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Abstract—The reaction of bivalves Modiolus modiolus to pulse (for 24 and 48 h) exposure with multiwalled carbon nanotubes (MWCNTs) (12–14 nm, MWNT ...
ISSN 1995-0780, Nanotechnologies in Russia, 2015, Vol. 10, Nos. 3–4, pp. 278–287. © Pleiades Publishing, Ltd., 2015. Original Russian Text © A.A. Anisimova, V.V. Chaika, V.L. Kuznetsov, K.S. Golokhvast, 2015, published in Rossiiskie Nanotekhnologii, 2015, Vol. 10, Nos. 3–4.

Study of the Influence of Multiwalled Carbon Nanotubes (12–14 nm) on the Main Target Tissues of the Bivalve Modiolus modiolus A. A. Anisimovaa, V. V. Chaikaa,b, V. L. Kuznetsovc, and K. S. Golokhvasta a

Far Eastern Federal University, ul. Sukhanov 8, Vladivostok, 690950 Russia Nevelsky Maritime State University, ul. Verkhneportovaya 50a, Vladivostok, 690059 Russia cBoreskov Institute of Catalysis, Siberian Branch, Russian Academy of Sciences, pr. Akademika Lavrentyeva 5, Novosibirsk, 630090 Russia e-mail: [email protected]

b

Received June 24, 2014; accepted for publication December 18, 2014

Abstract—The reaction of bivalves Modiolus modiolus to pulse (for 24 and 48 h) exposure with multiwalled carbon nanotubes (MWCNTs) (12–14 nm, MWNT concentration in sea water of 100 mg/L) is manifested in the ingestion of MWCNT aggregates formed in seawater despite their rapid sedimentation from the water column to the bottom of the aquariums. After 24 h, the MWCNT aggregates are observed in the intestinal lumen (size of 10 to 150 μm) and in the tubules of the digestive gland (10 to 50 μm). After 48 h, only large aggregates in contact with mucus and desquamated epithelium fragments are detected in the lumen of the intestine. The smallest aggregates seem to be inside epithelial cells. In the intestine, digestive gland, and gills, MWCNT aggregates induce histopathological changes in the epithelium (erosion, necrosis, trend towards increased vacuolization of the cells) and swelling of the connective tissue. In the gill epithelium after 48 h, patterns morphologically corresponding to apoptosis are observed. Despite significant organ damage, no change in the cellular composition of the hemolymph in mussels exposed to the MWCNTs is found. DOI: 10.1134/S1995078015020020

INTRODUCTION As a result of the increasing anthropogenic impact on the coastal areas of marine water, an enormous amount of pollutants, including water-insoluble xenobiotics, enter the oceans and seas. Nanomaterials are new and potentially hazardous pollutants entering coastal waters from industrial effluents [1, 2]; their accumulation can be a toxic threat to marine biota [3–6]. The evaluation of the real toxicity of nanoparticles in aquatic ecosystems is still ambiguous, because the ecotoxicological properties of nanomaterials, their behavior in water, and bioavailability are little studied [6, 7]. Over the last decade there has been a lot of works highlighting the potential risk of aquatic organisms being exposed to nanomaterials [3, 4, 6, 8–19], but the results of in vivo studies are often highly controversial [6] and, therefore, an analysis of the biological effects of different types of nanoparticles using model bioindicators remains a relevant scientific and practical problem. It is important to take into account and to describe as fully as possible physical and chemical properties of the tested nanomaterials and the behavior of the particles in the environment in which the exposure is carried out [6, 20–22].

Bivalves are a convenient test object in water nanotoxicology. Thanks to the way they eat, by the filtration of particles suspended in sea water, these animals are traditionally used in bioindication and environmental monitoring [3, 6, 23–26] and they are considered a unique model for studying the effects and mechanisms of action of nanomaterials [3, 6, 27]. The main target organs of mollusks in direct contact with foreign substances are gills and organs of the digestive tract [6, 27, 28]: the food particles are captured by the gills and sent to the labial palps, then through the mouth they fall into the intestine and reach the digestive gland, where they are trapped by digestive cell endocytosis and digested with the lysosomal system. This standard path seems to be utilized for accumulation of the nanoparticles as well as the food particles [3, 10, 12]. The further movement of nanoparticles inside the clam organism is believed to be associated with their transport from the digestive system into the hemolymph and circulating hemocytes [6, 9, 29], which are normally involved in the nutrient transfer and digestion and also provide cell-mediated immunity through phagocytosis and cytotoxic reactions [30, 31]. Among other things, the behavior of the Bivalvia hemocytes cell population reflects not only the direct

