Effects of Graphene on Larix Olgensis Seedlings and Soil Properties Of

Article Eﬀects of Graphene on Larix olgensis Seedlings and Soil Properties of Haplic Cambisols in Northeast China

Jinfeng Song 1 , Kai Cao 2, Chengwei Duan 1, Na Luo 1 and Xiaoyang Cui 1,* 1 Key Laboratory of Sustainable Forest Ecosystem Management-Ministry of Education, School of Forestry, Northeast Forestry University, 26 Hexing Road, Harbin 150040, China; [email protected] (J.S.); [email protected] (C.D.); [email protected] (N.L.) 2 Daqing Wanfang Economic Development Corporation, Daqing, Heilongjiang 163411, China; [email protected] * Correspondence: [email protected]; Tel.: +86-137-9660-8073

Received: 14 January 2020; Accepted: 24 February 2020; Published: 27 February 2020

Abstract: We investigated the impacts of graphene application at different concentrations on the growth and physiological characteristics of Changbai larch (Larix olgensis A. Henry) seedlings and the chemical properties and enzyme activities of Haplic Cambisols under these seedlings. The aim is to evaluate the environmental effects of graphene on the afforestation species and the zonal forest soils of 1 Northeast China. Seedlings receiving 0 (CK), 25, 50, 100, 250, or 500 mg L− graphene were incubated 1 for 30, 40, or 50 days. Low concentrations (25–50 mg L− ) of graphene increased the dry masses 1 of root, stem, and leaf; however, high concentrations (100–500 mg L− ) inhibited them. Compared 1 with those under 0 mg L− graphene, the root length, surface area, volume, and average diameter all increased during the early stages of incubation (i.e., 30 and 40 days) under low concentration of 1 1 graphene (<50 or 100 mg L− ) and decreased at higher graphene concentration (>100 mg L− ); at 50 days, they were significantly inhibited. At 30 days, graphene decreased superoxide dismutase (SOD) and peroxidase (POD) activities, as well as pigment, soluble protein, and proline contents, and the decline increased with increasing graphene concentration; at 40 and 50 days, the above parameters 1 increased initially and then decreased, reaching a maximum at 50 mg L− . The changes in relative conductivity and malondialdehyde (MDA), superoxide anion and hydrogen peroxide contents were the opposite of those in the physiological indexes mentioned above. Therefore, graphene caused different degrees of oxidative stress in L. olgensis seedlings. At 30 days, graphene generally increased the organic matter, hydrolytic nitrogen, and available phosphorus and potassium contents of Haplic Cambisols, but these parameters decreased at 40 and 50 days. Graphene generally decreased acid phosphatase, urease, dehydrogenase, and catalase activities. Therefore, when graphene reaches a certain content level in this soil, it may also affect nitrogen and phosphorus cycling.

Keywords: Graphene; Larix olgensis; Haplic Cambisols; soil enzyme activities; physiological eﬀects; ecological environment eﬀects

1. Introduction Nanomaterials are usually defined as particles with a particle size of 1–100 nm and are also known as ultrafine particles [1]. They have many excellent optical, magnetic, electrical, thermal, chemical and mechanical properties [2]. Graphene is the thinnest and toughest two-dimensional nanomaterial in the 2 1 world [3,4] and has a thickness of only 0.34 nm, a specific surface area of 2630 m g− , and thermal 1 1 conductivity of 5000 W m− K− [5–8]. These special properties make it appropriate for widespread application in environmental applications [9], electronics [10], information technology, optics [11],

