WST 19160, Catalase Assay Kit CAT100, Glutathione
Peroxidase Cellular Activity Assay CGP1, and Glutathione-S-Transferase Assay Kit
CS0410 (Sigma-Aldrich Co. LLC, USA). The manufacturer’s working instructions
were strictly followed.

Acetylcholinesterase (AChE) activity was assayed by the method of Ellman (EIl-
man et al. 1961). The reaction mixture contained 0.1 M K-PO4 buffer pH 8.0, 0.045
M acetylthiocholine iodide, 0.008 M 5,5’-Dithiobis(2-nitrobenzoic acid) (DTNB)
and the appropriate amount of fish’s tissue homogenate. The enzymatic hydroliza-
tion of acetylthiocholine produced thiocholine. The latter reacted with DTNB and
yield a yellow colored product, 5-thio-2-nitrobenzoic acid with absorbance pick at
412 nm. The enzyme activity was calculated as U/mg protein. Protein concentration

Table |. Trawling localities along the Bulgarian Black Sea coast with depth and bottom temperature.

Code —_ Trawling locality Trawling start point Trawling end point Depth Te §
N,E N, E [m] [°C]

S1 Maslen Nos 42.312452°N, 28.054628°E 42.296292°N, 28.089651°E 37 7.7

$2 St. Oryahovo 42.993074°N, 27.951590°E 42.960442°N, 27.948072°E 23 7.9

S3 Nessebar 42.599613°N, 27.791345°E 42.619495°N, 27.8267 10°E 20 7.4

120 Albena Alexandrova et al. / BioRisk 17: 117—126 (2022)

was measured according to Lowry et al. (1951) and was computed from a stand-
ard curve, obtained using bovine serum albumin (CAS Number: 9048-46-8, Merck
KGaA, Darmstadt, Germany).

Statistical analyses

For statistical analyses the software package STATISTICA 10 (StatSoft Inc.) was used.
The Kruskal—Wallis test was used to assess the significance of differences among the
values of the OS biomarkers. Post hock comparisons between variables were made us-
ing Mann-Whitney test. Multidimensional Scaling (MDS) was carried out to detect
patterns of similarities among the studied objects.

Results

Data from the measurements of the OS biomarkers in the liver and gills of the studied
fish species from the selected localities of the Bulgarian Black sea coast are presented
in Table 2. Our data showed that the measured OS bioindicators significantly varied
among the fish species, the studied organs and the localities. In general, the two fish
organs studied showed different susceptibility to oxidative changes, due to its unique
composition and metabolic profile. Significantly higher lipid peroxidation (LPO) was
found in the gills compared to the liver of the fish species, with the exception of the
round goby, where no differences were observed (Table 2). The gills of all studied fish
species had lower glutathione-S-transferase (GST) and higher acetylcholine esterase
(AChE) activities than the liver. The activity of superoxide dismutase (SOD) in the
liver was approximately twice as high as in the gills for all fish species studied, and the
activity of catalase (CAT) was two to five times higher in the liver of round goby, red
mullet and sprat, compared to gills. Glutathione peroxidase (GPx) activity was signif-
cantly higher in the gills than in the liver only in sprat. The glutathione (GSH) level in
the gills of red mullet was significantly lower than in the liver, while the opposite was
observed in sprat. In the round goby and horse mackerel no difference in GSH between
the two organs was present (Table 2). The gills of the studied fish species were more
affected by OS as a whole, showing high levels of LPO, with the exception of round
goby. In the liver of the studied fish species lower activity of CAT, SOD (except for the
fish species from Maslen Nos) and GST were present, compared to the gills (Table 2).

Significantly higher LPO and GST activities were established, together with the
lowest AChE activity in both liver and gills of round gobies and mullets from Nessebar
Bay, compared to those from Maslen Nos and Staro Oryahovo (Table 2), thus indi-
cating clear signs of OS. Round gobies from Maslen Nos had the lowest level of OS
as indicated by the lowest LPO levels and GST activities, and the highest concentra-
tions of GSH and SOD activity, together with highest activity of AChE (Table 2). No
significant differences were present in the measured OS biomarkers between mullets
caught from Maslen Nos and from Staro Oryahovo. The OS markers in sprat from

Oxidative stress of fish from Bulgarian Black Sea a

Table 2. Values (ME+SD) of the measured OS biomarkers in gills and liver of the studied fish species
from the different localities of the Black Sea coast (*statistical significance of differences at p<0.05: S"
indicate significant differences of the OS indicator in fish species between sites; letters (G, L) indicate

significance of differences of the OS indicator between organs — G = gills and L = liver).

