Effects of titanium dioxide nanoparticles on red swamp crayfish, Procambarus clarkii: Bioaccumulation, oxidative stress and histopathological biomarkers

Egyptian Journal of Aquatic Research 45 (2019) 11–18

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Effects of titanium dioxide nanoparticles on red swamp crayfish, Procambarus clarkii: Bioaccumulation, oxidative stress and histopathological biomarkers Mahmoud Abd El-Atti a, Mahmoud M.A. Desouky a,⇑, Amaal Mohamadien a,b, Radwa M. Said a a b

Department of Zoology, Faculty of Science, Zagazig University, Zagazig, Egypt Department of Biology, Faculty of Science, Tai’f University, Saudi Arabia

a r t i c l e

i n f o

Article history: Received 18 November 2018 Revised 6 January 2019 Accepted 13 January 2019 Available online 25 January 2019 Keywords: Red swamp crayfish Procambarus clarkii Titanium dioxide nanoparticles Antioxidant parameters Histopathology Hepatopancreas Gills

a b s t r a c t The widespread use of titanium dioxide nanoparticles (TiO2 NPs) in many applications has led to its significant release to aquatic systems. Hence, the present study was performed to evaluate effects of TiO2 NPs on crayfish Procambarus clarkii, which is often used as a bioindicator of water pollution. Adult male specimens were treated with 25, 125, and 250 mg/l of TiO2 NPs for 28 days. Mortalities were 0, 3.3, and 10% for animals treated with 25, 125, and 250 mg/l of TiO2 NPs, respectively. The level of titanium bioaccumulation in different tissues was as follows, gills > hepatopancreas > green glands > muscles. TiO2 NPsexposed crayfish showed significant increase in the levels of catalase, glutathione-S-transferase, glutathione peroxidase, gamma-glutamyl transferase, glutathione, and metallothioneins in the hepatopancreas. Exposure of the crayfish to 250 mg/l TiO2 NPs caused severe histopathological alterations. In the hepatopancreas, the most prominent pathological changes were tubular disruption and inflammatory infiltration. Under transmission electron microscope (TEM), ruptured microvilli, deformed mitochondria and fragmentation of rough endoplasmic reticulum (RER) were observed. In the gills, swelling of gill lamellae, disorganization and degeneration of epithelial cells were noted under the light microscope (LM). Under electron microscope (EM), gills of crayfish treated with 250 mg/l TiO2 NPs showed vacuolation, dense granules, and diminution in the number of apical plasma membrane infoldings. In conclusion, TiO2 NPs caused alteration in antioxidant activities and severe histopathological changes. Ó 2019 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction The production and utilization of nanomaterials for a wide variety of uses have resulted in their potential environmental impact, hence becoming an important topic of academic and industrial research. Nanomaterials have distinctive features such as very small size, high surface area, good mobility, penetration ability and high reactivity (Sahooli et al., 2012; Khashan et al., 2016). Metal oxide nanoparticles are of special concern due to their extensive industrial applications in solar cells and photocatalytic water purification systems (Usui et al., 2004) resulting in the potential contamination of both terrestrial and aquatic ecosystems. The extensive applications of nanomaterials are likely to result in an increase in their discharge into aquatic environments

Peer review under responsibility of National Institute of Oceanography and Fisheries. ⇑ Corresponding author. E-mail address: [email protected] (M.M.A. Desouky).

throughout manufacturing, usage and disposal (Mueller and Nowack, 2008). Once in the aquatic ecosystem, these nanomaterials may interfere with physiological processes, posing a potential risk to aquatic organisms. Hence, it is important to regulate the safe limits of nanomaterials for both environmental and occupational safety. Furthermore, the development of sensitive and effective analytical approaches for monitoring of nanomaterials in the environment becomes necessary (Miranda et al., 2016). Titanium dioxide nanoparticles (TiO2 NPs) are produced plentifully and used widely in industrial and consumer goods owing to their high stability, anticorrosion, and photocatalytic properties (Hao et al., 2009; Vena et al., 2017). TiO2 NPs are generally used in papers, plastics, paints, medicines, sunscreens, pharmaceuticals, food products, and toothpaste (Wang et al., 2006; Trouiller et al., 2009) due to their UV absorption and photocatalytic sterilizing properties. TiO2 NPs also show antibacterial properties under UV light irradiation (Montazer et al., 2011). However; research indicates that TiO2 NPs may show probable toxicity. TiO2 NPs caused high cytotoxicity in cell culture at high concentration (Zhao et al.,

