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828
ISSN 0001-4370, Oceanology, 2017, Vol. 57, No. 6, pp. 828–840. © Pleiades Publishing, Inc., 2017.
Original Russian Text © G.A. Kolyuchkina, D.F. Budko, V.K. Chasovnikov, V.P. Chzhu, 2017, published in Okeanologiya, 2017, Vol. 57, No. 6, pp. 919–933.
Influence of the Bottom Sediment Characteristics on the Bivalve
Mollusk Anadara kagoshimensis Histopathology’s Variability
in the Northeastern Coast of the Black Sea
G. A. Kolyuchkinaa, *, D. F. Budkoa, V. K. Chasovnikovb, and V. P. Chzhub
a Shirshov Institute of Oceanology, Russian Academy of Sciences, Moscow, Russia
b Southern Branch of Shirshov Institute of Oceanology, Russian Academy of Sciences, Gelendzhik, Russia
*e-mail: galka.sio@gmail.com
Received June 14, 2016
Abstract—With increasing anthropogenic impact on the environment, investigations of organism’s response
to the contamination of natural habitats, are especially relevant. In the present study, we sought to identify
the correlation between the bottom sediments and local variability in histopathology of Anadara kagoshimen-
sis (Bivalvia) in four sites of the north-eastern coast of the Black Sea. Bottom sediment grain size, redox
potential of pore water, heavy metals, benzo-α-pyrene and DDT concentrations have been used as charac-
teristics of bottom sediments. Analysis of the data revealed differences in the geochemical background of the
studied sites and the histopathological state of the molluscs from these areas. Among the 10 studied elements
as well as benzo-α-pyrene and DDT, only Ni has shown an exceedance of statutory limits of concentration
in bottom sediments. The study reveals a relationship between Ni concentration in the bottom sediments and
frequency of heavy histopathologies in the mollusсs. In addition to causes directly related to pollution, mild
pathology may be influenced by “natural” causes; in particular, the high content of brown cells in the con-
nective tissue of the digestive gland may be due to the age of molluscs.
DOI: 10.1134/S00014370170600 66
INTRODUCTION
Histopathological changes are an integrated result
of different biochemical and cytological processes.
Comprehensive characterization of the degree of
damage to the body and, in particular, the status of
gonads—important parameters for the prediction of
the reproductive potential of a population [42]—can
be obtained by histological examination [40]. Bivalves
are important aquaculture and environmental moni-
toring organisms, hence identification of their pathol-
ogies and dysfunctions is important for understanding
the causes of population changes [10] and the impact
of environmental contamination [39]. The histopa-
thologies of bivalves can be caused by the impact of
various factors: availability of food [32], infestation by
parasites [19], oxygen deficiency [30], the presence of
contaminants [38], or other negative factors [32].
Mollusks in areas of high anthropogenic impact are
characterized by pathological changes in tissues, in
particular, an increase in the content of lipofuscin,
vacuolization of the digestive cells of the digestive
gland (DG), atrophy of the epithelium of the DG,
hyperemia of gills, and vacuolization of oocytes [10,
18, 39, 43]. These disorders are indicators of environ-
mental contamination [40, 41, 43].
Bivalves are predominate group of macrozooben-
thos on soft bottom to a depth of 100–130 m in the
Black Sea [34]. Among them, the role of blood ark
Anadara kagoshimensis—an invader from the Western
Pacific—has increased in recent decades [7, 20]. This
relatively large (shell length up to 85 mm [35]) species
began to predominate [16] in some regions, and its
concentration became exploitable [7, 14]. blood ark is
a filter-feeder and feeds on seston [23]. Similar to
other filter-feeder bivalves, blood ark filters a large
amount of sea water and thus comes in contact with
various pollutants and plays a significant role in
biosedimentation [23]. Though the northeastern coast
of the Black Sea is a recreational area, there are
sources of contamination of the marine environment
with heavy metals and petroleum hydrocarbons: ports,
ship- and car-repair factories, oil refineries, and
municipal wastewater treatment facilities [11]. A study
of the variability of histopathologies of blood ark pop-
ulations on the northeastern coast of the Black Sea in
2003–2008 did not reveal any pathology of gonads,
but it proved the existence of a positive correlation
between the frequency of histopathologies and cad-
mium content in bottom sediments and soft tissues of
blood ark [10].
The present study is the follow-up to the previous
project of blood ark histopathologies studies and we
MARINE BIOLOGY
OCEANOLOGY Vol. 57 No. 6 2017
INFLUENCE OF THE BOTTOM SEDIMENT CHARACTERISTICS 829
tried to reveal correlations between local variations of
histopathologies and some parameters of bottom sed-
iments (grain-size composition; oxidation–reduction
potential of pore water; the content of heavy metals,
benzo-α-pyrene, and DDT) in four regions of the
northeastern coast of the Black Sea.
MATERIALS AND METHODS
The investigations were performed in the Russian
sector of the Black Sea in 2014. The object of study was
filter-feeding infaunal bivalves Anadara kagoshimensis
(Tokunaga, 1906) (Bivalvia: Arcidae). The sampling
sites were recreational areas and resorts in open parts
of the coast: the Anapa barrier beach near the Bugaz
Sandbar (Bugaz), the settlement of Divnomorskoe,
Inal Bay, and the settlement of Shepsi (Fig. 1). Besides
shellfish, we also sampled bottom sediments (BSs) to
determine the grain-size composition; oxidation–
reduction potential of pore water (ORP); and the con-
tent of heavy metals (HM), benzo-α-pyrene (BAP),
and DDT. Samples near the Bugaz Sandbar were man-
ually collected on June 16–18, 2014, using scuba diving
equipment. We took seven individuals of blood ark from
a uniform sandy bottom at a depth of 3 m and six indi-
viduals from the muddy bottom of a depression at a
depth of 10 m for histological analysis. Near Divnomor-
skoe and Shepsi and in Inal Bay, samples were taken on
July 2–10, 2014, on an expedition of the R/V Ashamba.
