Toxicity of Silver Nanoparticles to Marine Microalgae Chlorella Vulgaris Xue-jiao ZHANG 1,2,*, Ting-wan ZHANG 1,2 and Romana MANZOOR 1,2

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1 2017 Interntionl Conference on Energy, Power nd Environmentl Engineering (ICEPEE 2017) ISBN: Toxicity of Silver Nnoprticles to Mrine Microlge Chlorell Vulgris Xue-jio ZHANG 1,2,*, Ting-wn ZHANG 1,2 nd Romn MANZOOR 1,2 1 Key Lortory of Mrine Environment nd Ecology, Ministry of Eduction, Ocen University of Chin, Qingdo, , P. R. Chin 2 College of Environmentl Science nd Engineering, Ocen University of Chin, Qingdo , P. R. Chin *Corresponding uthor Keywords: Chlorell vulgris, Silver nnoprticles, Cysteine, Toxicity. Astrct. Silver nnoprticles (AgNPs) hs een widely used in vrious industries, thus inevitly relesed into the qutic environment, including the costl environment, which is under serious nthropogenic influences. This study ssessed the toxicity of AgNPs (0.1 nd 1.0 mg/l) in the sence nd presence of 1μM cysteine on the mrine phytoplnkton Chlorell vulgris exposed for 96 h. Results showed tht AgNPs hve negtive effect on Chlorell vulgris, s demonstrted y significntly decrese in chlorophyll content, increse SOD, CAT ctivity nd MDA content. However, these indictors were not significntly ffected y dding cysteine. Thus, these results imply tht cysteine llevited toxic effects of AgNPs. Further reserch on nnoprticles to qutic orgnisms re needed, in order to etter understnd the ecotoxicologicl effects of nnoprticles. Introduction Engineered nnoprticles, with vrying sizes tht rnge from 1 to 100 nm, re incorported into n incresing numer of products such s textiles, food storge continers, electronic products, ctlysts nd even food dditives due to the unique properties (such s high specific surfce re nd moility). Silver nnoprticles (AgNPs), with production volumes of 500 tonnes per yer, re one of the most populr nd fstest growing engineered nnomterils for their promising ntimicroil properties [1]. Becuse of their widespred ppliction in ll kinds of consumer products, it is inevitle tht NPs re relesed in the qutic environment, which could potentilly led to unexpected helth or environmentl hzrds. However, once NPs re relesed into the nturl environment, their fte nd ehvior re lrgely unknown. Toxic effects of nnoprticles on qutic orgnisms remin to e evluted. In prticulr it is uncler whether toxicity of AgNPs is specificlly relted to unique properties or is due to the effects of Ag + ions. Toxic effects of AgNPs my e relted to dmges to cell memrnes, to oxidtive stress, or to interctions of Ag + ions with proteins nd enzymes [2]. Less informtion is ville on the uptke nd toxicity of AgNPs to lge. The ojective of this work is to explore the mechnism of the toxicity of AgNPs to sewter lge. C.vulgris is selected s test orgnism to exmine the iochemicl nd ehviourl responses nd whether toxicity of AgNPs is relted to relese of Ag + from the AgNPs. A lignd (cysteine) is used to decrese the Ag+ concentrtion nd thus to disentngle the contriution of Ag + nd AgNPs to the overll toxicity. Therefore, 96 h experiment ws conducted. After the termintion of the experiment, the growth inhiition of C.vulgris nd the chnge in its ntioxidnt defense were investigted. Mterils nd Methods Silver nnoprticles (Ag NPs) nd Cysteine were otined from Sigm-Aldrich (Germny) with the prticle size declred s <100 nm, surfce re s 5.0 m 2 g nd density g cm. A stock suspension of 400 mg/l AgNPs ws prepred y dispersing the nnoprticles in the F/2 medium nd ultrsonicting for 30 min efore use. All regents were nlysis purity. All glsswre ws soked in nitric cid for 12 h nd sterilized in n utoclve. 433