https://doi.org/10.1016/j.ejar.2019.01.001 1687-4285/Ó 2019 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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2009) and hence such NPs released into the environment may also be toxic. The Red swamp crayfish, Procambarus clarkii is a native species from Mexico and the USA. It was introduced to Egypt in the early 1980’s for aquaculture, but escaped and spread through the River Nile (Ibrahim et al., 1995). Not only such invasive species harm the native biota but may also transfer accumulated contaminants _ ´ ski through the food chain to humans who consume it (Struzyn et al., 2013). Crayfish are often used as a bioindicator of water pollution and are considered as a good model organism for ecotoxicological studies of pollutant-induced changes (Serrano et al., 2000). Therefore, the aim of the current study was to examine the possible effects of TiO2 NPs on the red swamp crayfish, P. clarkii and to critically evaluate its utility as a bioindicator for TiO2 NPs toxicity in the aquatic ecosystem. Materials and methods Experimental design TiO2 NPs (crystalline form, average size 55 ± 8 nm, derived from the crystalline mineral rutile in crystal and with a purity >99%) was purchased from Lab Chemical Trading Co., Cairo, Egypt. Suspensions of TiO2 NPs were dissolved in distilled water and dispersed with waterbath sonicator for 20 min. The stock solution was diluted to desired concentrations before treatment. The prepared concentrations were 25, 125 and 250 mg/l. Concentrations used in this paper were similar to other ecotoxicological studies, such as Farlow. 2014. Red swamp crayfish, P. clarkii (about 120 individuals) were collected with a 0.7 cm mesh size diagonal net from the Bany-Helal irrigation Canal, Zagazig, Sharkia Governorate, Egypt, in August 2017. The collected specimens were transferred to the laboratory and maintained in glass aquaria (40
40
40 cm). The average water quality parameters were: total ammonia 0.28 ± 0.03; temperature, 27.8 ± 0.28; dissolved oxygen, 6.05 ± 0.35; pH, 7.6 ± 0.19. All these ranges are within the acceptable ranges according to Boyd (1984). Suffocation was avoided by ensuring a water depth of 7–10 cm and the specimens were exposed to air room temperature. Mature males weighing 25–34 g and 9–12 cm long were acclimated to indoor laboratory conditions for one week. Ten mature animals were stocked inside each aquarium and treated with either 0.0 (control), 25, 125, or 250 mg/l TiO2 NPs for 28 days in triplicates. Crayfish were fed with carrot and minced meat up to apparent satiation thrice a day at 9:00, 13:00, and 17:00 h. Water temperature was 25 °C and light regime was maintained to be 12:12 h using light tubes. The water was renewed daily with aerated tap water. At the end of the experiment, crayfish were collected from each aquarium and counted. Mortality rate (%) was calculated as follows:

and Remediation of Hazardous Solid Wastes, National Research Centre, Egypt. Calibration curves were performed using diluted solutions prepared from 1000 mg/l of element standard solutions from Merck, USA according to NIST. Biomarkers of antioxidant parameters Activities of catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST), gamma glutamyl transferase (GGT), value of glutathione (GSH) and metallothioneins (MTs) were determined according to the corresponding assay kit protocol of the manufacturer (Bio Vision-Milpitas, CA, USA). Light microscopy preparations Specimens of hepatopancreas and gills of both control and TiO2 NPs-exposed animals were dissected out, fixed in 10% formalin and dehydrated through an ascending series of ethanols, then, embedded in paraffin wax, sectioned at 4–6 lm thick and stained with hematoxylin and eosin. Transmission electron microscopy preparations Small pieces of hepatopancreas and gills were dissected out from control and TiO2 NPs-exposed animals. The dissected organs were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer and post-fixed in 1% OsO4 for 60 min. Samples were then dehydrated in an ascending ethanol series and finally embedded in Araldite Epon. Ultra-thin sections were contrasted with uranyl acetate and lead citrate stains, and examined using a Jeol Transmission Electron Microscope at the Regional Centre for Mycology and Biotechnology, El-Azhar University, Nasr city, Cairo, Egypt. Statistical analysis Statistical analysis was carried out using the SPSS version 20 (SPSS, Richmond, VA, USA) as described by Dytham (2011). Oneway ANOVA was used to compare the effect of TiO2 NPs concentrations. Two-way ANOVA was used to examine the effect of TiO2 NPs concentrations and exposure periods in different organs. Significant differences among treatments at P < 0.05 were performed using Duncan test as a post-hoc test. Results Crayfish mortality Percentage of dead crayfish after exposure to 125 and 250 mg/l of TiO2 NPs were 3.3% and 10%, respectively. No mortality was recorded after exposure to 0.0 (control) or 25 mg/l of TiO2 NPs for 28 days.

