Arsenic-induced genotoxicity in Nile tilapia (Orechromis niloticus); the role of Spirulina platensis extract

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1 Environ Monit Assess (2015) 187:751 DOI /s Arsenic-induced genotoxicity in Nile tilapia (Orechromis niloticus); the role of Spirulina platensis extract Alaa El-Din H. Sayed & Heba Allah M. Elbaghdady & Eman Zahran Received: 4 July 2015 /Accepted: 10 November 2015 # Springer International Publishing Switzerland 2015 Abstract Arsenic (As) is one of the most relevant environmental global single substance toxicants that have long been regarded as a carcinogenic and genotoxic potential. In this respect, we evaluated the cytogenetic effect of arsenic exposure in Nile tilapia (Oreochromis niloticus), in terms of erythrocyte alteration, apoptosis, and induction of micronuclei. Spirulina platensis (SP) is a filamentous cyanobacterium microalgae with potent dietary phytoantioxidant, anti-inflammatory, and anticancerous properties supplementation. The protective role of Spirulina as supplementary feeds was studied in Nile tilapia (O. niloticus) against arsenic-induced cytogenotoxicity. Four groups were assigned as control group (no SP or As), As group (exposed to water-born As in the form of NaAsO 2 at 7 ppm), SP1 (SP at 7.5 %+ As at the same level of exposure), and SP2 (SP at 10 %+ As at the same level of exposure). As-treated group had a significant increase in all cytogenetic analyses including erythrocyte alteration, apoptosis, and induction of micronuclei after 2 weeks with continuous increase in A. E.<D. H. Sayed (*) Zoology Department, Faculty of Science, Assiut University, Assiut, Egypt alaa_h254@yahoo.com H. A. M. Elbaghdady Department of Zoology, Faculty of Science, Mansoura University, Mansoura 35516, Egypt E. Zahran Department of Internal Medicine, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt response after 3 weeks. The combined treatment of Spirulina at two different concentrations of 7.5 and 10 % had significantly declined the induction of erythrocyte alteration, apoptosis, and micronuclei formation induced by arsenic intoxication. Keywords Sodium arsenite. Nile tilapia. RBC abnormalities. Micronucleus frequency. Spirulina platensis extract. Detoxification Introduction Aquaculture is currently one of the most rapidly growing production sectors in Egypt that offer fish and fish products for human consumption. Tilapia fish is one of the most important aquacultured species in Egypt and worldwide. Tilapia fish may be cultured in raceways, ponds, or net cages in open or coastal waters providing ideal conditions for aquaculture; hence, water quality is the major factor in aquaculture sustainability (Mokhtar et al. 2009). However, the current aquaculture practices promote threats of the environmental hazards. In this regard, heavy metal pollution in the aquatic ecosystem has been focused as a critical environmental issue; this is attributed to two main concepts, public health and the aquatic environment (Dural et al. 2007). Heavy metals enter the aquatic ecosystem mainly from effluents discharged from industries, sewage treatment plants, and drainage from urban and agricultural areas; these metals are of special concern due to their toxicity and potential to bioaccumulate at various trophic levels.

2 751 Page 2 of 10 Environ Monit Assess (2015) 187:751 They not only bring about morphological, physiological, and cytogenetical changes in the aquatic inhabitant but also cause mutagenic and carcinogenic reactions in living beings (Yadav and Trivedi 2009). Among the vast majority of heavy metal pollutants released into the aquatic environment, arsenic (As) has been recognized as one of the most relevant environmental global single substance toxicants (Gebel 2001). As exists in two forms, organic and inorganic ones; the inorganic particularly the trivalent (As +3 ) is the most toxic form (Ahmed et al. 2011). Higher As concentrations are often lethal while exposure to chronic sublethal concentration always results in increased risk of cancer and damage to various organs (Hughes 2002). As has long been regarded as a carcinogenic potential, genotoxic in both in vivo and in vitro conditions, besides causing chromosomal abnormalities (Lage et al. 2006). Several deleterious effects have been associated with As toxicity including induction of both reactive oxygen species (Barbosa et al.) and reactive nitrogen species (RNS) that are involved in the oxidation of cellular components particularly lipids, DNA, and proteins (Lushchak 2011); the increased levels of ROS can modulate gene expression and induce apoptosis while decreased levels can result in oxidative damage