2 Alge Cultures Alge cells were cultured in modified f/2 medium [3] t 22±1 with light illumintion of 5000 lux in 12:12 light-drk photocycle. The smples were mintined under sttic conditions nd shken y hnd twice ech dy to otin homogenous cell distriution. The cells were utilized for toxicity test t the mid-exponentil growth phse. Exposure Protocol C.vulgris in the logrithmic phse were exposed to 0.1nd 1mg/L AgNPs in the sence nd presence of 1μM cysteine for 96h. 200 ml of lgl culture were used for ech exposure tretment. The iomss of lge ws tested every 24h. The reltive growth rtes were clculted with the equtions s follows [4]: Reltive growth rte= ln N 1 ln N 0 (1) T 1 T 0 N 1 nd N 0 re the cell density t times T 1 nd T 0, respectively; After 96 h of exposure, chlorophyll of test lge ws extrcted in 90% ethnol in the drk for 24 h, followed y 10 min of centrifugtion t 4000 rpm. The light sornce of the superntnts ws mesured t 663 nm, 645nm with UV-vis scnning spectrophotometer (UV-2550.SHIMADZU, Jpn), nd 90% ethnol ws set s the control. The iochemicl indictors included totl protein, superoxide dismutse (SOD), ctlse (CAT), nd mlondildehyde (MDA) were mesured using dignostic regent kit (Nnjing Jincheng Bioengineering Institute, Nnjing, Chin) ccording to the mnufcturer s instructions. Sttisticl Anlysis Results were expressed s men vlue ± stndrd devition ( n = 3 ). All tests in this study were conducted in triplicte. The dt were nlyzed using Origin 9.2 softwre (OriginL, Northmpton, MA, USA). One-wy nlysis of vrince (ANOVA) ws conducted to evlute the significnce of different tretments. Differences were considered sttisticlly significnt when p < Sttisticl nlyses were conducted using IBM SPSS Sttistics Results nd Discussion Chrcteristics of AgNPs Figure 1. TEM imge of AgNPs dispersed in sewter (: Ruler is 100nm; : Ruler is 50nm). To gin etter understnding of AgNPs ehviors in the queous environment, physiochemicl properties of AgNPs were investigted. Aggregtion hs significnt influence on trnsport of nnoprticles in the environment [5]. Aggregtion nd dispersion cn chnge their toxicity in different environment. The TEM imge confirmed tht the ggregtion nd gglomertion. The men prticle size of AgNPs ws clculted to e ± nm. 434

3 Chl content(mg/l) MDA content( mol/10 10 cell) Reltive growth rte Growth Inhiition of Alge control 0.1 mg/l AgNPs 1 mg/l AgNPs 1mg/L AgNPs+1 M Cysteine Exposure time (h) Figure 2. The effects of different concentrtions of AgNPs (0.1, 1 mg/l) on the reltive growth rte of C.vulgris t 24, 48, 72 nd 96 h. Reltive growth rte of microlge C.vulgris under different concentrtions of AgNPs within 96 h ws shown in Fig.2. Under tretment of different AgNPs concentrtions, reltive growth rte hd significnt increse compred with control t initil 24 h, my e due to AgNPs stimulte growth. Reltive growth rte egn to decrese t 48 h under 1mg/L AgNPs concentrtion, then increse t 72 h. The reltive growth rte of 0.1 mg/l AgNPs on microlge ws clerly higher thn tht of 1mg/L AgNPs. The microlge nerly stopped growing under different concentrtions of AgNPs t 96 h. However, in generl, the tendency of reltive growth rte in tretment groups ws not oviously chnge thn tht of control group. This my e due to the formtion of lrger ggregtes in f/2 medium, then precipittion to the ottom, which cused lower opportunity for the contct etween AgNPs nd lge cells [6]. The stronger tolernce of C.vulgris to dverse conditions could lso e one of the resons [7]. Chl Content Determintion () 0.6 c () c AgNPs concentrtions (mg/l) 0.00 AgNPs concentrtions(mg/l) Figure 3. Chlorophyll- contents () nd MDA content () of C.vulgris when exposure to different concentrtions of AgNPs for 96 h. To further understnd the effects of AgNPs on C.vulgris, the chlorophyll content when exposure to different concentrtions ws determined. As shown in Fig.3(), the Chl content of C.vulgris in 0.1 mg/l AgNPs group ws not significntly different from tht of 1mg/L AgNPs group, nd the Chl content of C.vulgris in oth the 0.1 nd 1 mg/l AgNPs significntly decresed thn tht of the control ( p < 0.05 ). Interesting, Cysteine significntly incresed the Chl content of C.vulgris in 1 mg/l AgNPs group ( p < 0.05 ), the vlue ws out 2-fold of the control. This phenomenon indicte tht cysteine ws suitle lignd to decrese Ag + vilility. Cysteine reduced AgNPs toxicity. Zho et l [8] lso demonstrted tht AgNPs presented no cute toxicity to the dphnids when the relesed Ag + ws complexed with cysteine. 435

