To better understand the ecotoxicological effects of Zn oxide nanoparticles on aquatic workss, research lab experiments were conducted utilizing Salvinia natans All. as the trial being for 7-d exposure to the NPs. The activities of superoxide dismutase ( SOD ) , catalase ( CAT ) , peroxidase ( POD ) and the content of chlorophyll were determined at the terminal of the exposure to measure the toxicity of the NPs in the civilization medium on the workss. To investgate if the release of soluble Zn from ZnO NPs suspensions plays a cardinal function in the toxicity, we investigated its collection and disintegration in the medium. Our consequences indicate that ZnO NPs could convey harmful effects to S. natans when its concentration is above 50 mg L-1 in the civilization medium, and Zn2+ released from the NPs may be the chief beginning of its toxicity to this species.
Keywords: ZnO nanoparticles ; Salvinia natans ; Toxicity ; Aggregation ; Dissolution
Nanoparticles ( NPs ) have been referred to new stuffs with at least two dimensions under 100 nanometer. The alone physicochemical belongingss of NPs are attributable to their little size, chemical composing, surface construction, solubility, form, and collection. This makes them attractive for a broad scope of fresh applications in electronics, health care, cosmetics, engineerings and technology industries. Harmonizing to conservative estimations, the figure of consumer merchandises on the market incorporating NPs or nanofibers now exceeds 1300 and is turning quickly.
Harmonizing to “ The Nanotechnology Consumer Products Inventory ” , the most common metal oxide NPs stuff mentioned in the merchandise descriptions was Ti dioxide ( TiO2 ) , followed by Zinc oxide ( ZnO ) . ZnO NPs are widely used in pigments, semiconducting materials, sunblocks, and nutrient additives. Meanwhile, they may be released to the environment and go a menace to the ecosystem during the life rhythm of these commercial merchandises.
The extended usage of NPs will doubtless increase the possible hazard for homo and environmental exposures. Probes on the ecotoxicology of NPs are now emerging, with a end of measuring NPs ‘ harmful effects to the ecosystem. Because H2O environment is the most of import and possibly the ultimate finish of NPs released in the environment despite their beginnings, many surveies focused on toxicity of NPs on aquatic beings.
Toxicological effects of ZnO NPs upon a assortment of aquatic beings have late been reported. It has besides been shown to hold toxic effects and can suppress the growing of microalgae Pseudokirchneriella subcapitata, crustaceans Daphnia magna and Thamnocephalus platyurus and bacteriums Vibrio fischeri. However, research on the toxicity of ZnO NPs is far from complete and needs more work to measure its release hazard utilizing other beings, such as aquatic workss. In add-on, influences of collection and disintegration on toxicity of the NPs remain to be determined.
Salvinia natans ( L. ) All. is a free-floating, aquatic heterosporous fern that reproduces vegetatively and signifiers quickly spread outing mats of leaf on still H2O surfaces in tropical and semitropical parts. The works was antecedently selected for toxicity of heavy metals.
The present work aimed to measure the ecotoxicological effects of ZnO NPs on S. natans via alterations of photosynthetic pigments and responses of antioxidant enzymes. Meanwhile, influences of collection and disintegration on toxicity of the NPs are investigated.
2. Materials and Methods
2.1 Word picture of ZnO Nanoparticles
ZnO NPs were purchased from Aipurui Co. , Ltd. , Nanjing, China. The surface country of the NPs was further determined utilizing the multipoint Brunauer-Emmett-Teller ( BET ) method. The morphology of the NPs was examined utilizing transmittal negatron microscopy ( H-7500, HITACHI, Japan ) .
2.2 Cultivation and growing of S. natans
Salvinia natans ( L. ) All. was obtained from Linyi Fengheyuan Garden Co. , Ltd ( Linyi, China ) . The alimentary solution used for growing was prepared harmonizing to the OECD 221 guidelines ( 2006 ) and contains KNO3, Ca ( NO3 ) 2A·4H2O, KH2PO4, K2HPO4, MgSO4A·7H2O, H3BO3, ZnSO4A·7H2O, Na2MoO4A·2H2O, MnCl2A·4H2O, FeCl3A·6H2O, and EDTA Disodium-dihydrate. The pH was adjusted to 6.5 by add-on of 0.1N NaOH.