and cell death (Chen et al. 1998). Genotoxicity tests have been used successfully as indicators of the environmental pollutants, and fish is considered a good model for these types of analysis due to easier handling and monitored under controlled conditions, moreover low cost compared to other models (Barbosa et al. 2010). Using fish erythrocytes assure quick results and enables detecting clastogenic (that break chromosomes) and aneugenic agents (that induce aneuplody or abnormal chromosomal segregation), in vivo and in vitro trials (Galindo and Moreira 2009). The micronucleus (MN) test is the most used tool to evaluate chromosomal damage in different organisms and can be analyzed in different fish cell types; peripheral erythrocytes are one of them (Bolognesi et al. 2006). Other nuclear abnormalities that are found coincided with micronuclei may complement micronucleus scoring in routine genotoxicity surveys and give better wider view of genotoxic effects (Galindo and Moreira 2009). Many studies have been tried to alleviate the toxic effects of the heavy metals either through neutralization or metal hydroxide precipitation using variety of chemical compounds that succeeded in their reduction, but it costs a lot. Thus, there is a need for alternative approaches to managing heavy metals toxicity that are applicable to many toxicants and many species. In this regard, an interest in using Spirulina (SP), a group of blue-green algae, in diet has gained attention in recent years (Sayed and Fawzy 2014). SP is the nature s richest whole food source of antioxidants. Its powerful effects are related to their content of β-carotene, xanthophylls, zeaxanthin, echinenone, cryptoxanthin, and phycocyanin. β-carotene and others pigments have shown anti-oxidant and antiinflammatory activities and enhance pigmentation as well (Güroy et al. 2012). Phycocyanin might be the active ingredient responsible for the hypolipidemic activity; it shows activity on vegetable cell cultures with the production of secondary metabolites Banthocyanin^ that was able to inhibit the oxidative damage of the DNA. Its chlorophyll content also acts as a cleansing agent and detoxifying phytonutrient against the toxic substances (Sharma et al. 2012). Additionally, SP was found to alleviate the hepatic damage due to heavy metal exposure, drug misuses, and other inflammatory responses. The cell extract of SP has been shown anti-microbial activity against number of pathogens, SP incorporated in fish food for greater growth, and enhancing fertility and the immune response as well (Sánchez et al. 2003; Sayed and Fawzy 2014). To the best of our knowledge, few studies have been undertaken to evaluate the effect of SP against heavy metal pollution in aquatic ecosystem. Consequently, the present study was designed to assess the genotoxic effects of As in Nile tilapia erythrocytes using different test analyses including altered RBC morphology, apoptosis, and MN test and the role of SP dietary supplementation in ameliorating the induced toxicity. Material and methods Arsenic exposure Sodium arsenite (NaAsO 2 ;95%)manufacturedby MERCK ( , Art. 6287) was collected from the Beta Scientific Stores, Egypt, in original package. In the present study, fish were exposed to arsenic for 3 weeks. An exposure concentration corresponding to 7 ppm was selected according to previous toxicological assay in Oreochromis niloticus for 96-h LC50 value of Hwang

3 Environ Monit Assess (2015) 187:751 Page 3 of and Tsai (1993), which is equal to 1/10th of 96-h LC50 (71.7 ppm), were estimated and used for the experiment. Fresh daily stock solution of NaAsO 2 was prepared by dissolving the analytical grade NaAsO 2 in double-distilled water, then desired concentration in part per million (ppm) was prepared from the stock solution. Unexposed fish were maintained in separate tanks without arsenic under identical conditions. Fish Nile tilapia, O. niloticus were obtained from a private fish farm at Ad-Dakahliya province, Egypt. One hundred and twenty fish were distributed in 12 tanks in triplicate, giving 30 fish per treatment. The average weight of the fish was 85.91±3.03 g, and 3 % of their body weight was administrated daily as feed. They were maintained in aquarium tanks, which were provided with adequate aeration and under water internal power filter. About 50 % of the water was exchanged daily to maintain water quality. The fish were twice ad lib with a commercial diet at 25±2 C during the feeding period. Fish were acclimatized for 2 weeks and during this period, no clinical signs were ever observed. Spirulina platensis extract A dried powder of S. platensis cultures was purchased from the