Mortality rate ð%Þ ¼ 100 ðanimal No: at the start-animal No: at finalÞ=animals No: at the start:

Bioaccumulation analysis The hepatopancreas, gills, green glands, and muscles were dissected out, then oven-dried for 72 h at 80 °C. The tissues were ground to a fine powder using a pestle and mortar and weighted to obtain 0.1 g. Tissues were placed in an Xpress vessel containing 5 ml of acid digestion mixture (3 ml HNO3: 2 ml HCIO4). Chemical digestion was performed using a MARS Xpress Microwave (Tsoumbaris, 1990). Measurements were conducted by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES, Agilent 5100) at the Analysis and Consultation Unit, Domain of Evaluation

Bioaccumulation of TiO2 NPs in selected organs of crayfish The accumulation and concentrations of Ti in various tissues of TiO2 NPs-exposed animals for 28 days is shown in Fig. 1 and Table 1. Titanium accumulation was significantly affected by TiO2 NPs concentrations, exposure period, and the target organs. Moreover, Ti accumulation displayed time and dose-dependent patterns in all tissues. The highest Ti accumulation was recorded in gills followed by hepatopancreas, green gland, and muscles, respectively. The highest Ti residues were retained in gills and hepatopancreas after 7 days of exposure to 250 mg/l of TiO2 NPs (44.60 and 6.90 mg/g dry weight, respectively). The amount of Ti in both organs increased significantly (P < 0.05) to 133.31 and 26 mg/g dry weight respectively at the end of the exposure period (Fig. 1a

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Fig. 1. Accumulation of Ti (mg.g1 dry weight) in a): hepatopancreas, b): gills, c): muscles and d): green glands of the red swamp crayfish P. clarkii exposed to different concentrations of TiO2 NPs over 28 days. Values are mean of 3 samples ± SD. Different alphabetical superscripts for each parameter among different concentrations are statistically significant at P < 0.05. Mean values with the same alphabetical superscripts are not significant at P > 0.05.

Table 1 Concentrations of titanium (mg/g dry weight) accumulated in selected organs of red swamp crayfish P. clarkii after exposure to different concentrations of TiO2 NPs at different exposure periods. Organ

The used concentrations (mg/l)

Concentrations of TiO2 NPs (lg/g dry w.) accumulated in different organs of P. clarkii at different exposure periods (days) 7

14 a

21 a

28 a

Muscles

Control 25 mg/l 125 mg/l 250 mg/l Two-way ANOVA Time of exposure TiO2 NPs concentration Concentrations
Time of exposure

0.1 ± 0.00 0.15 ± 0.07a 0.25 ± 0.12a,b 0.4 ± 0.14c

0.05 ± 0.00 0.2 ± 0.14a 0.4 ± 0.14c 0.4 ± 0.28c F-Value 3.35 26.63 0.78

0.05 ± 0.00 0.35 ± 0.21b 0.35 ± 0.17b 0.55 ± 0.21c P-value 0.036 0.000 0.639

0.1 ± 0.00a 0.4 ± 0.14c 0.55 ± 0.07c 0.8 ± 0.28c

Hepatopancreas

Control 25 mg/l 125 mg/l 250 mg/l Two-way ANOVA Time of exposure TiO2 NPs concentration Concentrations
Time of exposure

0.43 ± 0.06a 0.9 ± 0.42b 4.1 ± 0.57b 6.91 ± 0.13b

0.35 ± 0.15a 1.05 ± 0.78a 6.05 ± 0.07b 5.65 ± 0.92b F-Value 41.67 207.46 37.60

0.15 ± 0.04a 1 ± 0.04a 6.85 ± 1.34b 9.25 ± 2.48b,c P-value 0.000 0.000 0.000

0.4 ± 0.22a 1.25 ± 0.92a 5.5 ± 0.91b 26 ± 4.38c

Gills

Control 25 mg/l 125 mg/l 250 mg/l Two-way ANOVA Time of exposure TiO2 NPs concentration Concentrations
Time of exposure

1.34 ± 0.18a 9.2 ± 1.7b 36.55 ± 2.9b 44.80 ± 5.37b

1.45 ± 0.35a 9.5 ± 3.25b 45.7 ± 3.08b 47.17 ± 2.74b F-Value 261.81 1227.13 115.99