Site Liver Gills
LPO GSH SOD CAT’ GPx GST AChE LPO GSH SOD CAT GPx GST AChE
Neogobius melanostomus
4.83 701 16.29 1.54 19.72 37.1 31.27 2.90 457 16.00 1.05 20.20 38.99 42.7

S1 +1.42 +134 +4485 +002 +£3.22 +121 +064 +018 +99 +2.8 +0.02 +143 +£0.87 +1.43
#623 #623 62,3 #5 #237 623 62,3 #5 #5 623 *23CG
13.81 295 3.60 1.35 19.73 40.3 17.94 15.49 287 1.15 1.09 1644 49.09 28.4
S2 +2.01 +7%*s' +0.26 +0.08 +286 41.8% +059 +4168 +39 +016 +015 41.21 +£1.22 +0.33
*gl *gl IT *ol *gl *S *g 13 *IG
19.26 304 3.50 184 23.76 2643 13.86 20.92 276 1.87 0.54 14.23 68.10 248
S3 +0.65 +£23%*s' 41.05 +0.03 +10.7 +406 +1.06 +004 +33 +038 +004 +184 +414 +0.85
*gl *ol ¥ol27 «IT *ol *gl *o1.2 *ol *oL2 *IG

SiteMean 12.63 433 7.80 1.58 21.07 113.9 21.02 13.10 340 6.34 0.89 16.96 52.1 31.9
+1.36 +54 +2.05 +0.04 +559 +182 +0.76 +063 +57 +113 +007 +149 +2.08 +0.87

Mullus barbatus

0.64 692 58.13 15.08 10.94 55.45 54.64 2.77 294 20.88 1.77 19.46 9.77 292.4
SI +£0.12 +217 +3.75 +42.112 +3.24 73 +120 +086 +51*L +518 +033 +5.07 +1.08 +7.60

*SBG * * 3G *SG *63 *SG *SG *SL al B *L, * ST *.,

0.53 459 45.89 17.86 4.19 76.69 73.11 2.64 350 23.08 1.88 16.32 14.97 355.8
S2 +0.05 +118 +827 +£3.65 +1.51 +104 +109 +019 +27%*8 +359 +011 +473 +262 +75.3

*9G *G *9SG *9G *9SG *9SG *SL i AL, oe *3L *SL,

2.09 383 23.71 4.07 24.57 102.33 30.24 10.26 218 12.17 2.55 17.74 68.00 168.8
S3 +060 +75G +124 +£1.64 +308 +472 +340 +1.25 +12*s +£7.16 +031 +007 +1.91 +19

*23C *ol *oL2 *12G *oh2C *oL2C *oh27 *[e ¥ol27 «eT

SiteMean 1.09 511 42.58 12.34 13.23 78.15 52.66 5.22 287 18.71 2.07 17.84 30.91 272.4
+0.26 +136 £8.14 £2.47 12.61 +749 +8.79 +0.77 +30*L +£5.31 +0.25 +3.29 +1.87 +28.3
*G *G *G *G *G *G *L Ts ee *L cau,
Trachurus mediterraneus
2.93 335 14.80 1.30 46.51 79.55 51.92 21.90 393 7.64 1.04 3149 3484 214.4

S1 +148 +68 +3.08 +0.72 +163 +182 +203 +039 +£21%*s) +£5.16 +0.27 +9.08 +105 +60.4
AL *9SG *G ‘LT *L cal
2.09 249 14.37 0.60 36.09 59.07 4405 22.32 368 7.57 116 27.36 35.68 199.6
S2 +0.58 +8 +7.73 +017 +146 +110 +135 +043 +49 +638 +033 +880 +1.55 +287
th, *9 *G *SG cle gL iB
3.57 227 9.64 1.73 3437 48.43 66.00 24.41 246 4.79 131 2848 39.17 203.3
S3 +1.25 +18 +086 +0.20 +542 +3.49 +3.68 +0.96 +19%*s' +0.95 +0.24 +3.12 +0.89 +7.81L
nal 4 *o2 *IG *2G *L El i al

SiteMean 2.86 270 12.94 1.21 38.99 62.35 53.99 22.8% 336 6.67 117 29.11 36.6 205
+110 +31 +3.89 +036 412.1 +109 +125 +059 +30 +416 +028 +7.00 +431 +32.3

wie *G *“T *L oh,
Sprattus sprattus

16.27, 376 =12.57. 12.25 26.66 899.89 112.6 22.56 896 4.28 116 39.41 20.56 218.0

SI +8.22 +863 43.28 41.97 +£11.2 £11.55 +591 +057 +343 +118 +044 +17.8 +5.16 +269
4G +i 4G #63 *3G 4G (9 Te i UF #5 *SL iy