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(directed to hemocytes themselves) but also indirect (aimed at other targets) effects of damaging factors: changes may occur in the number of circulating cells and hemocytes proportions of different morphotypes and their functional properties [26, 32]. Thus, the reaction of the hemolymph as a system involved in the development of adaptations and maintenance of organismic homeostasis must be considered when evaluating the effect of nanoparticles on the physiological status of the tested aquatic organisms. In the present study we investigated the effect of multiwalled carbon nanotubes (MWCNTs) on the histological structure of the target organs (gills and organs of the digestive tract) and the state of the cell population of hemocytes in bivalves Modiolus modiolus (Bernard, 1983) in conditions of short-term exposure in vivo. MWCNTs are widely used in modern industry and can be released into the environment in significant amounts, representing a potential threat, including for aquatic organisms. In the water area of the Sea of Japan Modiolus modiolus is a mass representative of macrobenthos, an important element of coastal ecosystems, and a convenient indicator for toxicological studies. MATERIALS AND METHODS Preparation of Nanoparticles and Analysis of Their Behavior in Seawater MWCNT synthesis was performed by chemical vapor deposition on the surface of the catalyst (active component Fe-Co alloy) in a tubular reactor at 680°C at the Boreskov Institute of Catalysis, Siberian Branch, Russian Academy of Sciences [33, 34]. The main physicochemical characteristics of the produced MWCNTs are as follows: diameter 12–14 nm, specific surface (SBET) 300 m2/g, type of processing: boiling in concentrated HCl for 8 h, and main impurities (wt. % × 102): Fe (16), Co (7), Mg (5), and Cl (1). To study the dynamics of aggregation of nanotubes in seawater, kinetic studies were performed by spectrophotometry using a Shimadzu UV 2550 two-channel spectrophotometer. The MWCNT suspension with a concentration of 100 mg/L was prepared in sea water taken from the area of the mussels collection, after which the suspension was dispersed for 3 min using ultrasonic disintegrator Bandelin Sonopulse HD 3100 (energy 6384 ± 120 kJ). The suspension was then poured in a 4 mL cuvette and placed in the spectrophotometer. Maximum light absorption of the investigated samples was observed at a wavelength of 195 nm. Light absorption was recorded for a week at a minute interval (in total, 10080 measurements). Aggregates of nanotubes were photographed using the Hitachi S-3400N scanning electron microscope. NANOTECHNOLOGIES IN RUSSIA

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Collection of Biological Material and Incubation with Nanoparticles Adult mollusks Modiolus modiolus (Bernard, 1983) (= M. kurilensis) were collected in July (water temperature 20°C) in a clean area (b. Vityaz, Peter the Great Bay, Sea of Japan) and placed in aerated tanks of 50 L with sea water from the environment. After 3day acclimatization, mollusks were divided into four groups of ten animals each: two control (1C and 2C) and two experimental (1E and 2E). Freshly prepared suspensions of MWCNTs were added in aquariums with experimental animals at 100 mg/L of seawater. In the 1E group the mollusks were kept in the presence of MWCNTs for 24 h and in the 2E group for 48 h. Animals of the control groups (1C and 2C) from the start of the experiment were kept under standard conditions for 24 and 48 h, respectively. After incubation, mollusks of control and experimental groups were removed from the experiment simultaneously at intervals of 30 min between individuals within each group. Histological Analysis of Organs For a histological examination in mollusks, the gills, intestines, and digestive gland were dissected. Organs were fixed in 10% neutral buffered Histoline formalin solution (Element, Russia), dehydrated in ethanol of ascending concentrations, and embedded in Histomix Extra paraffin (BioVitrum, Russia). Semithin sections were prepared from these blocks, which were stained with hematoxylin and eosin and examined under the AxioObserver A1 light microscope (Zeiss). The AxioCam 3 (Zeiss) camera was used to take pictures; the computer morphometry program AxioVision 4.2 was used for image processing. Histopathological changes in organs were recorded visually; the degree of vacuolization of cells in the epithelia was expressed as a percentage of vacuolated epitheliocytes in the cell population. Morphometric analysis included the measurement of the length, width, and area of cells and their nuclei. Statistical analysis included the determination of the mean values of the studied parameters and of their standard errors and the reliability of differences at a significance level of P < 0.05 using Student’s t-test. Investigation of the Hemolymph Cell Population by Flow Cytometry For flow cytometric analysis, hemolymph samples were taken from the posterior adductor into 0.3 M EDTA sodium salt solution to prevent the aggregation of hemocytes and fixed as a cell suspension with 4% paraformaldehyde for 1 h. Cell suspensions were washed of the fixing solution with phosphate-buffered saline medium (PBS), placed in 1% solution of the permeabilization agent Triton X-100, and stained with propidium iodide (50 μg per 1 million cells) in the presence of RNase A (100 μg per 1 million cells) for 2015