1.1 ± 0.14a 12.26 ± 2.63b 59.40 ± 1.13c 47.75 ± 9.12c P-value 0.000 0.000 0.000

1.3 ± 0.35a 18.08 ± 1.31b 72.81 ± 2.39d 133.31 ± 2.68d

Green glands

Control 25 mg/l 125 mg/l 250 mg/l Two-way ANOVA Time of exposure TiO2 NPs concentration Concentrations
Time of exposure

0.09 ± 0.03a 0.4 ± 0.14a 0.5 ± 0.28a 2.05 ± 0.35b,c

0.01 ± 0.00a 0.5 ± 0.28a 0.8 ± 0.28a 2.5 ± 0.71b,c F-Value 4.84 7.57 3.39

0.01 ± 0.00a 0.95 ± 0.21a 0.9 ± 0.42a 2.65 ± 0.64b,c P-value 0.009 0.001 0.008

0.01 ± 0.00a 1.25 ± 0.49b 1.7 ± 0.28b 6.1 ± 2.55c

-Data are represented as means of 3 samples ± SD -Mean values with different letters are significantly different at P < 0.05 (Two way ANOVA and subsequent post hoc multiple comparison with Duncan’s Multiple Range Test) -Mean values with the same alphabetical superscripts are not statistically significant at P > 0.05.

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& b). Titanium accumulation in muscles was 0.15, 0.25, and 0.4 lg/ g on day 7, then increased to be 0.4, 0.55, and 0.8 lg/g dry weight after exposure to 25, 125 and 250 mg/l TiO2 NPs for 28, respectively. Titanium accumulation in green glands was 0.4, 0.5 and 2.05 lg/g dry weight on day 7, then increased to 1.25, 1.7, and 6.1 lg/g dry weights after exposure to 25, 125, and 250 mg/l TiO2 NPs for 28 days respectively. Titanium accumulation in muscle and green glands was therefore low compared to the hepatopancreas and gill tissues (Fig. 1c & d).

degenerations of connective tissue between the tubules were observed (Plate 1e). In addition, conspicuous disruption of tubules of the hepatopancreas with lysis of their epithelial cells, cellular atrophy, lumen dilatation and extensive vacuolation were also noted (Plate 1f). The treatment of red swamp crayfish with 250 mg/l TiO2 NPs resulted in marked deterioration of the tubules of the hepatopancreas including, lysis of epithelial cells and appearances of pyknotic nuclei. The connective tissue showed signs of degenerations and displayed in some parts a high inflammatory infiltration (Plate 1g & h). The TEM examination showed many ultrastructural alterations in hepatopancreatic cells of red swamp crayfish, P. clarkii treated with 250 mg/l of TiO2 NPs for 28 days. These alterations include ruptured microvilli and deformed mitochondria with cisternae (Plate 2e). The nucleus of absorptive cell became pyknotic; heterochromatin appeared as dense masses distributed in the nucleoplasm and as electron dense aggregates along the inner nuclear envelope, which had an irregular shape. The cytoplasm of absorptive cells lost its identity, and became highly vacuolated with cellular debris (Plate 2f). The nucleus of secretory cell became pyknotic (Plate 2g). The fragmentation of RER and necrotic nuclei was also noted in the fibrillar cell (Plate 2h).

Effect of TiO2 NPs on antioxidant activity As shown in Table 2, crayfish treated with 25 mg/l of TiO2 NPs showed no significant changes (P > 0.05) in the levels of CAT, GPX, GST, GGT, GSH and MTs but their levels were significantly (P < 0.05) higher at 125 and 250 mg/l of TiO2 NPs as compared to unexposed controls. The activities of GPX, GST, and GGT were increased by 11.9–14.8%, 45.4–48.2%, and 4.6–16.9%, respectively compared to control levels. Upon treatment with 250 mg/l of TiO2 NPs; levels of CAT, GSH and MTs increased by 22.9%, 25.5% and 52.9%, respectively compared to controls. Histopathology of hepatopancreas

Histopathology of gills Hepatopancreas of control crayfish The hepatopancreas is formed of numerous tubules separated by connective tissues (Plate 1a). Each tubule has a central lumen and its wall is formed of three types of cells: absorptive cells, secretory cells and fibrillar cells (Plate 1b). The absorptive cell is the most abundant cell type with basely located nucleus and the cytoplasm contains an apical small vacuole. The secretory cell has a basely located nucleus and a large central vacuole filled with acidophilic secretions. The fibrillar cell is small, darkly stained with a larger basely located nucleus (Plate 1b). Electron micrographs showed that the apical surface of absorptive cell has numerous microvilli (Plate 2a). The basal portion of absorptive cells contains a spherical nucleus, Golgi complex, and parallel tubules of RER (Plate 2b). The secretory cells contain very thin layer of perinuclear RER and a small number of mitochondria (Plate 2c). The fibrillar cell has a basely located nucleus. The RER is massive and fills almost all of the cells (Plate 2d).