19.15 413 13.27 13.27 31.90 72.44 35.91 22.05 1044 6.73 2.01 49.49 24.87 228.9

S2 +415 +63*G +£0.72 +0.72+= +116 +673 +603 +0.80 +t41*L +10 +058 +4236 142.45 +28.0
*G *G *s3 *SG *s'G *L8 ele; *L ah,

14.11 312 8.67 15.30 12.71 40.66 52.78 22.00 341 5.17 2.83 7448 47.72 142.9

S3 +2.78 +100 +3.61 +£1.23 +£0.26 +3.02 146.20 +0.8 +49 +£0.90 +0.06 +674 +1.80 141.9
“i 4G ¥gl2G #12 a = BA 4, IL ¥g1,2 i

SiteMean 16.51 367 11.50 13.61 23.76 71.00 67.12 22.2 760 5.39 2.00 54.4 31.0 196

+5.05 +57*G 42.54 +1.3 +£7.73 +£7.09 +238 +073 +145 +105 +036 +161 +314 +323
*G *Gl *G *G lB *L oa le re

*LPO was expressed as nmoles MDA/mg protein; GSH was expressed as ng/mg protein, all enzymes were expressed as U/mg protei

122 Albena Alexandrova et al. / BioRisk 17: 117—126 (2022)

Nessebar Bay demonstrated significantly lower levels of GSH, higher activities of CAT,
GPx and GST, along with significantly lower AChE activities in the gills, compared to
those from Maslen Nos and Staro Oryahovo, thus indicating clear OS signs. No dif-
ferences in the values of the studied OS biomarkers between sprat from Maslen Nos
and Staro Oryahovo were found, indicating a relatively similar level of OS in the fish
from these localities. There were no significant differences in the levels of the studied
OS indicators in horse mackerel from the three studied localities.

In order to identify significant similarities (dissimilarities) between the values of
the measured OS biomarkers MDS was applied (Fig. 1). The MDS clearly demon-
strated the presence of differences in the OS level and the enzyme defense activation
between the fish species studied (Fig. 1A). The red mullet differed significantly from
the other studied species (separated at the right side of the two dimensional plane). The
round goby was the other species with specific and different OS reaction (clearly sepa-
rated from the other species in the two dimensional plane). Both the red mullet and
the round goby have similar benthic live style and hence should have been subjected
to similar environmental pressures resulting in OS. The level of OS in horse mackerel
and sprat appeared more similar to each other than to the other two species (Fig. 1A).

The activity of the pro/antioxidative processes in the studied fish species showed
some indicative similarities (and dissimilarities) (Fig. 1B). The different degree of acti-
vation of the fish antioxidant defense system, as a response to multiple environmental
stressors, showed two lines of defense (including ancillary factors). The first one reflected
the activation of the first line of defense by antioxidant enzymes, indicated by the activ-
ity of CAT and SOD (grouped together in the left part of the two dimensional plane).
The second one was reflected by the grouping (although loose) of GSH, GPx and AChE
at the right side of the factor plane, thus indicating the activation also of the detoxifica-
tion defense system of the fish species as additional response to toxic pollutants.

As a general measure of fish condition and health, we used Fulton’s condition
factor (K) (Fig. 2). The results strongly indicated the presence of differences of the K

A. 0 ; Bui
S. sprattus GST
06 = os es
a4 M. barbatus a6
a CAT
o4 o
o2
o2
oo} I mediterraneus SOD LPO
oo ° GPx
0.2 GSH f
= 9-02 o
Z Z
aot @ oa
os nN ne
08 N. melanostomus 08 AChE
-1.0 -1.0

10 08 06 04 092 00 O02 O04 O8 #08 10 12 “0.8 08 14 02 00 oz a4 08 os

Dimers ion 1 Dimension 1

Figure |. Multidimentional scaling of similarity (dissimilarity) between the studied fish species in their
level of OS (A) and the induced activity of the pro/antioxidant processes (B).