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Time course graph

0.6000

0.4000 200 µm

(a) 0.2000

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Day 1 −0.0639 0

Day 7 5000 Time, min

10079

Fig. 1. Changes in absorbance of the MWCNT suspension in time, reflecting the dynamics of nanoparticles aggregation and sedimentation of aggregates in sea water (slurry concentration of 100 mg/L). The ordinate shows the absorption coefficient; the abscissa shows the time in days.

the quantitative detection of DNA in hemocytes nuclei. Then the samples were analyzed using the flow cytofluorimeter BD Accuri C6 in the channels FSC (forward light-scattering), SSC (side light-scattering) and FL2 (fluorescence of propidium iodide). The single cells were differentiated from cell aggregates and debris in the two-parameter histogram of all events distribution by area and height of propidium iodide fluorescence signal (FL2-A vs. FL2-H). The hemocytes morphotypes were identified in the two-parameter histogram of the single cells distribution by forward and side light-scattering (FSC vs. SSC). The percentage of different cell morphotypes was counted and the mean values of FSC and SSC for granulocytes were measured. Statistical analysis included the determination of the mean values of the studied parameters and of their standard errors and the reliability of differences at a significance level of P < 0.05 using Student’s t-test. RESULTS Analysis of the Behavior of MWCNTs in Sea Water The results of the spectrophotometric analysis of the nanotubes slurry showed that MWCNTs sus-

10.0 µm

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Fig. 2. MWCNT aggregates formed in seawater. Scanning electron microscopy of secondary electrons: (a) panoramic view and (b) surface of one of the units. Magnification: (a) 200×; (b) 5000×.

pended in sea water at a concentration of 100 mg/L begin to settle rapidly to the bottom in the first hours and, 24 h later, they are virtually completely on the bottom of the cuvette and absent in suspension (Fig. 1). This is due to the rapid nanoparticles aggregation and the formation of agglomerates of various sizes: from 10 to 200 μm (Fig. 2), which settle to the bottom under its own weight. Histological Analysis Histological analysis showed MWCNTs in organs of the digestive tract of mollusks exposed to carbon nanotubes. Free lying aggregates with a diameter between 10 and 150 μm (Fig. 3) were observed in the intestinal lumen in all individuals (N = 20) of both experimental groups (1E and 2E). The absence of these structures in the control animals (Figs. 3a, 3b) and their presence in all animals treated with nanotubes (Figs. 3c–3f), as well as color, texture, and size of aggregates, clearly show that the structures are identified in the histological preparations are indeed MWCNTs.

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Fig. 3. Histological structure of the intestine and its contents (MWCNTs) in mollusks Modiolus modiolus in the control and experimental groups; (a, b) control groups (1C and 2C), (c) group 1E, and (d–f) group 2E. The arrows indicate the destructive changes in the tissues. Staining with hematoxylin and eosin. Magnification: (a, c, d) 200×; (b, e, f) 400×.

In the 1E group, MWCNT aggregates did not penetrate through the epithelium, but caused in it erosive processes and the peeling of the basement membrane, as well as the puffiness of the connective tissue (Fig. 3). The proportion of vacuolated epithelial cells was 47.5 ± 3.8%, which is not statistically different from the control (36.3 ± 3.3%) at a level of significance of P < 0.05 (Table 1). NANOTECHNOLOGIES IN RUSSIA

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In the 2E group, MWCNT aggregates in the intestinal lumen were in contact with the mucus and fragments of desquamated epithelium (Figs. 3d–3f). In the intestinal epithelium, destructive processes and necrotic phenomena were also observed (Fig. 3d), and the percentage of vacuolated cells has increased 1.55 times authentically compared with the control (Table 1). Sometimes small dark inclusions, presumably corre2015

