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Biotechnological Journal of Environmental Microorganisms(BJEM)1(3)2022 127-134AbstractKey words:*The Acute Toxicity of Tin Dioxide Nanoparticles on Chlorella vulgaris AlgaeFatemeh Shariati1*,Mahrooz Ziksari1,Zohreh Ramazapour21Department of Environment,Lahijan Branch,Islamic Azad University,Lahijan,Iran2International Research Institute of Sturgeons,Rasht,IranReceived:15 May 2023/Revised:30 June 2023/Accepted:1 July 2023 Corresponding Author E-mail:shariat_Nowadays,nanotechnology and the use of its components,including nanoparticles,have successfully im-proved the situation of industries in advancing production goals.Among these nanoparticles,SnO2,or tin dioxide nanoparticle,which was used in this study,can be mentioned.Tin dioxide is used in the manufacture of batteries and fuel cells,capacitors,and the negative effects of factory effluents entering rivers and other water sources will affect catalysts,and the health of living organisms.In this study,the biotoxicity of tin oxide nanoparticles on Chlorella vulgaris algae,which is one of the primary producers and most important levels of the food chain,was investigated.This research was conducted using the OECD acute toxicity test method(counting method for algae,method 201),and statistical probit analysis was performed in order to obtain tox-icity data using the probit method.The results of exposure for Chlorella vulgaris in 48 and 72 hours were EC50 and EC90 equal to 6.99,57.54,and 13.08 and 1.07 x 1010 mg L-1,respectively.The highest growth decrease after 48 and 72 hours was observed in 5.5 mg L-1 SnO2NPs.During the test period,no morphological changes were observed for any of the microorganisms,which are based on the toxicity of tin oxide nanoparticles.Algae,Chlorella vulgaris,Tin oxide nanoparticle,ToxicityArchive of SID.irArchive of SID.irBiotechnological Journal of Environmental Microorganisms(BJEM)1(3)2022 127-1341281.IntroductionNanotoxicology is one of the new branches of science that studies and investigates the toxicity potential of micromaterials and microparticles.Breaking the solid material and turning it into small particles causes the particles to shrink and increase their overall surface area,which can re-veal the emerging properties of such materials.As the particle size decreases to 0.1 nm,quantum effects also appear(Ranjbar et al.,2006).SnO2 is an n-type semi-conducting nanoparticle whose capacity to destroy colored environmental pol-lutants is known(Khedmati,2013).Metal oxide nanoparticles have recently been manufactured in industries at the engineering level with large-scale effects(Pendashteh et al.,2011).Tin oxide nanoparticles(SnO2 NPs)are one such material that has seen widespread use in a variety of ap-plications,including electronics,solar cells,and coatings.SnO2 NPs can act as a photocatalyst for the degradation of pigments in colored materi-als.These nanoparticles can also be used in the decontamination of dye-contaminated water in textile factory effluents.With the advancement of nanotechnology science and the increase in the use of nanopar-ticles,it is expected that the consequences of re-leasing wastewater containing nanoparticles into the environment will emerge.The first group of organisms to be affected by these events are al-gae,which form the first group in the food chain.Chlorella vulgaris is a group of green algae.The members of this group are very diverse in terms of morphological forms,reproduction methods,life cycles,and habits,and structurally they have very advanced examples(Kianmehr,2005).Chlorella vulgaris is a freshwater alga that has a wide distribution and is a good species for bi-otoxicity tests(Auffan et al.,2011).The studies reported a range of negative impacts of SnO2 on aquatic organisms,including reduced growth and survival,altered behavior,and changes in bio-chemical and physiological parameters(Ahamed et al.,2016;Chvez-Caldern et al.,2016;Park&Park,2009;Wang et al.,2019;Zhang et al.,2019).Bounnit et al.(2022)studied the effects of SnO2 NPs on Picochlorum maculatum and ob-served that these nanoparticles had a toxic effect on algae growth.Also,it was observed that low-er doses had more negative impacts than high-er doses because of nanoparticle agglomeration,which resulted in a reduced effect on cell mor-phology and appearance.Protein production was inhibited,too(Bounnit et al.,2022).The long-term effects of SnO2 exposure on aquatic organ-isms are not yet fully understood,but several studies suggest that chronic exposure to SnO2 can have negative impacts on growth,reproduction,and survival.It is mentioned that intracellular ROS accumulation decrease of photosystem II(PSII)in algae were observed in microalgae(P.subcapitata)exposed to SnO2 NPs.Also,different biological models have been described showing that SnO2 produced and ac-cumulated significantly more intracellular ROS than control with the consequent cell oxidative disturbances,including lipid peroxidation and cell membrane damage(loss of integrity),an overwhelmed antioxidant defense system,re-duced mitochondrial function,chromatin con-densation,DNA damage,and cell death through the apoptotic pathway.It was observed that the viability of yeast Saccharomyces cerevisiae cells was reduced in a dose-dependent way when ex-posed to SnO2(Soares&Soares,2021).A study by Poynton et al.