Oxidative stress of fish from Bulgarian Black Sea 123

25
mM Maslennos & Staro Oryahovo Nessebar
2.0
~
9
£ 1.5
OO
O
£1.0 I
ox
s |
; i i
0.0
N. melanostomus M. barbatus T. mediterraneus S. sprattus

Figure 2. Fulton’s condition factor (K) of studied fish species from the sampled localities of the Black
Sea coastal zone (Mean+SD).

factor among fish species and also among the trawling localities. As a whole, the values
of K for round goby and red mullet were significantly higher (also higher than 1) that
those of the other species for all localities. The highest K factor values were present in
round goby and red mullet from Maslen Nos. ‘The K factor of sprat and horse mackerel
was significantly lower (also less than 1) for all trawling localities studied.

Discussion

Changes in the balance of cell oxidative processes are a basic reaction of marine organ-
isms to the impact of environmental pressure. The increasing contamination of the sea
with metals, polycyclic aromatic carbohydrates and eutrophication leads to increase of
LPO and alters the activity of antioxidant protection in the tissues of marine fish and
other organisms (Regoli and Giuliani 2014).

In this study, we reported preliminary data on the OS level in the liver and gills of
four commercially important fish species (sprat, horse mackerel, round goby and red
mullet) from the Bulgarian Black Sea region. ‘The first overall finding of our study was
that in the gills and liver of fish different oxidative changes were present, most prob-
ably due to their unique composition and metabolic profile. Secondly, the studied fish
species differed in the level of their OS and antioxidant systems activation, which de-
pended on their life style and habitat (food preferences and habits, mobility and hence
metabolism levels). he round goby and red mullet are classified as slow-swimming
and carnivorous, while horse mackerel and sprat are fast-swimming and plankton feed-
ers (Rudneva et al. 2010). The more active species compared to low-mobile forms

124 Albena Alexandrova et al. / BioRisk 17: 117-126 (2022)

require higher oxygen consumption and hence, have a higher metabolic rate which
determines higher ROS production and OS, and induction of different levels of anti-
oxidant protection (Martinez-Alvarez et al. 2005; Filho 2007; Rudneva et al. 2010).
In line with these findings were our results indicating significantly higher levels of
LPO and GST in the gills of sprat and horse mackerel, i.e. the fish species with higher
mobility. On the other hand, the benthic and suprabenthic, more sluggish, fish spe-
cies (as round goby and red mullet), are assumed to live in more contaminated marine
environments, because many pollutants accumulate in low water layers and bottom
sediments. In support of this assumption, our data showed significantly higher OS
levels in round goby and red mullet individuals from Nessebar bay (a bustling tourist
center), compared to the individuals from Maslen Nos (protected area under the Eu-
ropean ecological network NATURA). Furthermore, the extremely high GST values
established, especially in the liver of the studied fish species from Nessebar Bay, indicat-
ed the presence of activated detoxification metabolism of xenobiotics, most probably
entering the fish body from the sediment. ‘This was confirmed by the performed MDS
which showed activation of detoxification processes and decreased neurotransmission
capacity (indicated by AChE activity). Our results corresponded with the observations
of Rudneva et al. (2010), who also demonstrated higher activity of GST in the liver
of slower swimming and sluggish fish species. It can be concluded that the studied
by us round goby and red mullet appeared to be more vulnerable to OS induced by
multiple marine environmental stressors than the horse mackerel and sprat. However,
their antioxidant defense systems allowed them to tolerate and adapt to the changing
environmental conditions of their habitats.

Our findings on the specific pro/antioxidative processes in the studied fish species
were confirmed by values of the Fulton’s condition factor (K). This biometric tool is
used to indicate the general health and wellbeing of the fishes (Datta et al. 2013), as
well as the quality of the marine environment. The value of K reflects the interactions
between feeding conditions, parasitic infections and physiological nutritional/energy/
reserves and hence, OS levels cannot alone determine the K factor variations. In our
study we found that the round goby and red mullet from all three localities were in
good condition according to K (K>1), whereas the condition of the sprat and horse
mackerel from all studied localities was not so good (K<1). These findings correspond-
ed, as a whole, to the estimated OS levels in these fish species.

Conclusion

It can be concluded that round goby and red mullet are more vulnerable to OS induced
by marine environmental factors than the horse mackerel and sprat. However, their an-
tioxidant defense system allows them to tolerate and adapt to the environmental condi-
tions of their habitats. Therefore, it could be assumed that demersal fish species are more
convenient for monitoring the state of the marine environment and the risk for impair-
ing the fitness of fish. Obviously, further studies are needed for more comprehensive
assessment of OS in fish of economic importance in the Bulgarian part of the Black Sea.

Oxidative stress of fish from Bulgarian Black Sea 125

Acknowledgements

This work was supported by grant KT1-06-H41/7/2020 of National Science Fund, Bulgaria.

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