Bottom sediments were sampled by an Ocean bottom
grabber with the sampling area of 0.1 m2 from a depth
of 7.3–30 m. Single samples of BSs were taken for
analysis from five depth levels in each region. Shellfish
were sampled manually at a depth of 7.3–15 m using a
diving equipment. In each of the three regions, 27–
31 representatives of blood ark were sampled for histo-
logical analysis. In general, we investigated 101 indi-
viduals. The exact depths of BSs sampling are given in
Table 1.
Weather conditions in the investigated regions. Con-
stant sources of freshwater discharge into the sea are
absent in the vicinity of Bugaz. The weather prior to
the sampling period near the Bugaz Sandbar was calm,
and the temperature of bottom water at a depth of 10 m
was 23°C. The sampling points in other areas were
located near the mouths of the Mezyb’ (in Divnomor-
skoe), M. Bzhid (in Inal Bay), and Shepsi (in Shepsi)
rivers. The weather prior to sampling here was stormy
with heavy rain. The temperature of bottom water in
Divnomorskoe was 26.9°C at the depth of blood ark
habitat, 24.4°C in Inal Bay, and 26.5°C in the Shepsi
River; the thermocline was not detected. Heavy rains
caused the formation of a mudflow in the Shepsi
River, in the estuary of which the sampling points of
shellfish were located.
The organs of mollusks were fixed with formalde-
hyde and then processed by conventional methods for
histological examination [8]. Slices were stained with
Carazzi’s hematoxylin and eosin and studied under a
Mikromed light microscope at a magnification of
×400–900. We determined the sex and development
stage of gonads according to [36], revealed the pres-
ence or absence of pathologies of each shellfish, esti-
mated the rate of pathologies according to [26], and
calculated the number and percentage of mollusks in
the sampling with each pathology.
Heavy metals, benzo-α-pyrene, and DDT in bot-
tom sediments were determined according to the
manuals of Environmental Regulatory Document
F 16.1:2.2:2.3.36-2002. The top 5 cm layer of BSs was
sampled by clean plastic sampler, placed in plastic zip
bags, and stored at –18°C until the analysis. Each
whole sample (without separation into fractions) was
decomposed by a mixture of nitric and hydrochloric
Fig. 1. Map of sampling stations of mollusks and BSs in 2014.
45.5°
45.0°
44.5°
44.0°
43.5°
43.0°
36.0°36.5°37.0°37.5°38.0°38.5°39.0°39.5°40.0°40.5°
Bugaz Anapa
Gelendzhik
Divnomorskoe
Inal Tuap se
Shepsi
Adler
BLACK SEA
N
E
200 m
830
OCEANOLOGY Vol. 57 No. 6 2017
KOLYUCHKINA et al.
acids at a 1 : 3 ratio (aqua regia). The contents of ele-
ments (Cd, Cu, Zn, Cr, Pb, Mn, Fe, Hg, Ni, and Co)
were determined on a KVANT-2A atomic absorption
spectrometer (AAS) (KORTEK, Russia) in samples
from the Divnomorskoe, Shepsi and Inal Bay, and by
mass spectrometry with inductively coupled plasma
(ICP-MS) on an Agilent 7500a spectrometer (Inter-
lab, United States) in samples from the area of the
Bugaz Sandbar. We also measured the contents of
BAP and DDT. The data are given for dry mass.
The HM content (except for Hg) was measured in
tissues mollusks sampled at Divnomorskoe and in Inal
Bay (eight specimens per station). The samples of
organs for analysis (gills, DG, and foot) were taken on
board immediately after sampling and stored at –18°C
until the analysis. The chemical composition of blood
ark tissues was analyzed at the Laboratory of Physical
and Geological Research of the Institute of Oceanol-
ogy, Russian Academy of Sciences. We measured the
contents of Mn, Cu, Co, Pb, Cd, Ni, and Cr by ICP-
MS and the Fe and Zn contents on the flame KVANT-
2A AAC. The samples were decomposed in a mixture
of 1.5 mL of double distilled concentrated nitric acid
(HNO3) and 0.5 mL of 30% H2O2 in teflon containers
at a temperature no higher than 70°C. The accuracy of
the analysis was controlled with the international stan-
dard sample of the National Institute of Standards of
Canada (NIST SRM): SRM IAEA MA – A2 Fish
flаsh homogen. The HM concentration was given for
dry mass for bioaccumulation calculation and for fresh
mass for comparison with the standards of Sanitary
Rules and Regulations [4].
The bioaccumulation coefficient of HM in tissues was
calculated individually for each organ (per dry mass) in
relation to the HM content in BSs.
The analysis of grain-size composition of the upper 5-
cm layer of bottom sediments was performed by collab-
orators of the Analytical Laboratory of the Institute of
Oceanology of the Russian Academy of Sciences using
the water-sieve approach. The contents of the main
fractions—from less than 0.01 to more than 10 mm—
were determined. For convenience, we used the four
standard gradations of grain-size composition of the
bottom: pelite (less than 0.01 mm), aleurite (0.01–
0.1 mm), sand (coarser than 0.1 to 1.0 mm), and pebble
(coarser than 1 mm) [17].
Table 1. Content of HM in BSs (μg/g of dry mass), benzo-α-pyrene (BAP, ng/g), and DDT (ng/g) of northeastern coast
of Black Sea in 2014 (dash means absence of data); excess of norm is given in bold type
* ICP-MS.