(2013)found that chronic exposure to SnO2 NPs reduced the growth and production of Daphnia magna,a common freshwater invertebrate.Similarly,Yu et al.(2020)reported that chronic exposure to SnO2 NPs causes significant damage to the gill filaments and liver tissues of Clarias gariepinus,a freshwater fish.Other studies have suggested that chronic exposure to SnO2 NPs can lead to changes in biochemical and physiological pa-rameters,indicating potential sublethal effects on aquatic organisms.For example,Lu et al.(2015)found that chronic exposure to SnO2 NPs caused oxidative stress and apoptosis in the liver of ze-brafish.Overall,the long-term effects of SnO2 NPs exposure on aquatic organisms are likely to depend on a variety of factors,including the dose and duration of exposure,the species and life stage of the organism,and the environmen-tal conditions in which the organism lives.The Archive of SID.irArchive of SID.irBiotechnological Journal of Environmental Microorganisms(BJEM)1(3)2022 127-134129mechanisms through which SnO2 impacts aquat-ic organisms include physical interactions,such as obstruction of gill filaments in fish,and chem-ical interactions,such as the release of toxic ions from SnO2 NPs.Navaro et al.(2008)and Adams et al.(2006)studied the toxicity of silver nanoparticles and nanooxides of titanium,zinc,and silicon,re-spectively.In their research,Mouivand and Fal-lahi investigated the effect of nanosilver on the growth rate and reproduction of blue-green al-gae(Anabaena flosaquae)for 3 months in 2010(Mouivand&Fallahi,2010).A study was carried out in 2012 with the aim of determining the acute toxicity of zinc oxide nanoparticles on two algae,Scenedesmus dimorphus and Chlorella vulgar-is(Pendashteh et al.,2011).In an investigation,freshwater algae(P.subcapitata)were exposed to 25 to 600 mgL-1 zinc oxide nanoparticles for 72 hours(Tsai et al.,2007).Undoubtedly,SnO2 NPs will be used in the wastewater of factories and industries during the waste production process,and considering the irreparable risks they have on water damage,especially at the initial levels of the food chain,this research related to the toxicity of tin dioxide nanoparticles on two species of algae that form the basis of the food chain was done.While the advantages of nanomaterials are numerous,their potential impacts on the environment and living organisms are not yet fully understood.In particu-lar,the impacts of SnO2 NPs on aquatic organisms have received relatively little attention compared to the extensive research on terrestrial organisms.This paper aims to study the impacts of SnO2 NPs on the algae Chlorella vulgaris,which is at the initial levels of the food chain.2.Materials and MethodsSnO2 NPs with a size of 40 nm were prepared by the chemistry department of Lahijan Azad Uni-versity.In the toxicity test,the working method and implementation of the steps were carried out according to the OECD method(201).Chlorella vulgaris algae was obtained from Anali Wetland and cultivated to make the main algae stock.Algae culture was done in the agar-agar medium on spe-cial culture plates under ultraviolet rays and then transferred to the liquid culture medium(Zinder)in a sterilized Erlenmeyer flask.Cultivation was carried out under controlled temperature condi-tions of 22C,12 hours of darkness,12 hours of 4000 lux light,and 14 days in a germinator.Before starting the exposure operation,range finding was done to determine the required con-centrations of tin dioxide nanoparticles.Exper-imental concentrations of 0.01,0.1,5,and 10 mg L-1 of nanoparticles were prepared in Zinder culture medium(Table 1),and 5103 cells of al-gae were added to each of the treated test tubes.After doing the range finding results,the main concentrations were calculated through loga-rithmic base,and their values were 0.01,0.08,0.66,5.50,50,and 100 mg L-1.All treatments were done with three replicates.Three control tubes without nanoparticles were also exam-ined as control samples.The amount of 5103 cells from the main stock of Chlorella vulgaris algae(according to the standard method of the OECD)was added to the test tubes,and the sam-ples were kept in the germinator for 24,48,and 72 hours under a temperature of 22 2 degrees Celsius and 12 hours of light and darkness.After 24 hours,the number of cells in the algae treat-ments was counted under a Nikon light micro-scope(model N-180M,Japan)by an improved Thoma-ruled hemocytometer consisting of nine 1 x 1 mm(1 mm2)squares.After counting and recording the data,the average number of cells in the top and bottom squares was calculated,and then the number of cells was calculated using the following equation:Cell density per ml=all cells counted in the large square104 (1)The specific growth rate(),inhibition percentage(%I),and doubling rate(G)were obtained using the following equations(Fogg et al.,1987):In equation(2),is the rate of specific growth,X=0 is the number of cells at time t0,and X=1 is the average number of cells at time t1.In equation(4),t,and c are defined as the growth rates in the treatment mean value of in the control,respectively.