Institute of National Research Center, Cairo, Egypt. Preparation of diets and experimental design Four groups were assigned as control group (no SP or As), As group (exposed to water-born As at 7 ppm), SP1 (SP at 7.5 %+As at the same level of exposure), and SP2 (SP at 10 %+As at the same level of exposure). SP powder was added to the diet as described in Table 1.All ingredients were mixed with oil and then adding water until stiff dough resulted. Each diet was then extruded through a mincer. The resulting strands were shadowdried, broken up, sieved into pellets, and stored in plastic bags at 4 C until use. Nile tilapia fish (O. niloticus) weighted 75.91±3.03 g were distributed in the aquaria and triplicate tanks were assigned per dietary treatment (total N=120 fish). Fish were fed ad lib twice daily in equal rations at and h for 21 days. Water changed daily at about 50 % and the water quality was measured (ammonia, Table 1 Basic components of the basal diet (air dry basis, %) Ingredient nitrite, nitrate, ph, and dissolved oxygen) twice weekly via water quality test kits (Aquarium Pharmaceuticals, Inc.). The fecal matter and other waste materials were siphoned off daily to reduce ammonia content in water. Sample collections Control group SP1 group Twofishfromeachaquarium(sixfish/group)were sampled at week 2 and 3. Each aquarium was sampled one at a time; the fish were sedated with a low dose of buffered tricane (30 mg/l tricane+60 mg/l sodium bicarbonate), and each fish was then euthanized one at a time in a separate container having a high dose of buffered tricane (200 mg/l tricane+ 400 mg/l sodium bicarbonate). Blood samples were collected from caudal veins, and the smear of the blood was prepared on a glass slide (three slides/fish) and then was subjected to different staining to assess the analyses tested. Micronucleus test and erythrocyte alterations SP2 group Yellow corn Soya bean meal Fish meal Wheat bran Corn gluten meal Sunflower oil Vitamins and mineral premixes a Salt SP a Trace minerals and vitamin premixes were prepared to cover the levels of the microminerals and vitamins for tilapia fish as recommended by NRC (1993). Vitamin premix (IU or mg/kg diet); Vit. A 5000, Vit. D3 1000, Vit. E 20, Vit. k3 2, Vit. B1 2, Vit. B2 5, Vit. B6 1.5, Vit. B , Pantothenic acid 10, Folic acid 1, Biotin 0.15, and Niacin 30. Mineral mixture (mg/kg diet); Fe 40, Mn 80, Cu 4, Zn 50, I 0.5, Co 0.2, and Se 0.2 Micronucleus test and erythrocyte alteration blood smears were obtained by the caudal incision on clean grease-free microscopic slides after completion of a desired exposure. The smears were fixed in absolute

4 751 Page 4 of 10 Environ Monit Assess (2015) 187:751 methanol for 10 min after drying at room temperature. Slides were stained with hematoxylin and eosin. It was followed by dehydration in ascending grades of alcohol (30, 50, 70, and 90 % absolute). Finally, the slides were cleared in xylene and permanently mounted by DPX (Pascoe and Gatehouse 1986). Many slides were selected on the basis of staining quality, then coded, randomized, and scored blindly. In each group, 10,000 cells (a minimum of 1000 per slide) were examined (Al-Sabti and Metcalfe 1995) at400 objective micronucleated and morphologically altered erythrocytes in separate studies. The established criteria for identifying MN (Schmidt 1975) were strictly followed to ensure authentic scoring. The morphologically altered erythrocytes were according to the description of (Mekkawy et al. 2011). Apoptosis detection Apoptotic erythrocytes were detected using acridine orange stain (Cat. No. A1031, Life Technologies, 5791 Van Allen Way Carlsbad, CA 92008, USA). The modified protocol according to Darzynkiewicz (1990) used to detect the apoptosis in RBCs was as, after the preparation of blood smears on clean glass slides, the slides were washed in 1X phosphate buffer saline (PBS) (ph=7.2). AO buffer (17 μg/l acridine orange in 1X PBS buffer) was added to the slides for 30 min in the dark. Decolorization process as washing the slides every 30 min with 1X PBS for four times. Fixation was in paraformaldehyde 4 % for 5 min. Finally, observation cells were under a Zeiss Axioplan2 fluorescence microscope (200 ). Statistical analysis Statistical analysis was performed with the statistical software SPSS 17 (SPPS Inc. Chicago, IL, USA). The effects of different treatments on induction of MN, altered erythrocytes, and apoptosis were tested using one-way ANOVA followed by Duncan s multiple-range test. The results were expressed as mean±sd. The P values <0.05 were considered significant. Ethical statement All experiments were carried out in accordance with the Egyptian laws and