The workss were rinsed with unfertile H2O and were allowed to acclimatise to the medium for 7 yearss before the beginning of the NPs exposure. Three workss were cultivated in 500 milliliter beakers incorporating 350 milliliter of the civilization medium. The beakers were placed in a thermostated chamber ( at 24 A± 2 oC ) with a photoperiod of 16 H of visible radiation ( 180 Aµmol m-2 s-1 ) and 8 H of darkness. The continuance of the exposure was 7 d. Exposures of the NPs were performed at four concentrations ( mg L-1 ) : 0 ( Control ) , 1, 10 and 50. A positive control which was exposed to ZnSO4 with the concentration of 44 mg L-1 ( Zn2+ 10 mg L-1 ) was besides performed.
A stock suspension of ZnO at 100 mg L-1, obtained by add-on of ZnO NPs to the civilization medium was used to fix assorted ZnO concentrations. The stock suspension was treated by a sonicator ( Vibra-Cell TM, USA ; 50 Hz, 10 s pulsation and 5 s interval ) for 10 min merely earlier used. The civilization media with different ZnO concentrations were prepared by adding the stock suspension, followed by sonication for 20 s.
After 7-d exposure, the workss were harvested, rinsed with distilled H2O, and their fresh weights were measured. The comparative growing rate ( g g-1 d-1 ) in each intervention was calculated by the expression: RGR= ( lnW2 a?’lnW1 ) /t, where W1 and W2 are the initial and concluding fresh weight ( g ) , and T is the incubating clip ( vitamin D ) . The dry weight of the workss was obtained by being oven dried at 80 oC for 48 H to constant weight.
2.3 Pigments assay
The contents of chl a, chl B and and carotenoid in the foliages of the workss were analysed harmonizing to Lichtenthaler. Freeze dried foliages were cut into little pieces and subsamples of about 5 milligrams were extracted with 8 milliliters 96 % ethyl alcohol in the dark at room temperature. After 24 H, the pigment infusions were, after smartly stirring, centrifuged for 3 – 5 min and the optical density of the infusions measured at 665, 648, and 470 nanometer with a VIS-7220 spectrophotometer.
2.4 Antioxidative enzymes assay
Superoxide dismutase ( SOD, EC 220.127.116.11 ) activity was determined by usage of the ferrous cytochrome degree Celsiuss method with xanthine/xanthine oxidase used as the beginning of super-oxide groups, and a unit of activity was defined as that described in McCord and Fridovich. Catalase activity ( CAT, EC.18.104.22.168 ) was assayed spectrophotometrically with a Hitachi U-3000 by mensurating the lessening of optical density at 240 nanometers due to H2O2 decomposition. Peroxidase ( POD, EC 22.214.171.124 ) activity was determined by usage of the methods described by Li et Al. .
2.5 Collection of the NPs and concentrations of Zn2+ assay
Prior to the trial, ZnO NPs were added to the civilization medium with the volume 350 milliliter in a 500 milliliter beaker and got a concluding concentration of 10 mg L-1. The suspension was treated by a sonicator as described above, and so the beakers were placed in the thermostated chamber ( at 24 A± 2 oC ) with no upseting. Particle size of the ZnO NPs in the suspension was determined with a Nanotrac 250 atom analyser ( Microtrac Inc. , USA ) . The 10 milliliter H2O samples were taken from the upper bed of the suspension carefully to avoid upseting at each hr of the initial 24 H for the trial of the atom distribution.
The concentrations of Zn2+ in the civilization media ( with workss ) at 7 vitamin Ds were detected by ICP-OES ( VISTA-MPX, USA ) .
2.6 Statistical analysis
All samples were triplicated and three independent experiments were run. The consequences presented are the arithmetic agencies with their corresponding standard divergences. The differences between groups were tested for significance utilizing one-way analysis of discrepancy ( ANOVA ) utilizing Origin 7.0, taking P & lt ; 0.05 as important and P & lt ; 0.01 as extremely important.
3.1 Features of ZnO NPs
The consequences show that the crystalline stage of ZnO NPs is monocrystalline, and the average size of the individual atom was about 30 nanometers ( Fig. 1 ) with the surface country 90 m2 g-1.
3.2 Chlorophyll and carotenoid contents
Both chlorophyll and carotenoid were influenced by ZnO NPs and ZnSO4 ( Table 1 ) . The contents of chl a and chl Bs decreased with the addition of the dose of ZnO NPs. The chl a content of the group 50 was about 2.57 milligrams g-1 DW ( 60 % of the control ) , significantly lower than that of the control ( P & lt ; 0.05 ) . When treated with ZnSO4, both chl a and chl B were significantly lower than those of the control ( P & lt ; 0.05 ) , and besides lower than the groups treated with ZnO NPs.