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Table 1. Morphometric parameters of the Modiolus modiolus intestinal epitheliocytes in the control and experimental groups Group

Length of the nucleus, µm

Width of the nucleus, µm

Area of the nucleus, µm2

Length of the cell, µm

Width of the cell, µm

Area of the cell, µm2

Share of vacuolated cells, %

1C 1E 2C 2E

5.72 ± 0.90 8.3 ± 2.23 7.75 ± 1.56 7.13 ± 1.19

4.7 ± 0.81 5.21 ± 0.72 4.93 ± 0.87 5.03 ± 1.07

19.75 ± 6.27 30.96 ± 7.13 24.84 ± 6.07 21.23 ± 6.68

12.69 ± 2.09 21.91 ± 6.51 18.79 ± 2.32 16.31 ± 2.31

10.46 ± 1.88 9.85 ± 2.53 10.44 ± 3.11 11.26 ± 2.15

84.23 ± 22.29 131.23 ± 41.2 99.64 ± 23.44 91.42 ± 13.87

36.3 ± 3.3 47.5 ± 3.8 38.2 ± 3.9 59.4 ± 5.7

Table 2. Morphometric parameters of the Modiolus modiolus cells of the digestive gland in the control and experimental groups Group

Length of the nucleus, µm

Width of the nucleus, µm

Area of the nucleus, µm2

Length of the cell, µm

Width of the cell, µm

Area of the cell, µm2

Share of vacuolated cells, %

1C 1E 2C 2E

8.76 ± 2.15 5.84 ± 0.76 4.97 ± 0.6 5.30 ± 0.66

5.01 ± 1.21 4.98 ± 0.7 4.20 ± 0.58 4.27 ± 0.56

23.98 ± 6.91 21.16 ± 4.63 15.17 ± 3.35 15.77 ± 3.05

15.68 ± 3.14 13.88 ± 2.34 12.88 ± 2.78 12.23 ± 1.62

9.24 ± 2 10.57 ± 1.9 9.40 ± 1.61 9.57 ± 1.19

78.36 ± 21.69 88.53 ± 16.07 73.97 ± 19.97 76.23 ± 14.02

54.1 ± 4.7 71.3 ± 7.2 56.7 ± 4.2 78.2 ± 7.3

Table 3. Morphometric parameters of the Modiolus modiolus gill epithelium cells in the control and experimental groups Group

Length of the nucleus, µm

Width of the nucleus, µm

Area of the nucleus, µm2

1C 1E 2C 2E

7.07 ± 1.13 5.90 ± 0.85 5.49 ± 0.94 5.28 ± 0.79

5.47 ± 0.96 4.41 ± 0.69 4.16 ± 0.64 3.74 ± 0.74

25.03 ± 6.46 18.22 ± 3.67 15.03 ± 2.88 13.39 ± 3.55

sponding to the smallest aggregates, were observed inside the epithelial cells (Fig. 3f). It should be noted that, on the second day (group 2E), only large aggregates of MWCNT were visible in the lumen of the intestine (Figs. 3d–3f); small aggregates and free slurry, which for a day earlier (group 1E) was visualized in the intestine as a fine gray substance (Fig. 3c), were observed to a much lesser extent. In the intestinal lumen of 2E group animals, one observed a picture that showed the participation of intestinal epithelial cells in the aggregation of MWCNTs (Fig. 3e). The average values of all morphometric parameters of epithelial cells of the intestine in mollusks from the experimental groups did not differ from each other or from the control values (Table 1). MWCNT aggregates and their associated histopathological changes were also observed in the tubules of the digestive gland of mollusks exposed to carbon nanotubes (Fig. 4). As compared with the control (Fig. 4a), the animals of both experimental groups (1E and 2E) were marked by damages in the structure of the digestive tubules, and also by a tendency to increase the level of vacuolization of digestive cells (Fig. 4b), although the differences are not considered reliable at a significance

Length of the cell, Width of the cell, Area of the cell, µm µm µm2 12.44 ± 1.69 10.26 ± 0.9 12.86 ± 1.63 13.37 ± 1.36