=()(2)3(1-2G=ln )4(c /t cI=%Archive of SID.irArchive of SID.irBiotechnological Journal of Environmental Microorganisms(BJEM)1(3)2022 127-134130Table 1.The composition of Zinder culture medium(Sakamoto et al.,2019)After determining the algae cell number,24-,48-,and 72-hour levels of EC10,EC50,and EC90 were derived from the Probit analysis table,and NOEC was calculated(Equation 5)(Finny,1971).To determine the significance of differenc-es among treatments in various concentrations of algae cells and control samples,a one-way ANO-VA was used.The Tukeys test was used to iden-tify differences between each level of treatment.NOEC=EC50/10 (5)3.Results3.1 Cell Count Test(cell ml-1)for Chlorella vul-garis algae The comparison of the control with each treatment revealed that an obvious reduction in algae cells was observed after 48 and 72 hours,but a difference was observed after 24 hours of 0.08 mg L-1 treatment(Fig.1).This reduction in cell density during 48 and 72 hours,especially at 5.5 and 100 mg L-1 concentrations,indicates the acute toxicity of SnO2 NPs and its effect on Chlorella algae growth.The results showed that growth-inhibitory effects and a reduction in cell density usually appear after 24 hours.The high-est decrease occurred at 5.5 and 100 mg L-1 with the control(Fig.1).Based on the ANOVA test,a significant difference was observed between the control and 5.5 and 100 mg L-1 exposures(p Al2O3 Mn3O4 SiO2 In2O3(Sosa et al.,2019).According to Table 2,72-hour EC90 has the highest amount.The large number of EC90 indi-cates the low toxicity of SnO2 NPs on the pro-liferation of Chlorella algae cells.A study con-ducted by Gong et al.(2011)on the toxicity of nickel oxide on Chlorella vulgaris algae indicat-ed the 72-hour EC50 was 32.28 mg L-1.The re-sults showed that the biotoxicity of nanoparticles and the bioavailability of nickel oxide(NiO)to marine algae are reduced by accumulation.In the present study,the 72-hour EC50 was 131.82 mg L-1,which indicated it is less toxic compared to nickel oxide.In another study,the effect of alu-minum nanoparticles on Dunaliella algae was tested,and 72-hour values were EC10=1.66 10-3,EC50=0.162,EC50=31.15,and EC90=162 mg L-1.The amount of cell density and growth inhi-bition between the algae of the control treatment and the studied concentrations had a significant difference(P0.05).Aluminum oxide nanoparti-cles had a significant effect on the shape and the cell topography,which has caused the swelling and enlargement of Dunaliella algae cells(Ayat-tallahzadeh Shirazi et al.,2013).In another test to determine the toxicity of na-nozinc oxide on Chlorella and Scenedesmus al-gae,it was found that this nanoparticle has a de-creasing effect on the growth rate of two species of algae,and a decrease in the number of Scened-esmus algae was observed in all concentrations after 72 hours(Meulenkamp,1998).Mouivand et al.(2011)investigated the effect of nanosil-ver on the growth rate and reproduction of blue-green algae(Anabena flosaquae)for 3 months in 2009.The results showed that the effective concentration of nanosilver was in the range of 0.005 to 0.050 mg L-1 in blue-green algae,and the maximum reduction in the growth of these algae was obtained at a concentration of 0.05 mg L-1 nanosilver.According to the results,the high-est concentration of exposure to nanosilver had a negative effect on the growth and reproduction of Anabaena algae.In a study conducted with the aim of deter-mining the acute toxicity of zinc oxide nanopar-ticles on Chlorella vulgaris algae,48-hour EC50 values were 2.830 mg L-1 when compared with the results of this study,showing that zinc ox-ide nanoparticles were somehow more toxic than SnO2(Pendashteh et al.,2011).Freshwater algae species(P.subcapitata)were exposed to 25 to 600 mg L-1 zinc oxide nanoparticles for 72 hours.The comparison of toxicity effects between ZnCl2 and ZnO(in powder form)and nano ZnO(in aqueous form)was done,and the EC50 value was calculat-ed as 60 mg L-1(Tsai et al.,2007).Also,in research on determining the size-de-pendent toxicity of silver nanoparticles on Chlorella algae,extreme changes in the chlorophyll content of the algae and an increase in lipid peroxidation were observed.The target algae were exposed to zero to 10 mg L-1 of silver nanoparticles for 24 hours(Oukarroum et al.,2010).Metal oxides of nanopar-ticles can have different toxicities,and the toxicity of nanoparticles is related to the nature of their con-stituent elements in addition to their nanostructure and surface-to-mass