influence of soil type and environmental conditions on zno, tio2 and ni nanoparticles phytotoxicity

Chemosphere xxx (2013) xxx–xxx

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Chemosphere

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Influence of soil type and environmental conditions on ZnO, TiO2 and Ninanoparticles phytotoxicity

0045-6535/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.chemosphere.2013.02.048

⇑ Corresponding author. Tel.: +48 81 5248160; fax: +48 81 5248150.E-mail address: [email protected] (P. Oleszczuk).

Please cite this article in press as: Josko, I., Oleszczuk, P. Influence of soil type and environmental conditions on ZnO, TiO2 and Ni nanoparticles phicity. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.02.048

Izabela Josko, Patryk Oleszczuk ⇑Department of Environmental Chemistry, Maria Curie-Skłodowska University, 3 Maria Curie-Skłodowska Square, 20-031 Lublin, Poland

h i g h l i g h t s

� Phytotoxicity of NPs in three differentsoils were tested.� The range of toxicity varied

depending on the soil and thenanoparticles.� The relationship between dose–effect

was only observed in the OECD soil.� NPs aging and increase of incubation

temperature caused a reduction oftoxicity.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 20 September 2012Received in revised form 13 February 2013Accepted 20 February 2013Available online xxxx

Keywords:Risk assessmentPhysico-chemical propertiesLepidium sativumNanoparticlesPhytotoxicity

a b s t r a c t

Intensive development of nanotechnology will result in releasing nanoparticles (NPs) to the environmentincluding soil. The objective of the study was the evaluation of phytotoxicity of inorganic nanoparticlesand their bulk counterparts (ZnO, TiO2 and Ni) in various soils using Phytotoxkit F™ method. The estima-tion of toxicity was conducted with relation to Lepidium sativum. The toxicity of NPs was also estimated inrelation to contact time between NPs and soil, effect of light and temperature and NPs–NPs interactions.In all tested variants no effect of NPs on seed germination was observed. NPs displayed varied effect oninhibition of plant root growth in relation to soil type. Only in the case of ZnO nanoparticles and theirbulk counterparts a dose–effect relationship was observed. That relationship, however, was observedonly in OECD soil. In a majority of cases, aging and increase of temperature caused a reduction of toxicityof NPs, while light conditions increased the toxic effect of NPs. The effect of the NPs interaction: ZnO withTiO2 or Ni had an antagonistic character, that was manifested in a reduction of the toxicity of ZnO.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Intensive development of nanotechnology is manifested inincreasing use of its products in various branches of the economy.The unique properties of nanoparticles (NPs), acquired thanks totheir ‘‘nano’’ size, permit an extensive range of applications. ZnO

(nZnO) and TiO2 (nTiO2) nanoparticles find an application in cos-metics, sunscreen products, pigments, solar cells and photocataly-sis (Ju-Nam and Lead, 2008). Ni nanoparticles (nNi), on the otherhand, are used in production catalysts, battery electrodes and die-sel–fuel additives (Gong et al., 2011). The growing scale of produc-tion of NPs involves the risk of their release into the environment(Gottschalk and Nowack, 2011), including the soil. Influx of NPs tothe soil may appear from various sources that may be point-type(incidental outflow from production sites) (Gottschalk and

ytotox-

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Table 1Physico-chemical properties of soils used in the experiment.

Properties Soils

OECD GL1 GL2

Sand 50 61 14Silt 31 36 76Clay 19 3 10pH 6 4.7 6.7TOC 4 1.6 11.7Nt 0.01 1.1 1.4TOC/Nt 400 1.5 8.4CEC 88.8 52.7 99.1TEB 70 19.2 86.6K2O 0.3 1.5 2.6P2O 0.2 0.7 5.5Mg 0.6 0.3 1.1Na – 0.2 0.8Ca – 5.5 27.9Zn n.d. 18.8 39.6Ni n.d. 1.1 5.66

pH, reactivity in KCl; TOC, total organic carbon content (g kg�1); Nt, total nitrogencontent (g kg�1); CEC, cation exchange capacity (mmol kg�1); TEB, the total of theexchangeable bases (mmol kg�1); P2O5, K2O, Mg, available forms of phosphorous,potassium and magnesium (mg kg�1); Na, Ca, Zn, Ni – content (mg kg�1).

2 I. Josko, P. Oleszczuk / Chemosphere xxx (2013) xxx–xxx

Nowack, 2011) or area-type, being an effect of use of nanoproducts(e.g. nTiO2 released from the walls of buildings) (Gottschalk andNowack, 2011). NPs can also be indirectly introduced into the soilas a sensor for detecting plant pathogens (Khot et al., 2012), as theconstituents of fertilizers, pesticides or herbicides (Gogos et al.,2012) or together with the application of sewage sludge in whichthey can be present (Kim et al., 2012a). Therefore, there is necessityof research aimed at the estimation of toxicity of NPs, not only inwaters but also in such matrices as soils or sediments.