The content of carotenoid increased somewhat when treated with low concentration of ZnO NPs ( 1 mg L-1 ) , and so decreased with the addition of the dose. The carotenoid contents of the group 50 and group ZnSO4 were significantly lower compared with that of the control.
3.3 Responses of the antioxidant enzymes
Responses of SOD, CAT and POD are shown in Fig. 2. The activities of SOD increased with increasing of the NP doses and merely the group 50 showed important differences ( P & lt ; 0.05 ) from the control. When exposed to 10 milligrams L-1 Zn2+ , the SOD activity of S. natans was significantly higher ( P & lt ; 0.01 ) than that of the control.
The responses of CAT were similar to SOD. When exposed to ZnO NPs for 7 vitamin D at the highest concentration ( 50 mg L-1 ) , the CAT activity of S. natans was significantly higher ( P & lt ; 0.05 ) than that of the control. The group ZnSO4 followed the tendency of SOD.
As for POD, it increased foremost with the addition of the dose of ZnO NPs, the dropped markedly ( P & lt ; 0.05 ) at the concentration of 50 mg L-1. When exposed to ZnSO4 for 7 vitamin D, the POD activity of S. natans was significantly lower ( P & lt ; 0.01 ) than that of the control.
3.4 Aggregation and disintegration of ZnO NPs
Figure 3 depicts atom size distribution ( average value ) of 30-nm ZnO NPs in the civilization medium ( with no workss ) over the initial 24 h. Figure 4 shows the size distribution at 0 H, 3 H, 6 H and 12 H after readying. Although the suspension was treated with sonication, the average value of the atom size was about 130 nanometers. Aggregation occurred instantly after readying, and the atom size could make more than 4 Aµm. Over the initial 6 H tested, there were no regular forms of collection could be detected in both solutions. After 12 H, the atom sizes reduced to lower than 1 nanometers, proposing that there were no ZnO NPs in the supernatants. The white collections could be found at the underside of the beakers.
The concentrations of Zn2+ in the civilization media were shown in Table 1. The medium of the control provided Zn2+ as one of the indispensable foods with the concentration 11.4 Aµg L-1 which could keep the normal growing of S. natans. The parts of disintegration were the chief beginnings of the Zn2+ in the group 1, 10, 20 and 50, with the average concentrations of 0.413, 1.874, 2.572 and 2.963 milligram L-1, severally. The concentration of Zn2+ in the group ZnSO4 was 8.712 mg L-1.
The consequences of the toxicological trials in this survey indicate that ZnO NPs could bring forth harmful effects to S. natans when its initial concentration was above 50 mg L-1 in the civilization medium. The consequences from collection and disintegration analyses suggest that Zn2+ released from the NPs may be the chief beginning of toxicity of ZnO NPs to the species.
Antioxidant enzymes of life beings possess antioxidative activities and can protect cells against inauspicious effects of reactive O species ( ROS ) . The enzymes SOD, CAT and POD can command cellular degrees of ROS. The activities of these antioxidants determine the steady province degrees of ROS in cells. POD plays a critical function in scavenging these ROS. SOD and CAT could work together to change over O2A· and H2O2 into harmless H2O and O2, cut down the formation of hydroxyl free extremist A·OH every bit good as lower the overall free extremist content of cells. In this survey, SOD and CAT showed increased activities in the workss treated with the ZnO NPs, bespeaking better ROS scavenging in the system. Harmonizing to the studies, Zn has a potency to increase the biogenesis of antioxidant enzymes in S. polyrhiza. The activity of POD was significantly inhibited at the concentration of 50 mg L-1. This may be that the emphasis from the NPs or the Zn2+ was beyond the protective ability of the enzymes.
Colloid is the generic term applied to atoms in the 1-nm to 1-Aµm size rangeIn aquatic systems. Therefore, ZnO NPs existed in the solutions as colloid foremost, and so aggregated to big atoms and deposited. It was reported that colloidal destiny and behaviour are dominated by collection, and colloids will finally aggregate to atoms ( & gt ; 1 Aµm ) that are sufficiently big that their conveyance is dominated by deposit. It is of import in the self-purification of H2O organic structures and consequences in pollutant loss from surface Waterss and accretion in the deposits and is correspondent to the likely behaviour of NPs, with collection and subsequent deposit an of import procedure in their ultimate destiny. Our consequences showed that the collection of the ZnO NPs is important in the civilization medium and that there were well no ZnO NPs in the supernatants over 12 h. It is consistent with the resent studies [ 10, 11 ] , which evaluated the collection of the ZnO NPs in a fresh water system.