Gills of control crayfish Photomicrographs of control gill lamellae are shown in Plate 3a & b. The gill is formed of uniform arrangement of gill lamellae with uniform intralamellar spaces (Plate 3a). The gill lamellae are enclosed by a thick cuticle, which covers the whole outer surface. Underlying the cuticle is a continuous layer of epithelial cells. The cells narrow towards the intralamellar space extending later to contain the nucleus (Plate 3b). The TEM examinations of gills showed that the fibrous basement membrane formed the basal boundary of the filament epithelium of the gills. The epithelium consisted of a layer of cells, which may be quite simple with large vacuoles or more complex with highly folded basal and apical plasma membranes, and covered with a cuticle (Plate 4a and b). The apical plasma membrane of these cells was highly folded in areas where basal folding was prevalent, and outward extension of the apical plasma membrane may approach to the cuticle (Plate 4a). The apical side of the epithelial cell was covered with irregular microvilli (Plate 4b).

Hepatopancreas of TiO2 NPs-exposed crayfish Light microscopical examination showed many cytological changes in hepatopancreatic tubules of crayfish treated with 25, 125, and 250 mg/l of TiO2 NPs for 28 days. After treatment with 25 mg/l of TiO2 NPs, the presence of deeply stained pyknotic nuclei and lysis of their epithelial cells in some parts of the tubules were noticed (Plate 1c & d). After treatment with 125 mg/l of TiO2 NPs,

Gills of TiO2 NPs-exposed crayfish Light micrographs of gills of red swamp crayfish treated with 25 mg/l of TiO2 NPs exhibited swelling and disarrangement of the gill lamellae (Plate 3c). The epithelial cells of the gill lamellae became disorganized (Plate 3d). After treatment with 125 mg/l of

Table 2 Antioxidant activity in hepatopancreas of red swamp crayfish, P. clarkii, exposed to different concentrations of TiO2 NPs for 28 days. Parameter

CAT (MU/mg) GPX (MU/mg) GST (MU/mg) GGT (MU/mg) GSH (mg/mg) MTs (mg/mg)

Concentrations of TiO2 NPs (mg/l) 0.0 Control

25

125

250

2.61 ± 0.21a 22.9 ± 11.11a 1.41 ± 0.36a 11.75 ± 0.08a 0.47 ± 0.06a 1.21 ± 0.06a

2.64 ± 0.02a 23.61 ± 0.57a 1.69 ± 0.09a 11.89 ± 0.37a 0.49 ± 0.13a 1.22 ± 0.05a

2.71 ± 0.21a 25.63 ± 0.93b,c 2.05 ± 0.15b 12.29 ± 0.83b 0.52 ± 0.05b 1.53 ± 0.29a,b

3.21 ± 0.15b 26.30 ± 0.64c 2.09 ± 0.06b 13.74 ± 0.11b 0.59 ± 0.06c 1.85 ± 0.18b

F-value

P-value

5.57 7.04 5.01 10.39 15.16 12.07

0.039 0.04 0.04 0.02 0.012 0.018

-Each value is mean of 4 samples ± SD -Means values with different alphabetical superscripts at each row differed significantly at P < 0.05 (one way ANOVA and subsequent post hoc multiple comparison with Duncan‘s Multiple Range Test). -Mean values with the same alphabetical superscripts are not statistically significant at P > 0.05. CAT: Catalase; GPX: Glutathione peroxidase; GST: Glutathione s transferase; GGT: Gamma glutamyl transferase; GSH: Glutathione; MTs: metallothioneins.