The effects of the presence of nanoparticles in the soil environ-ment have been widely discussed in studies conducted so far, butthose were concerned primarily with bacteria (Ge et al., 2011),crustaceans (Manzo et al., 2011) and earthworms (Shoults-Wilsonet al., 2011), while reports concerning the phytotoxicity of NPs inthe soil are scarce (El-Temsah and Joner, 2012; Du et al., 2011;Kim et al., 2011; Lee et al., 2012). Until now, a majority of studieson plants were conducted in hydroponic cultures (Geisler-Leeet al., 2012; Kim et al., 2012b) or on agar media (Lee et al., 2008;Lee et al., 2010). The phytotoxicity of NPs was manifested mainlyin the form of inhibition of germination and the growth of plantroots (Lin and Xing, 2007; El-Temsah and Joner, 2012; Manzoet al., 2011; Lee et al., 2012). Studies have also shown that nano-particles may undergo accumulation in plants. Geisler-Lee et al.(2012) observed accumulation of Ag NPs in Arabidopsis thaliana.Also Wang et al. (2012) noted accumulation of CuO NPs in maize(Zea mays). In those studies, transport of NPs within plants was ob-served, which permitted CuO NPs easier contact with various or-gans of the plants. The studies indicate the existence of threatsrelated not only with the direct effect of nanoparticles on soilorganisms and plants, but also with the risk of their accumulationin organisms and the related food transfer (Rico et al., 2011).Therefore, it is important to determine the toxicity of NPs withrelation to various plants in the first stage of the research, and toidentify the factors that affect that process.

The effect of nanoparticles on soil organisms and plants can bediverse, depending on the kind of nanoparticles, their concentra-tion, size, chemical composition or functionality (Ge et al., 2011;Rico et al., 2011; Shoults-Wilson et al., 2011). Studies focused pri-marily on the aquatic environment show that matrix propertiessuch as the pH, ionic strength, concentration and composition ofnatural organic matter (NOM) as well as different salts may havean effect on the level of toxicity of nanoparticles (Ju-Nam and Lead,2008). However, studies concerning the phytotoxicity of nanopar-ticles in relation to variable environmental conditions are stillscarce (El-Temsah and Joner, 2012; Du et al., 2011; Kim et al.,2011; Lee et al., 2012). The knowledge gap relates in particularto studies comparing the toxicity of NPs in various soils, i.e. in soilwith different physicochemical properties. Studies conducted todate concerning heavy metals demonstrate that such factors aspH, cation exchange capacity (CEC), NOM content may modifythe phytotoxicity of NPs (Kabata-Pendias and Pendias, 1993).

ZnO and TiO2 NPs are widely used in the consumer productswhich needs the detailed assessment of their potential toxicity toplants (Ju-Nam and Lead, 2008; Gottschalk and Nowack, 2011; Go-gos et al., 2012). Regarding to Ni NPs there is no data about its tox-icity to different groups of organisms including plants. Theobjective of the study was the estimation of phytotoxicity ofZnO, TiO2 and Ni nanoparticles with relation to Lepidium sativum.In the experiment L. sativum (cress) was chosen because of its highsensitivity to different contaminants. The scope of the study com-prised the estimation of (i) the toxicity of nanoparticles in relationto the type of soil, (ii) the effect of various conditions of soil incu-bation (contact time between NPs and soil, light conditions andtemperature, and the synergistic/antagonistic effect betweennanoparticles).

Please cite this article in press as: Josko, I., Oleszczuk, P. Influence of soil type aicity. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.02.0

2. Materials and methods

2.1. Materials

Nanoparticles nZnO, nTiO2, nNi and their bulk counterparts(bZnO, bTiO2 and bNi) were purchased from Sigma–Aldrich(USA). The primary particle size of nanoparticles was as follows:nZnO < 50 nm (nZnO50), nZnO < 100 nm (nZnO100); nTiO2 < 21 -nm (nTiO2); nNi < 100 nm (nNi). Transmission electron microscope(TEM) images of the tested nanoparticles were obtained by JEM-3010 TEM (JEOL, Ltd., Japan) (Fig. S1, Supporting information). Sur-face area (obtained from Sigma–Aldrich) of nZnO50, nZnO100,nTiO2, was 10,8; 15–25; 35–65 m2 g�1, respectively.

2.2. Soil preparation and characteristics

Three different soils with different physico-chemical propertieswere selected for the presented study: OECD, Haplic Podzol orginat-ing from sand (GL1) and Haplic Luvisol orginating from silt (GL2)(Table 1). Soils GL1 and GL2 were taken from the surface horizon(0–20 cm), from locations in the arable areas of south-east Poland(the area is not exposed to industrial and urban contamination).OECD soil was purchased from the microbiotest (Belgium). OECDartificial soil is a widely used substrate in soil toxicity tests. Ithas been recommended as a medium for ecotoxicological testsand it is a reference soil in the testing of complex solid samples.