university guidelines for the care of experimental animals. The protocol for this work was described in compliance with the guidelines of the ethical committee of Assiut University and Mansoura University. Results Induction of MN The MN induction showed a significant increase in As-treated group compared to the other groups after 2 and 3 weeks of Spirulina feeding. A significant decreasewasobservedinbothsp1andsp2groups compared to the As-treated group. After 3 weeks, the micronucleated cell percentage showed a nominal increase in As-treated group than after 2 weeks. There were no significant differences between the SP1 and SP2 or between them and the control one after 2 weeks; only, there was a significant increase in SP1 group compared to the control. There were significant differences between the control one and SP1 group and between the control one and SP2 group after 3 weeks (Fig. 1). Induction of erythrocyte alterations The results showed that after 2 and 3 weeks, a significant increase in deformed cell percentage was observed in As-treated group compared to the other groups. A significant decrease was observed in both SP1 and SP2 groups compared to the As-treated group. After 3 weeks, the deformed cell percentage showed a nominal increase in As-treated group than after 2 weeks. There were significant differences between the SP1 and SP2 or between them and the control one after 2 weeks (Fig. 2). Also, the presented data in Fig. 2 indicate the significant differences between SP1 group and the control one and between SP2 group and the control one after 3 weeks. There was a significant increase in SP1 group compared to the control. The blood smear of control tilapia and other groups showing normal erythrocytes (Er) and important alterations in erythrocytes, such as acanthocytes, tear drop-like cells, sickle cells, and enucleated erythrocytes are indicated by arrows (Figs. 4 and 5).

5 Environ Monit Assess (2015) 187:751 Page 5 of Fig. 1 MN induction levels in Nile tilapia fed with 7.5 and 10 % SP (mean±se) under NaAsO2 exposure by a rate (7 ppm) for 21 days. N=6 fish per sampling time/group. Values with different letters are significantly different between and within groups at weeks 2 and 3. As NaAsO2 only, SP1 As with Spirulina at 7.5 %, SP2 As with Spirulina at 10 %. Different letters a, b, c, and d indicate there is a significant difference at p 0.05 Induction of apoptosis After 2 and 3 weeks, significant increase in apoptotic cell percentage was observed in As-treated group compared to the other groups. A significant decrease was observed in both SP1 and SP2 groups compared to the As-treated group. The apoptotic cell percentage showed a nominal increase in As-treated group than after 2 weeks. There were significant differences between the SP1 and SP2 or between them and the control one after 2 and 3 weeks (Fig. 3). The blood smear of control tilapia and other groups showed normal erythrocytes (Er) and different micronucleated cells (Mn) with variation either in periphery or located near the main nucleus of erythrocytes (Figs. 4 and 5). The blood smear of control tilapia and other groups showed different apoptotic cells (AC) (Figs. 4 and 5). Discussion Using fish biomarkers as indicators in the ecotoxicological studies is of great concern that warrants early detection of the aquatic environmental problems (Nwani et al. 2010). MN are cytoplasmic chromatin-containing bodies formed as a result of chromosome breakage due to unrepaired or misrepaired DNA lesions, or chromosome malsegregation due to mitotic malfunction. They also appear as separate, small nuclei, in addition to main nucleus, in the cytoplasm of daughter cells (maturing erythrocytes). These events may be induced by oxidative stress, exposure to clastogens or aneugens, genetic defects in cell-cycle checkpoint and/or DNA repair genes, and by deficiencies in nutrients required as cofactors in DNA metabolism and chromosome segregation. Thus, micronuclei are considered as a biomarker of chromosomal instability, integrating acquired mutations and genetic susceptibility (Iarmarcovai et al. 2008). In the current study, all cytogenetic analyses including altered shaped erythrocytes, apoptotic erythrocytes, and MN formation were significantly peaked in the Astreated group compared to the other groups. While upon the feeding of SP, all investigated analyses were significantly declined again either at 7.5 or 10 % after 2 weeks; Fig. 2 Altered erythrocytes percentage in Nile tilapia fed with 7.5 and 10 % SP (mean±se) under NaAsO2 exposure by a rate (7 ppm) for 21 days. N=6 fish per sampling time/group. Values with NaAsO2 only, SP1 As with Spirulina at 7.5 %, SP2 As with Spirulina at 10 %. Different letters a, b, c,andd indicate there is a significant difference at p 0.05