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Plate 1. Photomicrograph of tubules of hepatopancreas of red swamp crayfish, P. clarkii. (a): T.S. of untreated tubules with normal cellular content (X100). (b): T.S. of untreated tubules showing different types of hepatopancreatic cells (X400). (c & d): T.S. of hepatopancreatic tubules after treatment with 25 mg/l TiO2 NPs for 28 days showing lysis of cells in some parts of the tubules (X 400). (e & f): T.S. of hepatopancreatic tubules after treatment with 125 mg/l TiO2 NPs for 28 days showing degenerations of connective tissue between the tubules and tubular disruption (X100 & 400), and (g & h): T.S. of hepatopancreatic tubules after treatment with 250 mg/l TiO2 NPs for 28 days showing deterioration of tubules (X100 & 400). AC: Absorptive cell; CL: Cell lysis; CA: Cellular atrophy; DT: Digestive tubules; FC: Fibrillar cell; HI: Haemocytes infiltration; L: Lumen; NC: Necrotic cells; PN: Pyknotic nuclei; RL: Reduced lumen; SC: Secretory cell; TD: Tubular disruption; YG: Yellow granules.

Plate 2. Electron micrographs of hepatopancreatic cells of red swamp crayfish, P. clarkii. (a): Apical portion of absorptive cell with numerous microvilli and number of mitochondria (X15000). (b): Basal portion of absorptive cell with Golgi complex, mitochondria, spherical nucleus, and RER (X8000). (c): Secretory cell (X12000). (d): Fibrillar cell with extensive RER (X5000). (e): Apical portion of absorptive cell after treatment with 250 mg/l TiO2 NPs for 28 days showing ruptured microvilli and deformed mitochondria (X15000). (f): Basal portion of absorptive cell after treatment with 250 mg/l TiO2 NPs for 28 days showing lytic and vacuolated cytoplasm (X8000). (g): Secretory cell after treatment with 250 mg/l TiO2 NPs for 28 days showing pyknotic nuclei (X8000), and (h): Fibrillar cell after treatment with 250 mg/l TiO2 NPs for 28 days showing fragmentation of RER and necrotic nuclei (X8000). CDB: Cellular debris; DM: Deformed mitochondria; FRER: Fragmentation of RER; GC: Golgi complex; LC: Lytic cytoplasm; L: Lumen; MI: Microvilli; M: Mitochondria; N: Nucleus; NN: Necrotic nuclei; PN: Pyknotic nuclei; RER: Rough endoplasmic reticulum; RMI: Ruptured microvilli; VA: Vacuolated cytoplasm.

TiO2 NPs, the tips of the gill lamellae seemed atypical with peculiar deformities. Epithelial cells became detached from the cuticle (Plate 3e). In some regions, epithelial cells became disorganized and others became necrotic, with necrotic and pyknotic nuclei (Plate 3f). Exposure to 250 mg/l of TiO2 NP caused disarrangement of lamellae with abnormal malformation of the tips (Plate 3g), and degeneration of epithelium, which became completely detached from the cuticle (Plate 3h).

Under TEM, it can be seen that the cells of gills treated with 250 mg/l TiO2 NPs became severely degraded with degenerated cellular debris and granules encapsulated within vacuoles in cytoplasm. Heterochromatin of the nuclei appeared as dense masses distributed in the nucleoplasm and as irregular clumps near the inner nuclear envelop (Plate 4c & d). Diminution in the number of apical plasma membrane infoldings and presence of dense granules were also noticed (Plate 4e).

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Plate 3. Histological sections of gill lamellae of red swamp crayfish P. clarkii. (a & b): L.S. of untreated gills showing uniform arrangement of lamellae with uniform intralamellar spaces and gill epithelial cells (X100 & 400). (c & d): L.S. of the gill lamellae after treatment with 25 mg/l of TiO2 NPs for 28 days showing swelling and disarrangement of the gill lamellae (X100 & 400). (e & f): L.S of the gill lamellae after treatment with 125 mg/l of TiO2 NPs for 28 days showing detaching of epithelial cells from the cuticle (X100 & 400), and (g & h): L.S. of the gill lamellae after treatment with 250 mg/l TiO2 NPs for 28 days showing necrosis of epithelial cell of lamellae and the cells became completely detached from the cuticle (X100 & 400). AM: Abnormal malformation of tips of lamellae; Cu: cuticle; DEGL: Degeneration of epithelial cells of gill lamellae; DGL: disorganization of gill lamellae; DC: detached epithelial cell from the cuticle, EC: Epithelial cell; GL: Gill lamellae; HC: Haemocyte; HP: Hyperplasia; IS: intralamellar spaces; LC: Lysis of cuticle; NN: Necrotic nuclei; PN: Pyknotic nuclei; SL: Swelling of gill lamellae.