Soils (GL1 and GL2) were air-dried and mixed to obtain repre-sentative sample. Soils were sieved through 1 mm sieve for chem-ical and ecotoxicological analysis. Soil OECD was used in the formas delivered by the manufacturer. The chemical properties of soilsstudied were determined by standard methods. The particle sizedistribution of the soils was assayed with the areometric method.The pH was measured potentiometrically in 1 M KCl after 24 h inthe liquid/soil ratio of 2,5, the total of the exchangeable bases(TEB) were determined in the 0.1 N HCl extract. The cation ex-change capacity (CEC) and concentrations of P2O5, K2O, Mg, Ca,Na were determined according to ‘‘Procedures for Soil Analysis’’(van Reeuwijk, 2002). Total organic carbon (TOC) was determinedby TOC-VCSH (SHIMADZU) with Solid Sample Module SSM-5000.The total nitrogen (Nt) was determined by the Kjeldahl’s methodwithout the application of Dewarda’s alloy (Cu–Al–Zn alloy-redu-cer of nitrites and nitrates).

nd environmental conditions on ZnO, TiO2 and Ni nanoparticles phytotox-48


I. Josko, P. Oleszczuk / Chemosphere xxx (2013) xxx–xxx 3

Soil GL1 had lower pH than other two soils (GL2 and OECD).Investigated soils varied significantly in TOC content. The lowestTOC content was observed for soil SL1. The content of TOC in soilsSL2 and OECD was markedly higher. The Nt content was at the sim-ilar level in soil SL1 and SL2 while in the soil OECD was much low-er. The CEC values determined for GL1 soil showed the lowestvalue (52.7 mmol kg�1), while for OECD and SL2 soils the CEC val-ues were almost twice high comparing to GL1 soil.

2.3. Experiments

In the experiment where the effect of nanoparticles on the tox-icity of different soils was explored, GL1, GL2 and OECD soils werespiked with nanoparticles as a powder of ZnO and TiO2 at the con-centration of 10,100, 1000 and 10,000 mg kg�1 and Ni at the con-centration of 10,100, and 1000 mg kg�1. The concentrations at thelevel of 10–100 mg kg�1 correspond with natural content of theseelements in various soils. The higher concentrations were chosento check the potential consequences of soil contamination byNPs. OECD, SL1, SL2 soils (without nanoparticles) were used ascontrol samples. Samples of soil contaminated by NPs or bulkcounterparts were thoroughly mixed with a glass spatula, rolledend over end for about 30 min and placed in the test plate (Sup-porting information). Each sample was prepared in three replica-tions, and the final results were the arithmetic means.

Further experiments investigated the effects of: (i) contact timebetween nanoparticles and the soil, (ii) light conditions, and (iii)temperature on phytotoxicity. NPs were spiked at the concentra-tion of 100 mg kg�1. Soil GL1, GL2 and OECD were spiked withNPs and mixed according to the procedure described above. Aftermixing, samples were incubated for 90 day. In the part of theexperiment concerning of the light conditions effect, samples wereincubated in darkness or exposed to daylight. In the part of theexperiment with the effect of temperature, samples were incu-bated in darkness at constant temperatures of 8 ± 0.5 �C and23 ± 0.7 �C. Next, in all the cases, the effect of nanoparticles wasdetermined at temperature of 25 �C in accordance with the proce-dure described in Section 2.4.

In the experiment where the influence of interactions betweennZnO and nTiO2 or nNi, and between bZnO and bTiO2 or bNi wastested, the particular components were introduced into OECD soilas a powder. The objective of that part of the experiment was theestimation of the effect of nNi and nTiO2 on the toxicity of nZnO50and nZnO100. Also the interactions between the bulk counterpartswere analyzed. The particular components were applied to the soilat the dose of 1000 mg kg�1.

2.4. Phytotoxicity determination

Toxicity of nanoparticles and their bulk counterparts was as-sessed with the commercial toxicity bioassay – Phytotoxkit™ Test(Phytotoxkit, 2004). The Phytotoxkit™ measures the decrease (orthe absence) of seed germination and of the growth of the youngroots after 3 day of exposure of seeds of selected higher plants tocontaminated matrix in comparison to the controls in a referencesoil. The bioassays were performed in three replicates. The percentinhibition of seed germination (SG) and root growth inhibition (RI)were calculated with the formula:

SG=RI ¼ ðA� B=AÞ � 100

where A – mean seed germination, root length in the control soil; B– mean seed germination, root length in the test soil.

Statistically significant differences between the results wereevaluated on the basis of standard deviation determinations andon the analysis of variance method (Statistica 5.0; StatSoft, Tulsa,OK, USA).


3. Results

In none of the variants of the experiment any significant effectof the nanoparticles under study on seed germination was ob-served. Therefore, further discussion of results is focused only onthe inhibition of root growth.