** According to [12].
*** According to [15].
**** Norm, according to [31].
Region Depth, m Fe Mn Zn Cu Cr Co Ni Pb Cd Hg BAP DDT
Bugaz* 3.0 3729 133 17.6 0.73 5.7 1.5 4.1 4.2 0.02 – – –
Bugaz* 10.0 14346 384 35.4 3.86 26.6 5.3 15.1 8.5 0.03 – – –
Divnomorskoe 10.0 15682 432 24.6 5.3 5.5 4.8 8.5 2.5 0.56 0.000 2.68 0.07
Divnomorskoe 15.0 13 444 421 21.0 4.1 4.8 4.2 7.0 2.3 0.20 0.000 1.35 0.10
Divnomorskoe 20.0 16515 492 31.8 6.9 4.6 5.7 8.9 5.0 0.05 0.003 1.58 0.25
Divnomorskoe 25.0 15995 455 40.5 11.6 7.7 7.3 12.5 11.4 0.00 0.033 3.19 0.46
Divnomorskoe 30.0 15940 379 46.8 16.3 12.2 7.7 16.2 7.7 0.09 0.035 4.99 0.16
Inal 10.4 11785 830 28.3 10.3 7.2 4.5 59.3 8.2 0.05 0.009 2.47 0.22
Inal 14.5 15370 841 36.9 12.7 9.7 5.8 13.5 4.9 0.09 0.063 2.01 1.22
Inal 19.1 7220 294 24.7 9.2 5.9 2.8 6.8 4.9 0.24 0.020 1.61 0.83
Inal 23.0 20509 668 52.8 18.1 13.8 9.6 19.7 8.0 0.11 0.028 1.57 1.04
Inal 30.0 20456 600 54.7 21.5 13.7 9.2 19.3 9.4 0.16 0.035 5.11 0.29
Shepsi 7.3 20790 683 51.7 21.3 12.9 9.0 18.1 8.8 0.00 0.041 5.92 0.28
Shepsi 13.3 18832 778 43.3 16.0 10.6 8.5 17.2 7.4 0.00 0.029 2.80 0.13
Shepsi 18.6 18165 808 38.0 10.0 7.1 8.6 12.9 6.0 0.05 0.024 0.00 0.00
Shepsi 24.3 20391 703 54.6 19.4 10.8 10.3 17.7 9.8 0.03 0.033 2.95 0.00
Shepsi 28.3 21915 723 59.6 19.6 11.4 11.1 19.2 9.8 0.00 0.045 5.32 0.17
Kerch Strait (2007–2008)** 37800 630 61 – 93 54 37 – ––––
Shelf of Black Sea*** 11800–32500320–660483145–90144215–25––––
Norm**** – – 140.0 36.0 100.0 20.0 35.0 85.0 0.80 0.300 25.00 2.50
OCEANOLOGY Vol. 57 No. 6 2017
INFLUENCE OF THE BOTTOM SEDIMENT CHARACTERISTICS 831
The ORP of pore water was determined in layers
(from 0 to 1, 1 to 5, and 5 to 10 cm thick) using a
SanXin SX630 portable ORP meter.
Statistical processing and analysis of data was per-
formed in Excel (Microsoft Corporation), Statistica
(StatSoft Inc.), and Primer (Primer-E Ltd.). The
number of stations in the analysis was insufficient for
application of parametric statistical analysis methods,
so we used the Spearman nonparametric analysis (p =
0.05) with the Holm–Boferroni correction (Statistica,
Excel) to determine pair correlations between contin-
uous variables (length of shells of mollusks and the BSs
parameters). The standardized content of each ele-
ment was analyzed with PERMANOVA software
(Permutational ANOVA/MANOVA, Primer) to iden-
tify the correlations of HM contents with environmen-
tal factors and geographical position. Statistically dif-
ferent HM concentrations for particular areas/depths
were determined by SIMPER analysis and the Princi-
pal Component Analysis (PCA) (Primer). The rela-
tionship between a histopathology and environmental
parameters were determined by nonparametric logis-
tical analysis (logistic regression) (Statistica).
RESULTS
Stage of the reproductive cycle. We investigated
101 mollusks, including 99 monosexual animals and
2 hermaphrodites (we found one specimen near Div-
nomorskoe and one at Shepsi). The ratio of sexes in
blood ark samples was almost equal. The mollusks
collected in the area of the Bugaz Sandbar included
58% of animals with gonads at the initial and devel-
oping stages (stage 2–3) and 42% of shellfish with
mature gonads (stage 4). The blood ark sampled in
the southern areas near Divnomorskoe and Shepsi
and in Inal Bay three weeks later were ready to spawn
(90% of animals): only mature eggs and spermato-
zoids were found in their gonads (stage 4–5), and the
progenitor cells were not revealed.
Histopathological characteristics of blood arks. The
examined blood arks were characterized by mild
pathologies: hyperemia of gills (their vessels and lacu-
nae were filled with hemocytes), hemocytosis (a higher
content of hemocytes in the connective tissue around
the digestive tract in comparison with normal), and
brown cells [18] (a higher number of cells filled with
lipofuscin-like pigment in connective tissue of the DG
than the normal amount [28]) (Fig. 2i). Moderate
pathologies included vacuolization of oocytes (Fig. 2h)
and epithelial atrophy of the DG (Fig. 2d). Some mol-
lusks had serious generalized pathologies: degeneration
of the DG (Fig. 2e) and tissue necrosis (Fig. 2f). Rick-
ettsia-like organisms were found in digestive cells of the
DG (Fig. 2c, inset) and in the connective tissue of the
DG and gonads in 3–26% of mollusks in each studied
area. According to the classification by Costa et al. [26],
they correspond to moderate histopathology. Multicel-
lular parasites were not revealed in the examined indi-
viduals of blood arks.
In the area of the Bugaz Sandbar, the blood ark
specimens were characterized by five types of pathol-
ogy at a depth of 3 m and by three types at a depth of
10 m. In the latter sampling, brown cells often
occurred (Fig. 2i). Hemocytosis, hyperemia of gills,
and vacuolization of oocytes was revealed for one indi-
vidual in addition to a high content of brown cells.
Serious pathologies were not revealed there. Near Div-
nomorskoe, six types of histopathology were revealed.