The disintegration of ZnO is extremely pH-dependent. Acid could ease and speed up the procedure, while alkalic hinders it. The pH value of the civilization medium used in this survey is 6.5. We observed that the Zn2+ concentration was 2.963 mg L-1 in the group 50, which was fundamentally close to a resent study with a mensural mean Zn2+ concentration about 2.6 ( 0.8 mg L-1 in civilization media ( pH 7.0 ; ZnO NPs ) 20 mg L-1 ) . It has been assumed that the predominant bioavailable part of the entire contamination was the soluble signifier. Meanwhile, Adams et Al. and Franklin et Al. besides reported the stopping point relationship between the dissolved part and the toxicity of the ZnO NPs. Although other surveies have shown that metal or metal oxide NPs may be more toxic than either their Attic signifiers or their parent compounds, solubility is likely to be an of import facet and should be considered in the farther ecotoxicological researches of the NPs.
The disintegration rate of NPs is influenced by several belongingss of NPs, including size, surface country, surface curvature and raggedness of the atom. Furthermore, other factors, such as pH, ionic strength and collection may besides impact the disintegration significantly. How aggregation affects the disintegration behaviour of atoms is non good understood. The formation of bunchs or bunchs of primary atoms consequences in increased hydrodynamic size and reduced specific surface country, and may impede disintegration by cut downing the mean equilibrium solubility of the atom system. The behaviour may be farther complicated by the aggregative volumes and packing factors, with larger, more dumbly jammed sums exhibiting a slower disintegration. We detected that the Zn2+ concentration in the group 10 was 1.874 mg L-1, while 2.963 mg L-1 in the group 50. The ground might be the above mentioned.
NPs and natural colloids will interact and this will impact NP behaviour in the natural environment. At present, no direct published informations are available on the concentrations of NPs in natural Waterss, but a recent study utilizing a simplified box theoretical account and known current utilizations suggested environmental concentrations is about 1 to 100 Aµg/L, whereas typical dissolved and colloidal organic affair in fresh waters may be found at 1 to 10 milligrams L-1 concentrations. Disturbing in the existent aquatic environment may besides act upon the collection of the NPs.
In general, toxicological effects of ZnO NPs to S. natans were investigated in the present survey, and the consequences show that important emphasis from the NPs at 50 milligrams L-1 could be observed. Because the NPs are disposed to collection and disintegration in the medium, Zn2+ released from the NPs may be the chief beginning of the toxicity. Research on collection in the natural aquatic environment, sediment toxicity and bioaccumulation of NPs may supply more valuable information.
Fig. 1 Transmission negatron microscopic image of ZnO nanoparticles.
Fig. 2 The comparative growing rate ( RGR ) of S. natans over 8 500 exposure.
Fig. 3 The activities of SOD, CAT, and POD of S. natans after 7-d exposure to ZnO NPs and ZnSO4 with different concentrations.
Fig. 4 Variation of the atom sizes ( average A± SD ) of ZnO in the civilization medium ( with no workss ) over the initial 24 H.
Table 1 Effectss of ZnO nanoparticles and ZnSO4 on chl a, chl B and carotenoid ( mg g-1 DW ) in the foliages of Salvinia natans.
Table 2 The concentrations of Zn2+ in Salvinia natans and the civilization media after 7 vitamin D exposure.
( mg/g DW )
( mg/g DW )
Roots ( without rinsing ) ( mg/g DW )
( mg/L )
0.015 A± 0.006
0.018 A± 0.005
0.016 A± 0.006
0.011 A± 0.002
0.45 A± 0.13
0.33 A± 0.19
0.49 A± 0.15
0.41 A± 0.08
2.61 A± 0.72
1.56 A± 0.38
3.45 A± 0.53
1.87 A± 0.32
3.17 A± 0.56
1.93 A± 0.66
6.88 A± 1.22
2.57 A± 0.19
3.65 A± 0.81
1.97 A± 0.71
8.18 A± 1.35
2.93 A± 0.29
4.28 A± 0.83
3.82 A± 0.67
3.64 A± 0.57
8.71 A± 0.56