Plate 4. Electron micrographs of the gills epithelia of red swamp crayfish P. clarkii. (a & b): Gills epithelia of untreated crayfish. Note the presence of microvilli and thick cuticle (X8000). (c, d & e): epithelial cells of gills treated with 250 mg/l of TiO2 NPs for 28 days. Note that epithelial cells became severely degraded with degenerated cellular debris and granules encapsulated within vacuoles in their cytoplasm (X 8000). APM: Apical plasma membrane; BM: Basement membrane; CU: cuticle; CDB: Cellular debris; DG: Dense granules; EDD; Electron dense deposits; MF: Dense membrane folding; M: Mitochondria; MI: Microvilli; N: Nucleus; V: Vacuole.

Discussion Due to the rapid development of nano-sized materials in industrial applications, a great deal of attention has recently been directed to their release into the aquatic ecosystem and their consequent toxicity to aquatic organisms. Nanoparticles would affect aquatic organisms via the uptake and accumulation in their bodies over time with subsequent gastrointestinal toxicity (Miranda et al., 2016). Titanium dioxide nanoparticles are produced in large

quantities and used widely owing to their high stability, photocatalytic properties and anticorrosion attributes (Hao et al., 2009). Red swamp crayfish, P. clarkii was used in the present study as bioindicator to determine the possible toxicity of TiO2 NPs in freshwater ecosystems. The current study showed that the order of Ti accumulation in different organs of the red swamp crayfish was gills > hepatopancreas > green glands > muscles tissues, and this accumulation showed a dose-dependent pattern. Moger et al. (2008) found that

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Ti accumulation increased in gills of rainbow trout, Oncorhynchus mykiss, treated with 5000 mg L1 TiO2 NPs. In the common carp, Cyprinus carpio, large amount of Ti was accumulated in the intestine, stomach, gill and muscle (Sun et al., 2007). Differences between organs in the degree of metal accumulation are mainly ascribed to the dissimilarities in the physiological role of each organ (Karuppasamy, 2004; Abdel-Tawwab and Wafeek, 2014). However, the relatively large amounts of Ti accumulation in the gills observed in this study might be due to the large surface area of the epithelium and the direct contact with water that renders them more predisposed to uptake of aqueous chemical pollutants (Nowrouzi et al., 2012; Maleki et al., 2015). Titanium also accumulates in the hepatopancreas which plays a vital role in pollutant storage, redistribution and detoxification process (Jaiswal and Sanojini, 1990), which may explain the significant accumulation of Ti in this vital organ. Muscles and green glands have the lowest amount of Ti accumulation because they were not in direct contact with pollutants and they are also inactive organs in accumulating metals (Alam et al., 2002). Assaying antioxidant enzymes can be used as a potential biomarker for contaminant-mediated oxidative stress (Livingstone, 2001). The CAT provides the first line of defence against reactive oxygen species and is used as a biomarker of oxidative stress (Fang and Zheng, 2002). In the present study, CAT activity showed a significant rise in the hepatopancreas after treatment with different concentrations of TiO2 NPs. Such increases in CAT activities may be explained as a response to the increased H2O2 levels and superoxide anions (John et al., 2001). These results, however, differ from Hao et al. (2009) who reported that CAT level decreased in the liver of common carp, C. carpio, after exposure to 100 and 200 mg/l of TiO2 NPs for 6 days. The GST plays a significant role in protecting cells and tissues from oxidative stress (Slatinská et al., 2008). Ana et al. (2015) found that GST activity increased in the intestine of the goldfish Carassius auratus when treated with 100 mg/l TiO2 NPs for 14 days. These results disagree with the findings of Disner et al. (2017) who reported that GST decreased in liver of the wolf fish Hoplias intermedius after treatment with nano-TiO2 for 4 days; however, the exposure concentrations (0.1 and 1 mg/l) were much lower. The GPX is one of the principal antioxidant enzymes that defend organisms against hydro peroxides (Orbea et al., 2002). The current study showed that GPX activity increased significantly in hepatopancreas of the red swamp crayfish, P. clarkii, after exposure to TiO2 NPs. Özgür and Rifat (2018) found that GPX activity increased in the gills of Nile tilapia Oreochromis niloticus when treated with 5 mg/l TiO2 NPs for 14 days. These increases in GPX may be a function of the protective role of GPX against cells damage induced by oxyradical production. The GGT plays a vital role in the production and breakdown of glutathione (GSH) and xenobiotic detoxifications where GSH is the most significant molecule in protecting the cells from oxidative _ ´ ski et al., 2013). In the current stress induced damage (Struzyn study, both GGT and GSH levels were significantly increased in hepatopancreas of red swamp crayfish P. clarkii after exposure to TiO2 NPs. These increases may be due to their defensive role in counteracting the effects of cellular oxidative stress (Monteiro et al., 2006). Metallothioneins (MTs) play a vital role in the detoxification, metabolism, and homostasis of toxic metals (Abdel-Tawwab and Wafeek, 2014). The present study showed that MT level was increased in TiO2 NPs-exposed crayfish. This increase may explain the role of MTs in the defence against toxicity of tested nanoparticles (Kelly, 1998). Similarly, Martín-Díaz et al. (2006) reported a rise in the MTs level in the hepatopancreas of crayfish, P. clarkii exposed to cadmium.