The samples were characterized by the maximum fre-
quency of gills’ hyperemia and epithelial atrophy of
the DG. The highest frequency of serious histopathol-
ogy’s was found for Inal Bay: 13% of mollusks were
characterized by tissue necrosis. The stage of gonad
development of one of the individuals with necrosis,
hemocytosis, and an increased number of brown cells
corresponded to the early maturation of oocytes con-
trary to all other studied individuals of Inal Bay char-
acterized by mature gonads. In Shepsi, the histopa-
thology level was slightly lower than in Inal Bay, but it
was generally higher than in Divnomorskoe. Tissue
necrosis was not revealed, but 15% of the mollusks
were characterized by DG degeneration.
Grain-size composition of BSs. Bottom sediments of
the studied areas were represented by muddy sand and
mud. The distribution of fractions with the depths was
specific for each investigated region (Fig. 3). In the
samples of BSs from a depth of 10–15 m, sands com-
prised to 60–70% of fractions. Sand fraction also pre-
dominated at a depth of 20 m in Divnomorskoe and
Shepsi, while it’s content did not exceed 30% in Inal
Bay. At depths of 25–30 m, sand content was lower than
40%, and aleuropelite (aleurite in combination with
pelite) predominated (up to 60–90%). At the stations in
the area of the Bugaz Sandbar, the sand fraction pre-
dominated in BSs samples from a depth of 3 m (93%).
At a depth of 10 m, the sediments were weakly sorted
(the contents of different fractions in them were simi-
lar), which due to this station’s being related to a
depression in terms of topography. The fractions of BSs
became less sorted from Divnomorskoe to Shepsi
(Fig. 3). Correlation analysis confirmed a significant
increase in the content of pelitic fraction with the depth
(R = 0.77, p < 0.008), while the correlation of other
fractions with this parameter was not significant.
ORP of BSs was positive at all the sites, except for
the depth of 10 m at the Bugaz Sandbar, which points
to oxidation conditions (Fig. 3). ORP (1–5 cm) was in
positive correlation with sand content (R = 0.58) and
in adverse correlation with depth (R = –0.59). Never-
theless, the verification by the test of Holm-Boferroni
showed that these correlations were not significant at
multiple comparisons, and only some trends existed
there.
HM, BAP, and DDT in bottom sediments. The HM
contents in BSs at the investigated depths are given in
832
OCEANOLOGY Vol. 57 No. 6 2017
KOLYUCHKINA et al.
Fig. 2. Histopathologies of blood arks. Lateral section of gills near lateral cilium junction: (a) normal (arrow indicates blood
sinus), (b) pathology: hyperemia (arrow indicates blood sinus filled with hemocytes). Successive stages of pathologic process in DG:
(c) normal status of acinus (pointed by arrow); inset: Rickettsia-like organism; (d) epithelial atrophy (arrow indicates detachment
of epithelial cells from basal membrane), (e) degeneration (arrow indicates acinus with degenerating epithelial cells separated
from basal membrane). Necrotic changes in connective tissue and digestive system of mollusks: (f) black arrow indicates necrotic
intestine with typhlosole without epithelial cells, gray arrow indicates clusters of hemocytes in tissue. Oocytes: (g) norm and
(h) vacuolization (arrows point to vacuoles). Brown cells (i) in loose connective tissue (gray arrow indicates normal hemocytes
and black arrow denotes brown cells).
50 µm50 µm
50 µm
50 µm
20 µm20 µm20 µm
50 µm
200 µm
200 µm
(a) (b)
(c) (d)
(e) (f)
(g) (h) (i)
OCEANOLOGY Vol. 57 No. 6 2017
INFLUENCE OF THE BOTTOM SEDIMENT CHARACTERISTICS 833
Table 1. The norms of organic contaminants in BSs
were not exceeded. The sole exception was Ni, whose
content at a depth of 10 m in Inal Bay was 1.7 times
higher than the European standards of concentrations
of elements in bottom sediments [29] and three or
more times greater than its concentrations at other
sites. Comparison of the content of elements with their
mean values on the shelf of the Black Sea showed
excess Ni at a depth of 10 m and Mn at depths of 10–
15 and 23 m in Inal Bay and at all the studied depths at
Shepsi. The correlations between the contents of HM
were significant, except for manganese, the content of
which showed only a low-reliable correlation with the
Ni content (Table 5). The pair correlation of Ni with
other HM in BSs was significant at most stations,
except at 10 m in Inal Bay. Nevertheless, the point
with the abnormally high Ni content was beyond the
95% significance interval with all HMs in BSs, e.g.,
with Cr (Fig. 4).
Nonparametric dispersion analysis (PERMANOVA)
showed that the regions differed in HM content in BSs
(Table 3). We detected an increased Cd content in BSs
at Divnomorskoe and Cr in BSs of the Bugaz Sandbar
compared to other regions (Table 1). The BSs of
Shepsi were characterized by higher contents of Pb,
Zn, Cu, Mn, Co, and Fe than in other areas. The HM
content in BSs of Inal Bay was intermediate. This pat-
tern is shown in the PCA diagram based on the nor-
malized contents of nine HMs in BSs (with the excep-
tion of Hg, the concentration of which was not mea-
sured in Bugaz) (Fig. 5a). The contents of BAP and
DDT did not depend on the studied region or depth
(Tables 1, 3).
Significant correlations between the parameters of
the grain-size composition, ORP of BSs, and the HM
content were not revealed, except for a high significant
inverse correlation between the contents of Cr and the
sand fraction in BSs (R = –0.73). We also detected
trends toward an increase in the Cu, Zn, Co, and Pb
contents with the depth; of Zn, Cu, Cr, Co, Ni, and Pb
contents with an increase in the amount of pelite in
BSs; and of Mn w ith an increase in ORP (1 cm). How-
ever, corrections for multiple comparisons showed
that these correlations were not significant.