Forests 2020, 11, 258; doi:10.3390/f11030258 www.mdpi.com/journal/forests Forests 2020, 11, 258 2 of 16 energy, materials science [12], and biomedicine [9,13–15]. Like other nanomaterials, graphene is stable in the environment and is not easily degraded, which makes it inevitably enter into the atmosphere, water, soil and other environments [16]. Therefore, its use may have some impacts or potential risks to ecosystems [17], especially for the soil and plants. Regarding the effects of graphene on plant growth, only a few studies have been done, and there is considerable debate about its effects since in some species (wheat and garlic), increases have been observed in root length, root dry mass, leaf length [18–20], adventitious root elongation and main root formation [21], while in other species (cabbage, tomato, coriander, wheat, Populus tremula Linn), decreases in seedling height, germination rate, biomass, adventitious root number and leaf area were observed [20–23]. For the studies of inhibition effects [20–23], the phytotoxicity of graphene to plants is closely related to the oxidative stress response of plants [22], and reactive oxygen species (ROS) contents in plants increase with increasing graphene concentration [20–23]. Studies have also shown that appropriate concentration of graphene can promote plant growth, while high concentration significantly inhibits it [21]. Therefore, the effects of graphene on plant growth are closely related to its concentration and treatment time as well as the plant type. Soil is the basis of agroforestry production, and its biological characteristics, such as enzyme activities, and nutrient cycling, are important and effective indicators to measure the impact of pollutants or foreign substances, such as graphene, on soil biological activity and metabolic capacity [24–26]. The response of soil properties to graphene, such as soil chemical properties, enzyme activities, and nutrient cycling, is rarely studied, but the effects of multiwalled carbon nanotubes (MWCNTs), which have similar properties as graphene, on soil enzyme activities have been reported. For example, 1 there was no significant change in glucosidase or phosphatase activity under 1000 mg kg− MWCNT treatment for three months [27,28], nor in catalase, phosphatase or glucosidase activity under 1000 mg 1 kg− fullerene for six months [29]. Similarly, MWCNTs had no significant effect on urease activity [30] but inhibited acid phosphatase activity [31]. It has been found that graphene in soils can interact with organic matter, which makes graphene less likely to enter into organisms [32], and its contact with enzymes, such as catalase, is limited; thus, graphene may not induce toxic effects on plants in a short period [27]. In addition, active oxygen is the toxic effect response of soil microorganisms to graphene, but graphene entering the soil does not cause an increase in active oxygen [33] that would affect enzyme activities. At present, the focus of the above researches has been on agricultural crops, while researches on forest soil environments and plantation-grown forest tree species are limited. Haplic Cambisols are zonal forest soils in Northeast China. Larix olgensis A. Henry is one of the most productive commercial forestry species and is a useful tree species for afforestation in this area. In some transition zone of agriculture and forestry, graphene may enter Haplic Cambisols under L. olgensis forests in various ways, thus influencing soil environments and L. olgensis growth. However, how graphene may affect L. olgensis and the properties of Haplic Cambisols is still unknown. Here, we applied different concentrations of graphene to Haplic Cambisols used to grow L. olgensis seedlings and monitored the resulting changes to the growth and physiological characteristics of L. olgensis seedlings, as well as the chemical properties and enzyme activities of the Haplic Cambisols. Our aim is to monitor the responses of L. olgensis seedlings and Haplic Cambisols to graphene to scientifically evaluate the potential toxicity or risk of graphene to the soil ecological environment of Northeast China.

2. Materials and Methods

2.1. Soil Collection and Treatments The study was conducted in the greenhouse of the Maoershan Forest Research Station of Northeast Forestry University, Harbin, Heilongjiang province, China. The station is located at 127◦300–127◦340 E and 45◦210–45◦250 N with a continental temperate monsoon climate. Here, annual average temperature is 2.4 C (range: 40 to 34 C, mean annual precipitation is 700 mm, and the frost-free period is about ◦ − ◦ 125 days with a growing season ranging between 120 and 140 days. The soils used here are the loam Forests 2020, 11, 258 3 of 16

A1 horizon of the Typic Bori-Udic Cambisols corresponding to Haplic Cambisols (Grayic, Dystric) [34], the zonal forest soil of Northeast China with high organic matter content and well-developed horizons. The soils were also collected from a L. olgensis forest in May 2019. Fresh soil samples were stored in hop-pockets and taken to the laboratory. Plant residues and other impurities in the soils were removed with tweezers. Then, the samples were powdered, suﬃciently mixed, sifted through 2 mm nylon screens, and air-dried for 1 day to sterilize. The properties of the soil samples were shown in Table1.

Table 1. Selected properties of the A1 horizon of Haplic Cambisols used in the experiment (mean, n c = 3).

CEC a Organic Hydrolytic Available Available Soil pH Soil (cmol Matter N P c K Horizon (H2O) 1 b 1 1 1 1 Texture kg− ) (g kg− ) (mg kg− ) (mg kg− ) (mg kg− )

A1 5.32 40.20 87.94 462.45 45.92 145.31 loam a b c 1 Cation exchange capacity; Centimoles of cation charge per kg soil; Extracted with 0.05 mol L− HCl–0.025 mol 1 d L− H2SO4; According to United States Department of Agriculture Soil Classiﬁcation.

Pure graphene was purchased from Suzhou Tanfeng Graphene Technology Co., Ltd. (Suzhou, 1 Jiangsu Province, China). Its speciﬁc parameters were as follows: concentration, 2 mg mL− ; thickness, 2 1 <1 nm; speciﬁc surface area, 1000–1217 m g− ; sheet diameter, 0.2–10 µm; layer number, 1–2; purity, >99 wt %; color, black.