10.8 ± 1.26 8.08 ± 0.95 8.82 ± 1.58 8.02 ± 1.96

86.7 ± 16.61 57.61 ± 8.95 74.85 ± 16.09 63.94 ± 9.33

level of P < 0.05 (Table 2). The mean values of morphometric parameters of cells in the digestive gland of mollusks from all studied groups did not differ significantly (Table 2). It should be noted that aggregates in the digestive tubules were significantly smaller than in the intestine: from 10 to 50 µm (Fig. 4c). In the gills of mollusks incubated with nanotubes for 24 and 48 h, MWCNT aggregates were not observed (Fig. 5); however, in the gill epithelium of the animals of both experimental groups (1E and 2E), one revealed erosion and necrosis (Fig. 5b). Furthermore, in the 2E group, pictures corresponding morphologically to apoptosis were detected (Figs. 5c, 5d, indicated by arrows). The expressed vacuolization of cells in the gill epithelium was not observed and mean values of morphometric parameters of cells in mollusks from all studied groups were not significantly different (Table 3). In general, the studied target organs (gills, intestines, and digestive gland) in all animals taken in the experiment responded to exposure to MWCNTs by typical histopathological processes: erosions and necroses of a local character, as well as a tendency to

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Analysis of the Cellular Composition of the Hemolymph

(d) (a)

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As we have shown previously (Anisimova, 2012) in the hemolymph of the mollusk Modiolus modiolus, there are three morphotypes of cells: hyalinocytes, semigranulocytes, and granulocytes. These cells are quite clearly identified by flow cytometry (by the forward and side light scattering) after the preliminary separation of the hemocyte population from debris and cell aggregates present in the samples (by the identical height and area ratio of propidium iodide signal from single cells with different DNA contents) (Fig. 6). The proportions of cells of different morphotypes within each group (1C, 1E, 2C, and 2E) varied in the same range; a comparison of the mean values of the relative number of hyalinocytes, semigranulocytes, and granulocytes in animals from different groups showed no significant differences (Table 4). Also, there was no difference in size (forward light scattering (FSC)) and degree of complexity (side light scattering (SSC)) of granulocytes (Table 4). DISCUSSION

(d) (b)

100 µm

(d) (c) Fig. 4. Histological structure of the digestive gland and the contents of the digestive tubules in mollusks Modiolus modiolus in the control and experimental groups; (a) the general morphology of the digestive gland in the control group (group 1 C); (b) histopathological changes in the digestive gland in experimental animals (group 2E); (c) MWCNT aggregate in the lumen of the digestive tubules (group 1E). Staining with hematoxylin and eosin. Magnification: (a, b) 100×; (c) 400×.

increase the degree of vacuolization of cells in the epithelium. No special individual responses in mollusks within each group were observed. NANOTECHNOLOGIES IN RUSSIA

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In this paper we tried to identify the effects of short-term (24 and 48 h) exposure of MWCNTs on the status of key systems interacting with foreign particles in the bivalve Modiolus modiolus. To assess the effect of MWCNTs on the body of Modiolus modiolus, we examined the histological picture of the gills, intestine, digestive gland, and hemolymph as the main tissue targets of the bivalve mollusks under increased load on the environment, including insoluble xenobiotics, in particular nanoparticles. It is known that nanoparticles falling into the water form more or less large aggregates, which facilitates their capture by clam gills when compared with freely suspended particles [7]. The results of the kinetic studies that we have conducted showed that MWCNTs suspended in sea water at a concentration of 100 mg/L intensively aggregate and settle to the bottom in the first hours and after 24 h they are almost completely absent in suspended state. According to the literature, various nanoparticles (NCB, C60 fullerene, TiO2, and SiO2) suspended in artificial sea water form aggregates of nano- and microsizes [35, 36] and, in the presence of clams, the aggregates associate with mucus, are deposited along the byssus thread, and settle on the bottom of the aquarium for 24 h [36]. Thus, the amount of nanoparticles captured by mollusks is significantly less than that expected on the basis of the initial concentration of the suspension and an evaluation of real internal concentration of nanoparticles in in vivo studies is extremely difficult [6, 7]. The active capture of nanoparticle aggregates is illustrated by histological analysis: after 24 h of incubation, free lying aggregates of up to 150 μm were observed in abundance in the intestinal lumen of the 2015

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20 µm

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100 µm

(c)

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20 µm

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20 µm

235948

4345458

100000

SSC 2000000

FL2-H

Fig. 5. Histological structure of the gills in mollusks Modiolus modiolus in the control and experimental groups; (a) control (group 1C), (b) group 1E, and (c, d) group 2E. Staining with hematoxylin and eosin. Magnification: (a, b, c) 400×; (d) 630×.