HMs in the tissues of bivalves. The HM content in
the organs of mollusks from Inal Bay and Divnomor-
skoe did not exceed the standards of Sanitary Rules
and Regulations (Table 2). Significant differences in
HM content (except for Cr) between organs of mol-
lusks did not depend on the sampling area (PER-
MANOVA; Table 4; Fig. 5b). Foot were characterized
by the highest Zn and Pb contents and the lowest Cu
and Fe contents (Table 2). The amount of Mn, Co,
Fig. 3. Ratio of grain-size fractions and ORP in BSs at a depth of 3–30 m in four regions of northeastern coast of Black Sea in
summer 2014. (1) pebble, %; (2) sand, %; (3) aleurite, %; (4) pelite, %; (5) ORP (0–1 cm); (6) ORP (1–5 cm).
100
90
80
70
60
50
40
30
20
10
0
600
500
400
300
200
–200
100
–100
0
1
2
3
4
5
6
Grain-size fractions, %
ORP, mV
Bugaz 10
Bugaz 3
Divnomorskoe 10
Divnomorskoe 15
Divnomorskoe 20
Divnomorskoe 25
Divnomorskoe 30
Inal 10
Inal 15
Inal 20
Inal 23
Inal 30
Shepsi 10
Shepsi 15
Shepsi 20
Shepsi 25
Shepsi 30
Fig. 4. Correlation between Ni and Cr content in BSs of
Bugaz, Divnomorskoe, Inal, and Shepsi in 2014. (1) abnor-
mal ly high Ni co ntent a t norm al amo unt of Cr in I nal Bay at
a depth of 10 m; (2) mean Ni and Cr contents on shelf of
Black Sea are within 95% significance interval (dashed line)
of regression (straight line).
2 4 6 8 10 12 14 16 18
Cr in BSs, µg/kg of dry mass
0
10
20
30
40
50
60
70
Ni in BSs, µg/kg of dry mass
1
2
834
OCEANOLOGY Vol. 57 No. 6 2017
KOLYUCHKINA et al.
Ni, Cd, and Cr in foot was similar to those in DG, but
lower than in gills (Table 2). Gills were characterized
by the highest Mn, Co, Ni, Cd, and Cr contents. The
Ni concentrations in a specimens from Inal Bay and
one individual from Divnomorskoe was 11.54 and
11.64 μg/g, respectively (three times higher than the
mean, Table 2). The DG was characterized by the low-
est Pb and Zn contents and the highest content of Cu
and Fe. The investigation areas did not differ with
respect to HM content in organs of blood ark. There
were significant correlations between the amounts of
some metals (Table 5).
Bioaccumulation. The contents of Fe, Mn, Cr, Co,
and Pb in BSs were higher than in mollusks, and those
Fig. 5. PCA diagrams plotted on basis of normalised concentrations of nine HM (a) in BSs of four regions and (b) in three
different organs of Anadara from two regions. (А): (1) Bugaz, (2) Divnomorskoe, (3) Inal, (4) Shepsi; (B): (1)–(3) Inal;
(4)‒(6) Divnomorskoe (1), (4) gills, (2), (5) DG; (3), (6) foot ofblood ark.
2
0
–2
–4
–6
–4 –2 0 2 4 6
(a) (b)
PC2 (19%)
PC1 (53%)
Mn
Fe
Cd
Co
Cu Ni
Zn
Pb
Cr
1
2
3
4
4
2
0
–2
–4
–6 –4 –2 0 2
PC2 (24%)
PC1 (35%)
Mn
Fe
Cd
Co
Cu
Ni
Zn
Pb
Cr
1
2
3
4
5
6
Table 2. Mean (1), standard deviation (2), and norm of HM content in blood arks tissues sampled at the northeastern coast
of Black Sea in 2014 (dry and fresh mass, μg/g)
* Norms are given according to [1].
** Norms according to [4].
Element
Digestive gland Gills Foot
NormInal Divnomorskoe Inal Divnomorskoe Inal Divnomorskoe
121212121212
Dry mass
Fe 378.5 103.3 523.2 264.0 335.3 84.2 265.2 93.1 270.3 113.1 176.2 28.2
Mn 3.4 1.0 6.1 4.0 10.1 2.6 6.6 2.1 7.6 4.6 5.3 2.4
Zn 12.6 3.1 11.9 4.1 26.3 17.1 24.3 16.9 38.9 8.5 38.9 7.2
Cu 12.0 2.7 14.4 4.8 7.0 0.9 9.2 1.3 4.5 3.9 3.2 3.9
Cr 0.50.43.32.93.36.93.06.51.84.40.81.7
Co 0.30.10.40.11.00.40.90.20.30.10.30.1
Ni 0.6 0.2 1.4 0.8 3.1 3.5 3.8 3.9 1.2 1.0 0.8 0.5
Pb 0.30 0.20 0.54 0.25 0.47 0.44 0.50 0.25 2.37 2.66 0.89 0.47
Cd 1.40 0.37 1.81 0.69 5.27 3.16 6.82 2.89 0.41 0.15 0.40 0.23
Fresh mass
Zn 2.62 0.70 3.61 0.87 2.44 1.70 3.08 2.22 7.54 1.81 7.90 1.51 200*
Cu 2.48 0.56 4.51 1.62 0.64 0.08 1.11 0.20 0.90 0.85 0.68 0.86 30*
Pb 0.06 0.05 0.18 0.11 0.04 0.04 0.06 0.03 0.60 0.65 0.18 0.10 10**
Cd 0.29 0.08 0.58 0.27 0.50 0.32 0.82 0.38 0.08 0.02 0.08 0.05 2**
OCEANOLOGY Vol. 57 No. 6 2017
INFLUENCE OF THE BOTTOM SEDIMENT CHARACTERISTICS 835
Table 3. The results of nonparametric dispersion analysis (PERMANOVA) on content of HM and organic contaminants
in BSs (main test and pair comparison of regions with respect to each HM or organic contaminant). Significance at p < 0.05
is given in cells. Dash means impossible comparison
Regions Fe Mn Zn Cu Cr Co Ni Pb Cd Hg BAP DDT
Bugaz-Divnomorskoe – – –
Bugaz-Inal –––
Bugaz-Shepsi 0.003 0.041
Divnomorskoe-Inal
Divnomorskoe-Shepsi 0.012 0.007 0.008 0.018 0.005 0.005 0.012 0.015
Inal-Shepsi
Main test 0.005 0.038 0.006 0.016
Table 4. The results of nonparametric dispersion analysis (PERMANOVA) on HM content in organs of blood arks.