3.1. Effect of soil type on nanoparticles toxicity

The toxicity of nanoparticles was significantly determined bythe soil type (Fig. 1). Only in OECD soil a correlation was found be-tween the dose of nZnO100 and the observed toxic effect. Whereas,no such relation was observed for GL1 and GL2 soils. In OECD soilthe lowest doses of nZnO (10, 100 mg kg�1) had a stimulating(10 mg kg�1) or no effect (100 mg kg�1) effect on root growth. Insoil GL1 also no toxicity of nZnO100 (10–100 mg kg�1) was noted,and its presence significantly stimulated root growth of L. sativum.At higher concentrations of nZnO100 (1000–10000 mg kg�1) con-siderable inhibition of root growth was observed both in OECD soiland in GL1. The values of that inhibition, however, were statisti-cally significantly lower in GL1 soil compared to OECD soil(Fig. 1A). The addition of nZnO100 to soil GL2, with the exceptionof the concentration of 100 mg kg�1, had a stimulating effect onroot growth of L. sativum (Fig. 1A).

Among the three soil studied, to which nTiO2 was added, thehighest nTiO2 toxicity was characteristic of soil GL2 (Fig. 1B). Rootgrowth inhibition in that soil, irrespective of the concentration ofNPs, was at the level of 20–44%. In soil GL1, the addition of nTiO2

at concentrations of 10,100 and 10,000 mg kg�1 caused stimulationof root growth in relation to the soil without any addition of NPs. Itshould be mentioned, however, that the tendency observed for thelowest concentrations of nTiO2 was not statistically significant,indicating rather a lack of any effect (Fig. 1B). Only nTiO2 concen-tration at the level of 1000 mg kg�1 caused a distinct toxic effect,and at the highest concentration (10,000 mg kg�1) nTiO2 had againa stimulating effect on root growth. As mentioned earlier, in thecase of OECD soil, within the range of concentrations from 10 to1000 mg kg�1, a decrease of toxicity with increasing concentrationof nTiO2 was observed, while the highest concentration caused atoxic effect.

The degree and character of the effect of nNi on root growth of L.sativum was varied and depended on its concentration and on the soiltype (Fig. 1C). In soil GL1, irrespective of its concentration, nNi had astimulating effect, the strongest positive effect being noted for thelowest concentration (10 mg kg�1). In the case of soil GL2, the lowestconcentration of nNi also had a positive effect on root growth of L. sat-ivum in relation to the soil with no addition of NPs. At higher concen-trations of nNi (>100 mg kg�1) an inhibition of root growth wasobserved in soil GL2. As opposed to the other two soils, the presenceof nNi in the amount of 10 mg kg�1 in OECD soil caused an inhibitionof root growth of L. sativum (at the level of 22%). In OECD soil a positiveeffect of nNi on L. sativum was also observed, but that took place afterthe application of the highest dose (1000 mg kg�1). The concentrationof 100 mg kg�1 of nNi in soils OECD and GL2 caused the highest toxiceffect, at 34% and 39%, respectively.

3.2. Effect of light on nanoparticles phytotoxicity

In the case of all nanoparticles and soils under study a signifi-cant effect of light on their phytotoxicity was observed (Fig. 2).The effects of nZnO50 in the different soils on root length variedwith relation to the light conditions (Fig. 2A). In soils OECD andGL1, with access of light, a 17% inhibition of root growth was ob-served, while with a lack of exposure to sunlight nZnO50 had astimulating effect on L. sativum. In soil GL2 the effect of light



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Fig. 2. The root growth inhibition of nZnO 50 (A), nZnO 100 (B), nTiO2 (C) i nNi, and (D) in the various light conditions depending on the soil. Error bars represent standarderror (SE, n = 3 tests).

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Fig. 1. Influence of nZnO 100 (A), nTiO2 (B), nNi, and (C) on root growth inhibition of Lepidium sativum in the different soils: OECD, GL1, GL2. Error bars represent standarderror (SE, n = 3 tests).


conditions displayed an opposite tendency to that observed in theother soils. With access of light nZnO50 caused a stimulation ofroot growth of L. sativum, while in darkness they showed a declinein root length. The effect of light was also notable in the case ofnZnO100, but here distinct differences were observed only for soilsGL1 and GL2 (Fig. 2B). In OECD soil, both with access of light and indarkness, negative effects of the presence of nZnO100 were ob-served, and no significant differences were found between the par-ticular variants. Root growth inhibition was at the level of 20%(light) and 13% (darkness). Access of light reduced the positive ef-fect of nZnO100 in soil GL1, while in soil GL2 it was less toxic.

The effect of nTiO2 on root growth of L. sativum was also relatedto the availability of light (Fig. 2C). In soils OECD and GL1 a similartoxic/stimulating effect was observed in relation to light


conditions. Lack of light in the presence of nTiO2 caused a slightstimulating effect, at the level of 5–8%. Under the effect of light asignificant increase of toxicity was observed, to the level of 20%(Fig. 2C). In soil GL2, on the other hand, access of light had a totallydifferent effect on the toxicity of nTiO2 compared to soils GL1 andOECD. In soil GL2, with access of light, nTiO2 caused a stimulatingeffect at the level of 13%, while in darkness the root length de-creased by 13%.