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In the current study, marked histopathological changes in different organs of red swamp crayfish induced by TiO2 NPs were recorded. Low TiO2 NPs concentrations caused slight alterations in the hepatopancreatic tubules such as the appearance of deeply stained pyknotic nuclei and the lysis of their epithelial cells in some parts of the tubules. Higher concentrations of TiO2 NPs resulted in the lysis of epithelial cells, cellular atrophy and extensive vacuolation. Similar alterations were observed in the liver of common carp (C. carpio) exposed to TiO2 NPs (Hao et al., 2009; Borhan et al., 2016). These histological alterations may be ascribed to direct toxic effects of toxicants on hepatopancreatic cells because it is the chief site of detoxification and hence a sink for potential toxins such as trace metals (Jaiswal and Sanojini, 1990). Under TEM, TiO2 NPs produced severe degeneration of cellular organelles, including ruptured microvilli, deformed mitochondria, pyknotic nuclei, lytic and vacuolated cytoplasm, and fragmented endoplasmic reticulum. The disintegration of RER might be a result of hyperactivity before cell necrosis (Roncero et al., 1992). Lysis of the cytoplasm may result of the interaction of nanoparticles with enzymes resulting in oxidative stress and reactive oxygen species formation, which may cause necrosis of cells (Chio et al., 2010). The crustacean gill is an important organ as it plays a key role in diffusion, gas exchange, and osmoregulation. As uptake from water is the most significant route for most contaminants, gills represent a principal target organ and may be one of the first organs to display symptoms of subchronic toxicity. The current study reveals that histopathological alterations in the gill epithelium of the crayfish occurred upon treatment with different concentrations of TiO2 NPs. The most prominent changes were the swelling of gill lamellae and disorganization of epithelial cells at low concentrations. High TiO2 NPs concentrations caused degeneration of gill epithelia. Under TEM, many pathological alterations were also noticed including the presence of degenerated cellular debris and granules encapsulated within cytosolic vacuoles and diminution in the number of plasma membrane infoldings. Moreover, heterochromatin condensation and marginalization in the nuclei were noticed. Similar results were also described in the gills of two fishes; common carp C. carpio (Hao et al., 2009; Borhan et al., 2016) and juvenile Prochilodus lineatus (Miranda et al., 2016; Talita et al., 2018); after treatment with TiO2 NPs. These changes may be signs of either nanoparticles intake or adaptation to prevent contaminant’s entry through the gill surface (Olurin et al., 2006). Vacuolation of epithelial cells may increase the diffusion distance for respiratory gases and ions, which will probably affect the physiological processes in the gill. The osmoregulatory function of gills is disturbed due to reduction in the number of basal plasma membrane infoldings (Desouky et al., 2013). As a result of treatment with TiO2 NPs, vacuoles containing cytoplasmic debris possibly arise to digest the damaged cellular components. As suggested by Asztalos et al. (1988), focal development of empty vacuoles might be the starting point of a cellular autolysis process. Heterochromatin condensation and marginalization may be due to progressive inactivation of nuclear constituent (Braunbeck, 1990). The magnitude of the histopathological destruction in the present study in the different organs was in related to the Ti concentration determined in hepatopancreas and gills and seemed to be concentration-dependent.

Conclusion It can be concluded that TiO2 NPs are not lethal to red swamp crayfish at low concentrations over 28 days’ exposure period. It has potential bioaccumulated in different organs, and serious

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changes indicative of oxidative stress and severe histopathological changes were observed at high TiO2 NP concentrations. Therefore, these nanoparticles must be used carefully and their discharge into the aquatic ecosystem should be monitored and controlled. Moreover, the present study revealed the efficacy of red swamp crayfish, P. clarkii to be used as a bioindicator for TiO2 NPs toxicity.

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