R3

R2 R1

10

(b) 0

10

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416371

0

2000000 4000000 FSC

6842068

Fig. 6. Identification of the cell population of hemocytes and of different morphological types of cells in the hemolymph of the Modiolus modiolus by flow cytometry; (a) differentiation of single cells, debris, and cell aggregates in a histogram of distribution of all events by area (FL2-A) and height (FL2-H) of propidium iodide fluorescence signal; single hemocytes with DNA content 2c and 4c are highlighted with frame; (b) identification of three morphotypes of hemocytes in the histogram of the distribution of single cells according the parameters of the forward (FSC) and side (SSC) light scattering: (R1) hyalinocytes, (R2) semi-granulocytes, and (R3) granulocytes. NANOTECHNOLOGIES IN RUSSIA

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Table 4. Proportion of cell morphotypes and morphological parameters of granulocytes in the hemolymph of Modiolus modiolus in the control and experimental groups Group

Share of hyalinocytes, %

Share of semigranulocytes, %

Share of granulocytes, %

Size of granulocytes (FSC, cond. units)

Complexity of granulocytes (SSC, cond. units)

1C 1E 2C 2E

8.6 ± 1.6 7.0 ± 0.8 11.7 ± 3.1 6.8 ± 0.5

52.4 ± 2.5 49.2 ± 1.7 45.6 ± 2.1 46.2 ± 1.6

37.2 ± 2.5 43.8 ± 2.1 42.4 ± 2.0 45.8 ± 1.6

4322.4 ± 70.6 4329.5 ± 64.9 4635.8 ± 91.8 4402.5 ± 116.4

2455.7 ± 78.4 2408.1 ± 71.8 2618.7 ± 112.1 2513.5 ± 119.4

modiolus. After two days, the MWCNT aggregates in the intestine were surrounded by mucus and desquamated epithelium fragments, which apparently is a protective mechanism to quickly evacuate the aggregates from the body. For a number of other marine invertebrates (freshwater crustacean Daphnia magna, marine polychaete Arenicola marina, and sea urchin Paracentrotus lividus), the intake of various nanoparticles (single-walled nanotubes and MWNTs and nanoparticles C60, TiO2, SnO2, CeO2, and Fe3O4) from an aqueous suspension in the digestive tract followed by the elimination of their aggregates has also been shown [4, 13–15, 37]. As a result of the interaction of MWCNT aggregates with the gut surface, already a day later the erosive processes were marked in the modiolus intestinal epithelium, which were accompanied by the swelling of the connective tissue and by a trend towards the increased vacuolization of cells, which is likely due to the development of injury due to physical and chemical irritation and mechanical damage to the intestine wall. Small aggregates, apparently, are able to penetrate the epithelial cells: on the second day of the experiment, dense inclusions were observed in the intestinal epithelial cells, but we do not undertake to assert unequivocally that these are indeed nanoparticles. Note that, in Arenicola marina, nanoparticle penetration from the gut lumen into the epithelial cells was not found [13]. After 24 h of incubation, small (up to 50 μm) MWCNT aggregates were observed in the lumens of the digestive gland tubules and pathological changes similar to lesions observed in the epithelium of the intestine were marked in the histological structure of the digestive epithelium. It is shown that only the smallest aggregates really penetrate into the digestive gland tubules [7, 10]. We did not observe any nanoparticle aggregates inside the digestive cells, although there is plenty of evidence of their penetration into the endosomal and lysosomal compartment [10, 12]. It is known that nanoparticles contacting digestive cells provokes lipid peroxidation [9–11] and a reduction in the stability of lysosomal membranes [10, 12]. The capture of nanoparticle aggregates accompanied by oxidative stress is described by some authors also for the gill epithelial cells [9–11]. Our study NANOTECHNOLOGIES IN RUSSIA