Light conditions played also an important role in the variationof toxicity of nNi (Fig. 2D). Irrespective of the soil type, in darknessa stimulating effect on root growth of L. sativum was observed (7–11%). In soils OECD and GL1 containing nNi, access of light causedan increase in their toxicity, to the levels of 21% and 24%,respectively.




3.3. Influence of temperature on toxicity of nanoparticles to L. sativum

Temperature of incubation of soils with nanoparticles played animportant role in the effect of NPs on L. sativum, dependent on thesoil type and the kind of NPs (Fig. 3). Only in the case of nNi aninvariable tendency was observed for all the soils (Fig. 3D). Lowtemperature (8 �C) incubation of soil samples containing nNicaused their toxic effect on root growth (root growth inhibitionin the range of 4–19%). At higher incubation temperatures a stim-ulating effect was observed.

In soil GL1, all of the NPs under study displayed the same effecton L. sativum with relation to temperature. At low incubation tem-perature an inhibition of root growth was observed, while increaseof the incubation temperature to 23 �C caused a stimulation of rootgrowth (Fig. 3A–D). In OECD soil temperature influenced the ef-fects of NPs as in soil GL1, with the exception of nZnO100. In thatsoil, incubated at higher temperature, nZnO100 caused aninhibition of root growth at the level of 13% (Fig. 3B). Increase ofincubation temperature of soil GL2 caused in increase in toxicityin the case of nZnO50, nZnO100 and nTiO2, by 20%, 24% and 32%,respectively. As mentioned above, the influence of temperatureon the effect of nNi in soil GL2 was expressed by the following rela-tion: the higher the temperature – the lower the level of inhibition.

3.4. Effect of interactions between nanoparticles on their phytotoxicity

The toxicity of nanoparticles present in soil concurrently dif-fered from the levels of toxicity caused by the nanoparticlesappearing individually (Fig. S2, Supporting information). In thestudy, estimation was made of the effect of nNi and nTiO2 on thetoxicity of nZnO50 and nZnO100. Both nNi and nTiO2 caused a sig-nificant reduction in the toxicity of nZnO.

The toxicity of nZnO50 was reduced by nNi to a greater degreethan that of nZnO100, by 4% and 15%, respectively, as compared to

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Fig. 3. Effect of temperature on the root growth inhibition of L. sativum in soil with nastandard error (SE, n = 3 tests).


nZnO without an addition of nNi (Fig. S2A, Supporting informa-tion). The addition of nTiO2 to a soil containing nZnO also resultedin a reduction of inhibition of both nZnO50 and nZnO100. Theaddition of nTiO2 to nZnO50 and nZnO100 caused a reduction oftoxicity by 12% and 17%, respectively, with relation to nZnOappearing individually. Comparing the effect of nNi on the toxicityof nZnO with that of nTiO2, it should be noted that the presence ofnNi had a more favorable effect on the reduction of toxicity ofnZnO50, while nTiO2 reduced the toxicity of nZnO100 to a greaterdegree than nNi (Fig. S2B, Supporting information).

A reduction of toxicity was also observed in the case of jointappearance of bZnO and bNi. That effect is described in greater de-tail in the Supporting information. Their simultaneous presence inOECD soil brought a positive effect in the form of reduction of tox-icity at the level of 6% (Fig. S2C, Supporting information). Whereas,joint presence of bZnO and bTiO2 resulted in an increase of thetoxic effect, by 7% and 13%, respectively, with relation to bZnOand bTiO2 occurring individually.

3.5. Effect of NPs aging on the phytotoxicity

In OECD soil, in the case of almost all of the NPs studied (withthe exception of nZnO100), extension of the contact time betweenthe soil and the NPs resulted in a reduction of their toxicity (Fig. 4).In this case, a distinct stimulating effect on root growth of L. sati-vum was observed. Likewise, as positive effect of aging was notedin soil GL1, enhancing the stimulating effect of time on rootgrowth. The exception, as in OECD soil, was nZnO100 for whichalso in soil GL1 a weakening of the positive effect on root growthwas observed after the extension of the time of contact with thesoil (Fig. 4B). In soil GL2 an opposite tendency was observed rela-tive to soils OECD and GL1 (Fig. 4). Also, in soil GL2 aging dependedto a greater extent on the type of nanoparticles. After a 90-day

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Fig. 4. The aging effect on the root growth inhibition of L. sativum in different soils with nanoparticles nZnO50 (A), nZnO100 (B), nTiO2 (C) and nNi (D). Error bars representstandard error (SE, n = 3 tests).


period of incubation nZnO50 displayed a toxic effect, while in thecase of immediate testing a slight stimulating effect was observed(Fig. 4A). In the case of nZnO100, extension of the time of its con-tact with soil GL2 amplified its negative effect on root growth of L.sativum. In the case of nTiO2, the time of contact had a positive ef-fect on root growth of L. sativum in all the soils, stimulating rootgrowth (in soils OECD and GL1) or reducing its negative effect(from 20% to 12%) (Fig. 4C).