Differences between HM groups are significant at 0.001 (ANOSIM global R = 0.984)
HM Probability of significance of differences between parameters, p
gills–DG gills–leg DG–Leg Inal–Divnomorskoe station–organ (note)
Zn 0.003 0.007 0.001 – –
Pb – 0.014 0.003 – Differences were significant
only in Inal
Cu 0.001 0.002 0.001 – –
Fe 0.004 0.013 0.001 0.046 (only in leg) Differences between organs
were significant
only in Divnomorskoe
Cr – – – – –
Mn 0.002 – – – –
Co 0.001 0.001 – – –
Ni 0.001 0.002 – – –
Cd 0.001 0.001 0.001 – –
Table 5. Coefficients of pairwise correlations of HM in BSs (top right part of table) and of HM in blood arks tissues
(bottom left part of table). Data significant at p < 0.05 with correction by Holm–Bonferroni for multiple comparisons
are given in bold type. Correlations are given for dry mass
ТМ Fe Mn Zn Cu Cr Co Ni Pb Cd Hg
Fe 0.46 0.89 0.81 0.54 0.95 0.63 0.61 –0.35 0.51
Mn 0.13 0.43 0.49 0.17 0.52 0.60 0.28 –0.29 0.36
Zn –0.53 0.23 0.92 0.75 0.96 0.75 0.78 –0.34 0.73
Cu 0.53 0.00 –0.67 0.64 0.86 0.77 0.67 –0.21 0.81
Cr 0.35 0.18 –0.32 0.43 0.64 0.73 0.67 –0.27 0.73
Co 0.24 0.55 –0.01 0.28 0.20 0.70 0.69 –0.34 0.62
Ni 0.14 0.54 0.17 0.01 0.34 0.70 0.68 –0.22 0.46
Pb –0.19 0.28 0.50 –0.28 –0.01 –0.03 0.15 –0.56 0.56
Cd 0.44 0.33 –0.38 0.55 0.23 0.75 0.46 –0.41 –0.49
836
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KOLYUCHKINA et al.
of Cd, Cu, Zn, and Ni were lower. The bioaccumula-
tion coefficient of Cd was 100 and 30 in gills, 30 and 10
in the DG, and 10 and 2 in foot in Inal Bay and Div-
nomorskoe, respectively. The Cd concentrations in
blood arks were similar in the two studied areas, but its
content in BSs differed considerably, which resulted in
a threefold difference in the bioaccumulation factors
between the two areas. In Divnomorskoe, the bioac-
cumulation factor of Cu was 3 in the DG and to 2 in
foot, while in Inal Bay, it was less than 1. The bioaccu-
mulation coefficient of Zn was high for foot and was
greater in Divnomorskoe (1.8) than in Inal Bay (1.4).
Bioaccumulation of Ni was revealed in one specimen
sampled near Divnomorskoe: the content of this ele-
ment in gills was higher than its content in BSs
1.7 times. In the other 15 specimens from the both
regions, Ni bioaccumulation was not seen. It should
be mentioned that the concentrations of Ni in gills of
specimens from Inal Bay and Divnomorskoe were
similar, but this element bioaccumulation was found
only at Divnomorskoe, because Inal Bay was charac-
terized by its high content in BSs.
Analysis of correlations of histopathologies with abi-
otic and biotic parameters. There was no significant
correlation between serious pathologies and the inves-
tigation regions, because the percentage of blood arks
with serious pathologies was low in all regions. The
frequency of mild pathologies significantly differed in
the studied regions (PERMANOVA: p < 0.05) with
the exception of Shepsi and Inal Bay. In contrast to
other areas, the pair comparison showed a difference
between these regions in the frequency of moderate
pathologies (p = 0.048).
There were no significant correlations of the fre-
quency of histopathological changes with the grain-
size composition of the bottom and ORP of BSs. The
contents of some HMs in BSs correlated with partic-
ular pathologies. The frequency of necrosis of mol-
lusks correlated with Ni content in BSs, but the sig-
nificance of the correlations corrected for multiple
comparison was low (p = 0.051). The frequency of an
elevated number of brown cells in mollusks cor-
related with Cd content in BSs, but its significance
was also low (p = 0.048).
We also revealed a highly significant correlation
between the development stage of gonads and DG
degeneration (p = 0.001). At the Bugaz Sandbar, only
the initial development stages of gonads (immature
gametes) were seen, but no degeneration of the DG. In
the remaining areas, individuals with this pathology
were present and gametes were mature. A highly sig-
nificant correlation (p < 0.001) was observed between
the length of mollusk shells and the presence of brown
cells in them. Brown cells were not found in bivalves
with a shell length less than 30 mm, while we did not
find any mollusks with shells longer than 55 mm with-
out brown cells. Significant correlations between his-
topathologies were not revealed.
DISCUSSION
Histological examination of blood ark from the
four regions of the northeastern coast of the Black Sea
showed that the condition of most mollusks was good.
Only a few individuals from Inal Bay had necrosis of
tissues, which could have resulted in their death. Sta-
tistical analysis of the frequency of histopathology’s
revealed the differences in the condition of shellfish
from the four regions of the northeastern coast of the
Black Sea. The variability and severity of pathological
changes within the studied region became greater from
the northwest to the southeast. The correlations
between the frequency of histopathologies and the
number of abiotic and biotic factors were analyzed for
the taken samples.
ORP and grain-size composition of BSs. There were
no correlations between histopathologies of blood arks
and ORP. For example, at a depth of 10 m near the
Bugaz Sandbar, where the ORP of the subsurface layer
of BSs was below zero, which indicates a lack of oxy-
gen, serious irreversible pathological changes were not
seen. This is probably because the studied species of
mollusks is characterized by a high resistance to
hypoxia due to the presence of red blood cells with
hemoglobin, which are capable of storing oxygen.