6 751 Page 6 of 10 Environ Monit Assess (2015) 187:751 Fig. 3 Apoptosis percentage in Nile tilapia fed with 7.5 and 10 % SP, under NaAsO2 exposure by a rate (7 ppm) for 21 days (mean±se). N=6 fish per sampling time/group. Values with different letters are significantly different notably, feeding SP at 10 % was nominally near the level detected in the control group. The genotoxic potential of arsenic has also been observed by (Ahmed et al. 2011) in terms of the induction of DNA damage in tilapia exposed to three different concentrations (3, 28, and 56 ppm) of arsenic for 192 h in liver, gills, and blood tissues. The 4-day exposure of fish to 3 ppm of NaAsO2 increased the MN frequency between and within groups at weeks 2 and 3. AsNaAsO2 only, SP1 As with Spirulina at 7.5 %, SP2 As with Spirulina at 10 %. Different letters a, b, andc indicate there is a significant difference at p 0.05 up to 2.2 %, exposure to 28 ppm to 4.4 %, and the treatment of fish with 56 ppm of NaAsO2 increased the MN frequency to 5.8 %. Similar observations have also been made by Ramírez and García (2005) in zebra fish; Danio rerio, exposed to arsenic revealed that both duration and dose dependence increase in the induction of MN in gill cells. Vuyyuri et al. (2006)have also reported the genotoxic potential of arsenic by Fig. 4 Blood film showing the rounded shape of the nucleated erythrocytes (Er), micronucleus (Mn), apoptotic cells (AC), and erythrocyte alterations (arrows) in Nile tilapia fed with 7.5 and 10 % SP, under NaAsO2 exposure by a rate (7 ppm) for 2 weeks. N=6 fish per sampling time/group. a Control. b As only, c SP1, As with Spirulina at 7.5 %. d SP2, As with Spirulina at 10 % (400 )

7 Environ Monit Assess (2015) 187:751 Page 7 of Fig. 5 Blood film showing the rounded shape of the nucleated erythrocytes (Er), micronucleus (Mn), apoptotic cells (AC), and erythrocyte alterations (arrows) in Nile tilapia fed with 7.5 and 10 % SP, under NaAsO2 exposure by a rate (7 ppm) for 3 weeks. N=6 fish per sampling time/group. a Control. b. As only. c SP1, As with Spirulina at 7.5 %. d SP2, As with Spirulina at 10 % (400 ) estimating the frequency of MN in the buccal cells in workers occupationally exposed to this metal. Yadav and Trivedi (2009) reported a significant level of micronuclei frequency in fishes exposed to As (III) at level of mg/l for 24, 48, 72, 96, and 168-h time periods. Two different species of fishes, Channa punctatus and the goldfish (Carassius auratus), showed a significant increase in the frequency of micronucleated erythrocytes in a dose-dependent manner in both fish after exposure to different levels of arsenic trioxide (0, 10, 50, 500, and 1000 μg/l) for 15 days (Kumar et al. 2013). Also, cytotoxicity in fish cell lines induced after exposure to sodium arsenide (Seok et al. 2007; Wang et al. 2004). Akter et al. (2009) described the death of fish cells involving fragmentation of chromosomal DNA due to arsenic toxic effects. In the same trends, Ahmed et al. (2008) showed the apoptotic cell death of C. punctatus due to arsenic toxicity. Our results corroborate with the previous finding of MN induction in fish erythrocytes in response to a large number of experimental carcinogens, such as aflatoxins, benzidine, ethyl methanesulfonate, methylcholantrene, chlorinated hydrocarbons, heavy metals (Al-Sabti and Metcalfe 1995), cyclophosphamide (Ayllon and Garcia-Vazquez 2000), and also to the most common carcinogenic environmental pollutants, such as 4-nonylphenol (Mekkawy et al. 2011), pesticides (Grisolia 2002), and xenobiotics (Bolognesi et al. 2006). Our results of the alteration of fish erythrocytes coincided with previous studies with other agents such as alteration of fish erythrocytes that were observed in a hypoxic condition (Sawhney and Johal 2000) and by factors that induce alterations of blood cells like radiations (Sayed et al. 2014). Apoptosis induced by low-level xenobiotic exposure is considered a useful cellular marker of xenobiotic stress; moreover, it provides mechanistic insight into chemical action and bioavailability (Cantrell et al. 1998). Arsenic is known to induce apoptosis in different cell types; Zhang et al.( 2003) reported that arsenic-induced apoptosis in certain cancer cell lines