The effect of aging on the phytotoxicity of nNi was different insoil GL1 than in soils OECD and GL2 (Fig. 4D). Extension of the timeof contact of nNi with soils OECD and GL2 caused a notable reduc-tion of the toxicity of nNi, by 43% and 46%, respectively. In soil GL1,on the other hand, a negative influence of aging on the effect of nNion L. sativum was observed. Extension of the time of nNi-soil GL1contact caused a reduction of root growth stimulation by 15%.

Depending on the soil type, varied influence of aging on the ef-fect of bulk counterparts of NPs was observed (Fig. S3, Supportinginformation). In OECD soil aging had a positive effect in the reduc-tion of toxicity caused by bTiO2, while the effects caused by bZnOand bNi displayed small differences with relation to the time ofcontact with soil. In soil GL1 aging had a negative effect on thestimulation caused by bZnO and bNi. After 90 day of incubation,in the case of bZnO a notable reduction of root growth stimulationwas observed, while nNi, after extended time of contact with soilGL1, caused a toxic effect (8%). The time of contact of bTiO2-soilGL1 had a favorable effect on the stimulating character of bTiO2.In soil GL2, aging had a distinctly positive effect on the reductionof toxicity caused by bZnO and bNi. Whereas, 90-day period ofincubation of bTiO2 with soil GL2 caused an increase in the toxicityof bTiO2 by 21%.

4. Discussion

Estimation of the toxicity of nanoparticles is an extensivelystudied problem which takes into account various test organismsinhabiting diverse environments (Ju-Nam and Lead, 2008;


El-Temsah and Joner, 2012; Ge et al., 2011; Manzo et al., 2011).Plants are an extremely important object of research, as they con-stitute the base of animal and human nutrition. So far studies onthe phytotoxicity of nanomaterials have been focused primarilyon the estimation of inhibition of germination and root growth inhydroponic cultures (Lin and Xing, 2008; El-Temsah and Joner,2012; Zhou et al., 2011; Geisler-Lee et al., 2012). Whereas, thereis a lack of data concerning the effect of nanoparticles on plantsin the soil environment. It should be emphasized that tests withthe use of water solutions, in spite of the plentiful information thatthey provide, do not reflect the real conditions of the environmentof land plants. In this situation the information about the risk in-volved in the presence of nanoparticles in the environment maybe insufficient. In this study, the effect of ZnO, TiO2 and Ni nano-particles on plant was studied not only in the aspect of soils withvaried physicochemical properties, but also taking into accountother important factors (environmental conditions – temperature,light, contact time between NPs and soil, synergistic/antagonisticeffect between NPs). As demonstrated in the study, the above fac-tors affect the toxicity of nanoparticles in every case, significantlymodifying its levels.

The study showed that the NPs affected on the growth of L. sat-ivum roots, however no impact on seed germination was observed.Research carried out by Lin and Xing (2007) showed that the seedgermination in presence of NPs depend on the type of NPs andplant species. Probably in this case the crucial is seed coats con-struction. It has been showed that the pores exhibit selective per-meability to various substances (Wierzbicka and Obidzinska,1998).

Basically, the degree of toxicity of NPs and their bulk counter-parts towards L. sativum (within the range of the doses applied)can be presented as the following sequence: ZnO > Ni > TiO2. Instudies conducted so far on the phytotoxicity of various NPs, inmany cases nZnO was also characterized by the highest toxicity(Lin and Xing, 2007; Lee et al., 2010). It is noteworthy that Niwas not strongly toxic in the study presented here. Kabata-Pendias




and Pendias (1993) report that plants, especially cruciferous plants(and thus L. sativum) have the ability of accumulation of notableamounts of Ni, without causing any toxic effects. Other studiesconcerning the effect of heavy metals on L. sativum indicated con-siderably higher toxicity of Ni than Zn (Montvydiene and Marciu-lioniene, 2004). In our study, however, this was not confirmedneither in the case of the NPs nor of their bulk counterparts.