4 SOD ctivity (U/mgprot) CAT ctivity(u/mgprot) Lipid Peroxidtion The level of MDA cn revel the cell memrne lipid peroxidtion nd reflect the degree of cell dmge from ROS indirectly. In the current study, the chnge in MDA content during exposure is shown in Fig 3(). MDA content incresed with the AgNPs concentrtion incresing in the sence of cysteine. At 0.1 mg/l AgNPs did not exert significnt influence on MDA content (p > 0.05 ), however, chnges t 1.0 mg/l AgNPs showed significntly difference (p < 0.05 ). Aprt from tht, MDA content in presence of cysteine returned to the similr level s the control. Oxidtive Stress Assessment 180 () c d () AgNPs concentrtions (mg/l) 0 AgNPs concentrtions(mg/l) Figure.4 SOD ctivity () nd CAT ctivity () of C.vulgris fter 96 h of eing exposed to the different concentrtions of AgNPs. Oxidtive stress hs een proposed s the primry mechnism of AgNPs toxicity. When SOD ctivity is stimulted, its ility to eliminte free rdicls is improved t low concentrtion of the toxicnt, therey protecting lge from oxidtive dmge [9]. To determine whether the ntioxidnt system tht ws induced y AgNPs, SOD ctivity nd CAT ctivity were determined. As shown in Fig.4(), SOD ctivity were significntly incresed with AgNPs concentrtion incresing (p < 0.05). The SOD ctivity of exposure to 1 mg/l AgNPs showed 2.15-fold increse compred to tht of the control group. Moreover, Cysteine significntly decresed SOD ctivity. Menwhile, the CAT ctivity possessed similr trend s SOD ctivity [Fig.4()]. Similrly, the incresed ctivities of CAT indicte potentil protection ginst oxidtion y these ntioxidnt enzyme. Moreover, concerning the influence of cysteine, CAT ctivity returned to the similr level s the control nd SOD ctivity ws even lower thn the control. This indicted tht cysteine llevited the oxidtive stress of AgNPs towrds C.vulgris with regrd to the functions of SOD nd CAT. Conclusions In summry, exposure to AgNPs cuse oxidtive stress in microlge C.vulgris s evidenced y the rekdown of the ntioxidnt defense system, the cuse of lipid peroxidtion. Menwhile, the existence of cysteine decresed the toxicity of AgNPs in microlge C.vulgris. Cysteine, strong silver lignd, proved useful to exmine the contriution of Ag + to the overll toxicity of AgNP. Our study confirmed tht AgNPs toxicity to C.vulgris is due to the relesing of Ag +. Understnding these phenomen would e eneficil in the effective ecologicl risk ssessment nd mngement of nnomterils. As consequence, future investigtions re urged considering the diversity of nnoprticle chrcteristics, their fte nd ecotoxicologicl potentil under vrying, field relevnt environmentl conditions. Acknowledgements This study ws supported y the Ntionl Nturl Science Foundtion of Chin (Grnt No ), Nturl Science Foundtion of Shndong Province, Chin (Grnt No. ZR2011DM013) 436

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