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demonstrates the presence of histopathological changes in the gills of the mussels exposed to MWCNTs. Furthermore, phenomena morphologically related to apoptosis were observed in the gill epithelium. It should be noted that the effect of nanoparticles in the bivalve Elliptio complanata was manifested also in damage to DNA molecules in the cells of gills and digestive gland [9]. Thus, the availability of the genotoxic effect implies the stimulation of apoptosis as a mechanism which protects the tissue from the adverse effects of nanoparticles. As for the reaction of the hemolymph of animals exposed experimentally to MWCNTs, we did not observe any changes in the general morphological pattern of this tissue. As is known, hemocytes of bivalves represent a heterogeneous cell population comprising two main cell forms: agranulocytes (hyalinocytes) and granulocytes [26, 30–32]; in some species one distinguishes cells with intermediate morphological characteristics [38–41]. For the clam Modiolus modiolus (= M. kurilensis), by flow cytometry, three “subpopulations” of hemocytes—hyalinocytes (agranulocytes), semigranulocytes, and granulocytes—are quite clearly differentiated in the hemolymph [41], which allows us to consider all three morphotypes as successive stages of maturation within the same cell line, where granulocytes represent the terminal differentiation step. Unlike agranulocytes, granular hemocytes of bivalves are characterized by a low nuclearcytoplasmic ratio, contain a large number of lysosomes and secretory granules, and possess a high level of phagocytic activity and free radicals production [30, 32, 40]. When evaluating the effects of stress, including those of toxicological character, as a rule a change in the proportion of cells of different morphotypes is observed. In particular, bivalves were kept in the presence of toxic microalgae, accompanied by the appearance in the hemolymph of a large number of young cells and, consequently, by a decrease in the proportion of highly differentiated large granulocytes [42– 44]. The authors attribute these effects either to the activation of proliferative processes in response to a toxic effect [42, 43] or to the migration of granulocytes from circulation into the internal organs to be involved in the process of phagocytosis of damaged cells [44]. We expected that the effect of nanoparticles on the 2015

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hemolymph of the Modiolus modiolus will be manifested by similar effects because, as in the case of microalgae toxins, gills and the digestive tract are direct targets of the toxic effect of nanoparticles. However, neither the proportion of cells of different morphological types nor sizes and degree of complexity of granulocytes in the control and experimental animals differed, despite the obvious damage in the structure of the internal organs. Galimany et al. (2008) states that the foci of infiltration of hemocytes in the connective tissue surrounding the tubules of the digestive gland were observed on the 3rd and 6th days of the experiment. Thus, the lack of effect in this case may be associated with the exposure time not being long enough (less than 3 days) or with the high individual variability of the investigated parameters within a population of modiolus. ACKNOWLEDGMENTS The authors are grateful to D.A. Anisimov for providing laboratory premises for research in the area of the mollusks collection (the Vityaz base) and to V.B. Durkin for carrying out underwater work. This work was supported by the FEFU Science Foundation (projects: 13-06-0318, m_a; 14-08-01-4_i) in parts of the studies on the nanoparticles properties and the histological analysis, and by the Russian Science Foundation (project 14-50-00034) in parts of the hemolymph research. REFERENCES 1. A. Vianello, A. Boldrin, P. Guerriero, et al., “Microplastic particles in sediments of Lagoon of Venice, Italy: first observations on occurrence, spatial patterns and identification,” Estuarine, Coastal Shelf Sci. 130, 54– 61 (2013). 2. J. A. Ivar Do Sul and M. F. Costa, “The present and future of microplastic pollution in the marine environment (review),” Environ. Pollut. 185, 352–364 (2014). 3. M. N. Moore, “Do nanoparticles present ecotoxicological risks for the health of the aquatic environment?,” Environ. Int. 32, 967–976 (2006). 4. A. Baun, N. B. Hartmann, K. Grieger, and K. O. Kusk, “Ecotoxicity of engineered nanoparticles to aquatic invertebrates: a brief review and recommendations for future toxicity testing,” Ecotoxicology 17 (5), 387–395 (2008). 5. K. Tiede, M. Hassellov, E. Breitbarth, et al., “Considerations for environmental fate and ecotoxicity testing to support environmental risk assessment for engineered nanoparticles,” J. Chromatogr. 1216 (3), 503– 509 (2009). 6. L. Canesi, C. Ciacci, R. Fabbri, et al., “Bivalve mollusks as a unique target group for nanoparticle toxicity,” Marine Environ. Res. 76, 16–21 (2012). 7. J. E. Ward and D. J. Kach, “Marine aggregates facilitate ingestion of nanoparticles by suspension feeding bivalves,” Marine Environ. Res. 68, 137–142 (2009).

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Translated by G. Naumova

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