The response of plants to NPs can be determined by the matrix.As shown by numerous studies (Zheng et al., 2005) the environ-ment can play an important role in the effect of NPs on organisms(Lee et al., 2012). Unfortunately, there is a lack of research concern-ing that problem in relation to the nanoparticles used in this study.In this respect, the best studied group are the nanoparticles of sil-ver (nAg). Studies by Lee et al. (2012) on the phytotoxicity of nAgdemonstrate that in the case of agar being used as the matrix, thegrowth of mung bean (Phaseolus radiatus) and sorghum (Sorghumbincolor) was significantly inhibited in the presence of the NPsstudied (EC50 13 and 26 mg/L, respectively). Whereas, applicationof nAg directly to the soil, within the range of concentrations of100–2000 mg kg�1, did not cause any significant effect of the NPson the growth of the plants. Soil is an environment in which themultitude of components may affect the behavior of NPs. Amongthose the most important include organic matter, soil colloids,and nutrients. These components may have a particular effect onthe aggregation of NPs, which significantly regulates their behaviorin the environment. The susceptibility of nanomaterials to homo-aggregation (NPs–NPs) causes a weakening of the mobility of thosestructures, due to which their bioavailability is reduced, and thus atoxic effect is exerted (Phenrat et al., 2009; Hotze et al., 2010). Theprocess of aggregation is subject to the effect of such factors as(Hotze et al., 2010): pH, ionic strength, presence of organic matter,and kind of cations present in the soil. All of those parameters varyin relation to the type of soils, so they also have an indirect effecton their phytotoxicity. Studies have shown that high ionic strengthand low dissolved organic carbon content increased the suscepti-bility of nTiO2 to aggregation in soil solutions (Fang et al., 2009).French et al. (2009) observed greater homo-aggregation of nTiO2

in the presence of bivalent ions than of monovalent ions. Thatstudy, however, was concerned with water solutions.

However, homo-aggregation in the soil environment can belimited by hetero-aggregation, as a result of which NPs-biomole-cule conglomerates are produced (for example, with NOM, col-loids) (Hotze et al., 2010). NOM, covering the surface of NPs,prevents their homo-aggregation (repulsive forces), but it also re-duced their availability to living organisms (Lee et al., 2011), indi-rectly reducing also their toxicity. In addition, NOM, rich in variousfunctional groups, can form complexes with NPs, which may alsocontribute to a reduction of their bioavailability (Lee et al., 2011).In the study presented here, nZnO (the most toxic on the NPs stud-ied) did not cause any inhibition of root growth of L. sativum in soilGL2. This was probably related with the higher concentration ofTOC than in the other soils (Table 1). That relation, however, didnot reappear in relation to soils OECD and GL1. In spite of highercontent of TOC, OECD soil was characterized by higher toxicitythan soil GL1. Another factor that could have a significant effecton the aggregation of NPs, and indirectly also the differences inphytotoxicity among soils, are soil colloids (e.g. clays). In a studyby El-Temsah and Joner (2012) germination inhibition was consid-erably lower in a clay soil than in a sandy soil. This is confirmed inour study in soil GL2, but again does not find support in soils GL1and OECD (Hotze et al., 2010). The toxicity of NPs can be influencedby such properties of soils as cation exchange capacity (CEC) andthe content of P or Ca. Higher value of CEC may determine greaterexchangeable sorption of NPs, and thus their immobilization. SoilGL2, characterized by the highest level of CEC compared to theother soils under study, very often did not display any toxic


properties after the addition of nZnO. However, in OECD soil, whichhad a higher value of CEC than soil GL1, a reverse tendency was ob-served. In that latter case, that could have been determined by thepresence of P. Soil GL1 was characterized by a notably higher con-tent of P2O5 (7.1 mg kg�1) as compared to OECD soil (1.9 mg kg�1)which, as demonstrated by study (Kabata-Pendias and Pendias,1993) may inhibit phytoavailability of Zn ions and their toxic effecton plants. The differences in the levels of toxicity between soilsOECD and GL1 may be also determined by the origin and composi-tion of organic matter, as well as by the mineral composition of thecolloidal fraction (differences in specific surface areas of minerals),that may have a potential importance in the limitation of availabil-ity of NPs. That problem requires further research.

Changing environmental conditions may be another factordetermining the toxicity of nanoparticles in various soils (Maet al., 2012). So far no studies of this type have been conductedwith relation to nanoparticles occurring in soils. Our study demon-strated that light conditions significantly determine the responseof L. sativum to the presence of nanoparticles. In the case of soilsOECD and GL1, solar radiation caused an increase of toxicity of soilscontaminated with nanoparticles as compared to the variant withlimited access of light. This may be related with the photocatalyticproperties of ZnO or TiO2, and with the production of reactiveoxide species (ROS). Increased toxicity of NPs under the effect ofsunlight was confirmed in a study concerning Daphnia magnaand Oryzias latipes (Ma et al., 2012). In soil GL2, however, an oppo-site tendency was observed (with the exception of nickel). Again,this may be related with higher content of organic matter whichreduces the reactivity of NPs through adsorption.

As indicated by studies conducted so far, incubation tempera-ture has an effect on the toxicity of heavy metals. Research by Khanet al. (2006) demonstrated an increase of toxicity of heavy metals,including Zn, with increase of temperature (17–27 �C). This foundsupport in this study with relation to nNi, irrespective of the typeof soil. In the case of the other NPs, the above tendency was ob-served also in soils OECD and GL1, while in soil GL2 increase oftemperature resulted in an increase of toxicity of the NPs.