8 751 Page 8 of 10 Environ Monit Assess (2015) 187:751 in vitro. Datta et al. (2007) observed apoptotic death of hepatocytes in fish exposed to low concentrations of arsenic (0.50 μm) in Indian catfish (Clarias batrachus) for 30 days. The numbers of apoptotic and/or necrotic cells in the liver C. punctatus (Bloch) exposed for 14 days to nonlethal concentrations (3.8 and 7.6 mg/l) of As 2 O 3 increased significantly until day 7 in the 3.8-mg/L dose (39 %) treated fish and until day 2 in the 7.6-mg/L (23 %) treated fish (Roy and Bhattacharya 2006). The present results indicate the cytogenetic toxic effect of arsenic on tilapia blood erythrocytes and micronucleus test as a sensitive indicator of arsenic genotoxicity that could be attributed to the toxic effect of arsenic on oxidative stress following the production of reactive oxygen species (Barbosa et al.). DNA damage and micronucleus formation caused by ROS may also potentiate cancer by enhancing cell proliferation (Hei et al. 1998). Another effect of mediated arsenic toxicity suggests that oxidative methylation of arsenic during its biotransformation was found to be associated with oxidative damage to DNA. The methylated arsenic species can release redox-active iron from ferritin and subsequently promotes the conversion of superoxide radical and hydrogen peroxide into highly reactive hydroxyl radicals (Ahmad et al. 2000). Thus, both mechanisms drastically affect the cellular protective mechanisms against ROS which further contributed to the increased level of oxidative damage in cells, exacerbating arsenic genotoxicity (Frechet et al. 2001). In the current study, diet supplementation with SP alleviates the arsenic genotoxic effects. Its antigenotoxicity and anti-carcinogenic properties inhibit cell proliferation, deformations, induction of MN, and apoptosis in tilapia blood erythrocytes. The percentage of all cytogenetic analyses investigated was of lower frequencies compared to the As-treated group and particularly, the high level 10 % SP showed a decline in their levels near the control levels. Recently, great attention and extensive studies have been devoted to evaluate its therapeutic benefits, including anti-oxidant, anti-bacterial, and anti-parasitic properties, and for several medical conditions such as allergies, ulcers, anemia, heavy-metal poisoning, and radiation poisoning (Deng and Chow 2010; Ibrahem et al. 2013; Sayed and Fawzy 2014). Our results indicate that SP was remarkably effective in ameliorating the higher induction of MN formation, apoptosis, and deformations in blood erythrocytes caused by arsenic, suggesting its potential therapeutic effect in our experiment. This was in accordance with other studies concerning SP. Mathew et al. (1995) showed the chemopreventive role of SP against oral cancer. Khan et al. (2005) investigated that SP inhibit carcinogenesis due to its anti-oxidant properties that protect tissues from cell damage. The potential hepatoprotective role of SP was associated with its anti-oxidant constituents such as selenium, chlorophyll, carotene, gamma-linolenic acids, tocopherol, phenolic compound content, and vitamin E and C working individually or in synergy; SP has been shown to be effective against free radical-induced cellular transformation; phycocyanin, the main pigment present in SP, can inhibit cytochrome P450-mediated reactions involved in the formation of reactive metabolites of the hepatotoxins (Linjawi 2011). Previous studies have shown the effect of diet supplementation with SP at different levels of 2, 4, 6, and 10 % on the hematological parameters in carp (Labeo rohita) exposed to copper (James et al. 2009). However, James (2010) reported that feeding SP at level of 4 % was optimum but in carp, Cirrhinus mrigala, since this dose produced the maximum elimination of copper from the body and better physiological and biochemical parameters. In conclusion, this study showed the relevance of using the peripheral blood MN, apoptosis, and erythrocyte alteration criteria as potential indicators of arsenic genotoxicity with emphasis on its potential risks to the aquatic organisms. Additionally, it also showed the important role of Spirulina dietary supplementation in ameliorating the genotoxic damages induced by arsenic in Nile tilapia fish. Compliance with ethical standards Ethical statement All experiments were carried out in accordance with the Egyptian laws and University guidelines for the care of experimental animals. The protocol for this work was described in compliance with the guidelines of the ethical committee of Assiut University and Mansoura University. References Ahmad, S., Kitchin, K. T., & Cullen, W. R. (2000). Arsenic species that cause release of iron from ferritin and generation of activated oxygen. Archives of Biochemistry and Biophysics, 382, Ahmed, K., Akhand, A. A., Hasan, M., Islam, M., & Hasan, A. (2008). Toxicity of arsenic (sodium arsenite) to fresh water

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