Blood arks are able to survive to 12 days at an oxygen
content less than 14% saturation [25]. No correlations
of histopathology’s with grain-size composition of
BSs were revealed.
HMs and organic contaminants. The studied areas
were characterized by smaller anthropogenic impact
[11] in comparison with, e.g., the Kerch Strait [12] or
the northwestern Black Sea [27]. This is obviously the
reason for the fragmentary data on HM content in BSs
near the Black Sea coast of the Caucasus. They mainly
referred to the most contaminated areas of ports and
large cities [11]. The present study provides more com-
prehensive data on the modern HM contamination of
the studied regions.
The content of mercury—the most toxic element—
in the BSs of the studied areas was an order of magni-
tude lower than the European standards [29]. The
amount of BAP and DDT was also 5 and 2–35 times
below the standards. It can be concluded that the sta-
tus of the studied areas was satisfactory with respect to
the content of these substances, in contrast to, e.g.,
port areas [11].
Fe and Zn are widespread in the Earth’s crust and
at the same time are essential elements [37]. The accu-
mulation of Zn in foot of shellfish was small (bioaccu-
mulation coefficient <2), and its content in tissues of
mollusks was two orders of magnitude lower than the
standards of Sanitary Norms and Regulations. There-
fore, Zn accumulation in foot of mollusks may be
related to the role of this element in metabolic pro-
cesses [37]. In addition, Zn and Fe contents in BSs
varied widely but did not exceed the European stan-
dards for Zn [29] and the mean Fe content in BSs of
OCEANOLOGY Vol. 57 No. 6 2017
INFLUENCE OF THE BOTTOM SEDIMENT CHARACTERISTICS 837
the shelf [15]. The content of Zn in BSs in the studied
area exceeded the mean content in BSs on the shelf,
but was lower than in the more contaminated waters of
the Kerch Strait [12]. Since no correlations between
histopathologies and the content of these metals were
revealed, and they mutually correlated with other
HMs, it can be preliminarily concluded that the origin
of these elements in BSs is natural, and their role in
histopathological changes of blood arks in the studied
area is insignificant.
The content of Cd in BSs correlated with the
increased amount of brown cells in the connective tis-
sues of blood arks. It is known that brown cells are able
to accumulate this element [43]. Nevertheless, analy-
sis of the Cd content in BSs of the studied regions did
not reveal any excess of the European standards [29];
its content in soft tissues of blood ark was below the
standards of Sanitary Rules and Regulations and the
amounts of this element in tissues of other animal spe-
cies sampled in uncontaminated areas [21]. Thus, the
correlation between the contents of Cd and brown
cells requires further investigation. According to our
data, the bioaccumulation coefficient of Cd in tissues
of blood arks reached 100. It is known that mollusks
are able to accumulate Cd in amounts exceeding its
content in the environment by several orders of mag-
nitude. This capability is explained by the fact that
their tissues contain peptides (metallothioneins) that
fix this toxic element in insoluble complexes [24].
Brown cells may be also involved in the neutralization
of this element [43].
Bioaccumulation coefficients of Cr, Co, and Pb
were not higher than 1. The content of Pb did not
exceed the permissible rates of Sanitary Rules and
Regulations, and Co content was lower than that in the
bodies of shellfish from uncontaminated habitats [33].
These significant differences between the bioaccumu-
lation coefficients of these HM and Cd may be related
to the fact that Cr, Co, and Pb predominate clay and
detrital minerals (from 50% of the total content for Pb
to 90% for Cr) and as a result, are characterized by
limited bioavailability [2, 5]. This is confirmed by the
tendency for a rise in the content of cobalt and lead
with the depth and of chromium, cobalt, and lead with
the increase in the content of pelite in BSs. On the
contrary, Cd is characterized by high adsorption
capacity at migration in BSs, and its amount is not
related to the content of the pelite fraction.
No significant correlation of Cu content in BSs with
histopathology was observed. In the DG and foot of
blood arks sampled near Divnomorskoe, the bioaccu-
mulation coefficient of Cu was higher than 1, and this
area was characterized by the greatest frequency of atro-
phy of DG epithelium (27%). Similar changes were
observed in blood arks after the experimental pollution
habitats by copper sulfate. Atrophic changes in the DG
were seen on the third day of exposure, when the copper
concentration in tissues rose to 150 μg/g of dry mass. In
the control samples (7 μg of Cu/g of dry mass), changes
were absent [9]. In the present study, the Cu content in
DG of blood arks was an order of magnitude lower (10–
23 μg/g of dry mass for Divnomorskoe and 7–16 μg/g
of dry mass for Inal Bay). It can be assumed that, in
addition to the impact of Cu, atrophy of the DG of
blood arks may be related to other factors. As well, the
Cu content in BSs of the studied area was lower than
its mean content in BSs of the shelf and correlated
with the content of other HMs (Table 5). This indi-
cates that there were no additional sources of this ele-
ment in the sea.
The content of Ni in BSs correlated with the fre-
quency of necrosis of blood arks. In Inal Bay, where
the Ni content in BSs exceeds the European standards
[29] and its mean content in sediments of the shelf
[15], tissue necrosis was detected in 13% of mollusks
and gills of 26% of specimens were characterized by
hyperemia. An increased Ni content in gills was seen
for 13% of shellfish in this area. It exceeded 10 μg/g of
dry mass, the threshold concentration of the develop-
ment of pathological processes in tissues of mollusks
[41]. It should be mentioned that the background Ni
content for the more contaminated Romanian sector
of the Black Sea is lower than 5 μg/g of dry mass of soft
tissues of blood arks [31]. According to our data, the
Ni concentration at a depth of 10 m in Inal Bay is out-
side the 95% confidence interval of regression for its
pair comparison with other elements. Thus, it can be
assumed that there was an additional source of Ni at
this site. The distribution pattern of this element was
obviously mosaic, and/or its biological availability was
limited, because the share of mollusks with patholo-
gies and high Ni concentration in tissues was small.