The transfer of NPs to the soil environment is inseparably con-nected with permanent exposure to interactions with the compo-nents of the soil. Apart from the interactions with NOM or clays,mentioned earlier, it is interesting to identify the mutual interac-tions of the particular NPs. The more so as the study presented hereindicates that the mutual effects of the NPs may be different thanthose observed for their bulk counterparts. The interactions ofnZnO100-NPs (nNi and nTiO2) had an antagonistic character whichwas expressed in a reduction of phytotoxicity. The effects of themutual interactions among the NPs may depend on the characterof Zn, Ti, Ni and their affinity to NOM and other soil components(Kabata-Pendias and Pendias, 1993). However, so far no researchhas been conducted on the effect of NPs interactions on plants orother living organisms. Studies performed so far have concentratedmainly on analyzing the effect of NPs on the toxicity of other pol-lutants, indicating in most cases an increase of the toxic effect (Jos-ko and Oleszczuk, in press).

The study of the effect of aging permits the estimation of howNPs may behave in the environment, as longer time of contact ofNPs-soil induces various interaction of the NPs with the soil com-ponents. This may have a bearing on the mobility and bioavailabil-ity of NPs (Lee et al., 2011). Extended time of contact of NPs–soilcreated a greater chance for interactions of the NPs with soil com-ponents, NOM or clays, which could have reduced their negativeeffect on L. sativum. In many cases the extension of the time of con-tact between the NPs and the soils caused a reduction of toxicity.There were also exceptions where aging resulted in an increaseof toxicity. In soil GL2, the toxicity of nZnO after a 90-day periodof incubation increased. This is particularly noteworthy as Zn




forms permanent complexes with organic matter (Feizi et al.,2012), which was confirmed by the reduction of toxicity of bZnO(Fig. S3A, Supporting information). No similar phenomenon wasobserved in the case of nZnO, which suggests different mecha-nisms of binding between the NPs and the soil matrix. This indi-cates the need for further research concerning with the behaviorof NPs in soil and the related effects of aging.

5. Conclusion

As demonstrated by the study, the effect of nanoparticles onplants is determined by numerous factors that operate within thenanoparticles–soil–plant system. Detailed understanding of thoserelations is of extreme importance in the estimation of risk in-volved in the occurrence of nanoparticles in the environment.The results obtained revealed considerable differences in the levelsof phytotoxicity observed in hydroponic experiments and thoseobtained in this study. Presented studies may indicate a smallthreat to plants, which from the viewpoint of agriculture doesnot create a risk of yield losses. However there is a risk like uptakeof the NPs, genotoxicity, cytotoxicity, etc. which was not investi-gated in this study. Moreover, NPs accumulated in the soil environ-ment constitute a threat to other organisms (bacteria, fungi,earthworms), not only to plants. NPs can be leached from the soiland migrate to ground waters, which also carries serious environ-mental consequences.

The choice of soil as the matrix was dictated by the richness ofthat environment in components that may have an effect on thebehavior and destiny of NPs in the environment. The use of soilas the matrix permits the recognition of the scale of the problem.The soil has its ‘‘defense mechanisms’’ in the form of various com-ponents that alleviate the toxicity of NPs (NOM, clays, microele-ments). Whereas, contrary to earlier reports, factors that canaffect the level of toxicity of NPs (aging, temperature, light) displayvarious effects in different soils. Therefore, discussing the toxicityof NPs one should keep in mind not only the character of the NPsas such, but also a number of environmental variables. The envi-ronmental variables and soil properties enumerated above arethe cause of the limited possibility of predicting the fate of NPsin the soil, as well as in other elements of the environment. How-ever, it is necessary to undertake efforts aimed at putting togetherthe most important external factors that could determine the effectof NPs under various conditions – which may bring us closer to areliable estimate of the potential consequences.

Among the NPs under study, only in the case of ZnO a significantrelation was observed between the dose and the toxic effect. No suchrelation was observed in the case of the other nanoparticles understudy. Also the range of toxicity varied with relation to the soil typeand to the nanoparticles tested, indicating that depending on the soiltype there may be significant differences in the observed levels oftoxicity. The lack of the dose–effect correlation will make it difficultto determine permissible concentration of NPs in the soil, signaling apotential consequences to agricultural cultivations and to yieldquality, constituting a risk to animal and human health.

The results presented here relate to the first research that takesinto account not only the effect of soil type on phytotoxicity, butalso of other important factors that may have an effect on the tox-icity of nanoparticles. Further studies are necessary, with the use ofvarious soil types and NPs, and taking into account external factorsaffecting the toxicity of NPs.

Acknowledgment

The work was funded in the frame of Grant no. N523 616639 fi-nanced in 2010–2012 from the budget of Ministry of Science andInformation Society Technologies (Poland).


Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.chemosphere.2013.02.048.

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influence of soil type and environmental conditions on zno, tio2 and ni nanoparticles phytotoxicity

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