According to our data, there was a tendency toward an
increase in Ni content with increasing Mn content in
BSs. In addition, there was a highly significant positive
correlation between the Ni and Mn contents in tissues
of blood arks. In Inal Bay—the site of the maximum
Mn concentration—an extremely high ORP of BSs
was seen at a depth of 10 m. These oxidation condi-
tions usually result in the transformation of soluble
Mn2+ into the oxidized form of Mn4+. These insoluble
amorphous hydroxides are adsorbents of HMs, in par-
ticular, Ni [2, 15]. A high Ni concentration at the
10-m-deep station in Inal Bay and the tendency
toward a correlation between the Ni and Mn contents
in BSs are obviously explained by Ni adsorption by
amorphous Mn hydroxides, which significantly
decreases its bioavailability and is responsible for the
correlation between the Ni and Mn contents [2, 15].
Anomalous concentrations of Mn were seen in BSs
of Inal Bay at a depth of 10–15 m and near Shepsi at
all studied depths. They exceeded by two times the
mean content of this element on the shelf [15] and by
25% its amount in the area of the Kerch Strait [12]. It
is known that the Mn concentration is the highest in
river mouths and is especially large in suspended par-
ticulate matter [15]. Since the sampling period was
838
OCEANOLOGY Vol. 57 No. 6 2017
KOLYUCHKINA et al.
preceded by heavy rains and there were no liman sed-
imentation basins on the rivers near the mouths of
which the sampling points were located, it can be
assumed that this local maximum is a result of transport
of terrigenous material enriched in Mn by flood waters
after heavy rains [6, 13, 15]. High ORP and its correla-
tion with the Mn content in BSs indicate the predomi-
nance of low bioavailability of this element [2, 15]. Sim-
ilar Mn concentrations were determined for gills, foot,
and DG of other species of mollusks in uncontaminated
waters [33].
Infestations with Rickettsia-like organisms. In addi-
tion to abiotic factors, histopathology may be related
to biotic factors: infestation of tissues and organs of
mollusks by parasites. In this study, we revealed obli-
gate intracellular parasites—Rickettsia-like organ-
isms—in tissues of blood arks. Similar Rickettsia-like
organisms were also determined in other species of
mollusks on the northeastern coast of the Black Sea
(Mytilus galloprovincialis and Chamelea gallina) [3, 9].
The presence of these microorganisms is normal for
most mollusks and very rarely correlates with inflam-
matory processes in their bodies [22]. Since the fre-
quency of infestation of mollusks with Rickettsia-like
microorganisms did not correlate with other patholo-
gies, we assume that it does not affect the histopatho-
logical condition of blood arks in the studied region.
Possible causes of histopathologies unrelated to high
concentrations of pollutants. Mild pathologies may be
explained by natural causes. For example, brown cells
are store kidneys [19, 43]. Their brown granules con-
tain significant amounts of the insoluble pigment lipo-
fuscin—a product of lipid peroxidation [28]—that
accumulates in cells with age [43] or in inflammatory
processes caused by parasitic infestations [19], as well
as by contamination of water basins with HMs [18].
Blood arks can reach the age of nine years or older at
the Black Sea, and their growth does not stop during
their lifetime; thus, shell length correlates with mol-
lusk age [35]. According to our data, the frequency of
occurrence of brown cells shows a highly significant
positive correlation with the size of blood arks’ shells
and a slightly significant correlation with Cd content
in BSs. Brown cells likely accumulate in mollusks
with age.
Severe irreversible pathologies were revealed for
less than one-quarter of mollusks, which testified to
an insignificant decrease in the reproductive poten-
tial of the population; thus we could not predict a
drop in the abundance of blood arks in the studied
regions. Vacuolization of oocytes revealed for some
mollusks may be an indicator of reduced reproduc-
tive potential of the population. Nevertheless, in this
study, it was only seen for 4–14% of individuals.
These were mainly mollusks at the final stages of the
generative cycle. This pathology did not correlate
with any of the factors studied. In our case, vacu-
olization of oocytes in gonads after spawning is prob-
ably one stage in the resorption of unspawned gam-
etes. The sole exception was a mollusk found in the
area of Bugaz, whose gonads were in the initial stages
of the generative cycle (stage 3). We suppose that vac-
uolization of oocytes in its organism was the result of
gerontological changes, because it was accompanied
by some other pathologies (hemocytosis and hyper-
emia of gills), and the large size of the shell testified
to the considerable age of the mollusk [35].
Epithelial atrophy and degeneration of the DG
may occur in mollusks not only from environmental
contamination, but also due to a smaller amount of
available food [32]. These DG pathologies were only
revealed for shellfish in areas affected by floods and
were probably caused by a lower availability of seston
as food as a result of a great amount of particulate
inorganic matter.
CONCLUSIONS
Our analytic data testify to the high probability of a
relationship between Ni content in BSs and an
increased frequency of serious histopathologies. Mild
pathologies are related to natural causes; in particular,
there is probably a correlation between the age of mol-
lusks and the elevated level of brown cells in the con-
nective tissue of the DG.
ACKNOWLEDGMENTS
This work was supported by the Russian Science
Foundation, project no. 17-14-00382.
The authors are grateful to the participants of the
Black Sea-2014 expedition, T.D. Prokhorova,
S.V. Sukhov, A.B. Basin, A.K. Zalota, and V.L. Semin
for assistance in the collection and initial treatment of
samples, and to A.I. Azovsky for consultations on sta-
tistical processing of the data. This work could not
have been performed without the comprehensive aid
of A.G. Zatsepin, supervisor of the Black Sea-14 expe-
dition; S.B. Kuklev, director of the Southern Branch
of the Institute of Oceanology, Russian Academy of
Sciences; the crew of the R/V Ashamba, and collabo-
rators of the Yamaika diving center on the Bugaz
Sandbar.
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Translated by I. Bel’chenko
SPELL: OK