2.2. Seedling Culture and Graphene Addition L. olgensis seedlings (1a) were purchased from Maoershan Forest Research Station of Northeast Forestry University, Harbin, Heilongjiang Province, China, at the end of May 2019. The average seedling height was 13 cm, and the average ground diameter was 2.5 mm. These seedlings were 3 planted in pots (volume: 10237.5 cm ) filled with the loam A1 horizon of the Haplic Cambisols free from impurities (3.3 kg per pot, Table1). Twenty seedlings were planted in each pot, and 180 pots and 3600 seedlings in total. After preculturing the L. olgensis seedlings for two weeks, the soils in pots were treated with 1 graphene solution at concentrations of 0 (Ck), 25, 50, 100, 250 and 500 mg L− (with reverse osmosis (RO) water, pH 5.8)). The addition procedure went as follows: the graphene solution was slowly added until it had evenly mixed with the soil. The amount of graphene added to the soil was estimated based on the concentration of graphene in solution, the amount of solution, and the soil weight in each pot to 1 achieve different graphene contents of 0, 7.6, 15.2, 30.3, 75.8 and 151.5 mg kg− , respectively. Then, the seedlings and soils were incubated at 25 ◦C. The soil water content was maintained at 70% of field water capacity by adding high-purity water weekly. The experimental layout is described in Table2.

Table 2. The experimental layout. Each of the treatments below was determined after 30, 40, or 50 days.

Treatment Ck T1 T2 T3 T4 T5 Graphene concentration 0 25 50 100 250 500 (mg L 1) · −

Because of the cold climate, the experiment was set up in late May; the tree leaves begin to turn yellow and fall oﬀ in late August, precluding an accurate determination of the relevant indicators. At 30, 40, and 50 days after graphene treatments started, the sampling and analysis were conducted. There were three sampling and analysis times (i.e., 30, 40, and 50 days) after graphene treatment. For each sampling time, there were six treatments, with 10 pots employed per treatment and 180 pots in total. The harvests included 10 pots for each treatment at each time. Forests 2020, 11, 258 4 of 16

2.3. Plant Assays

2.3.1. Growth Observation, and Survival Rate and Biomass Measurements At all graphene treatment times, seedling growth was observed twice a day, i.e., at 8 o’clock in the morning and at 8 o’clock in the evening. At 50 days, dead seedlings in each pot for all the treatments were counted, and survival rate was calculated. At 30, 40, and 50 days after graphene application, a total of 15 random seedlings per treatment from 10 pots were harvested, washed with running tap water, and divided into leaf, stem, and root. The dry masses of all parts were determined after drying to a constant weight at 80 ◦C.

2.3.2. Root Morphology At 30, 40, and 50 days after graphene application, three seedlings were randomly and carefully harvested using a shovel to avoid root damage across the 10 pots within each treatment. The seedlings were washed thoroughly with running tap water, dried with ﬁlter paper, and divided into root, stem, and leaf. Root (<2 mm) morphological indicators (length, volume, surface area, and average diameter) were measured using Root System Analyzer (Win-RHI Z0-2004a, Canada).

2.3.3. Physiological Parameter Measurements Fresh leaf samples from middle needles were randomly collected from each treatment across 10 pots (approximately 2.0 g) for immediate physiological assays. First, leaf samples were fully mixed together, and the relative conductivity was measured using a Shanghai Leici Magnetic DDS-6700 conductivity meter. Then, the other samples were frozen and ground with liquid nitrogen to perform the following physiological assays immediately. Malondialdehyde (MDA) contents that measure lipid peroxidation level were determined as 2-thiobarbituric acid (TBA) reactive metabolites, superoxide anion contents using the pyrogallol method, hydrogen peroxide contents using Ti (IV)-H2O2 colorimetry, superoxide dismutase (SOD) activity using a nitro blue tetrazolium (NBT) photochemical reduction, peroxidase (POD) activity by a guaiacolassay, proline contents by the acidic ninhydrin reaction, soluble protein contents by coomassie brilliant blue G-250 staining, and chlorophyll and carotenoid contents by 80% acetone extraction-spectrophotometry. All procedures were carried out according to the methods of Li [35]. Each assay was repeated three times.

2.3.4. Determination of Soil Chemical Properties and Enzyme Activities At 30, 40, and 50 days after graphene treatment, soil samples were randomly collected from each pot (approximately 200 g), air-dried, sifted through 2 mm nylon screens, mixed, and analyzed for their chemical properties and enzyme activities. Organic matter (OM) content was obtained using a TOC (total organic carbon) analyzer; hydrolytic nitrogen (HN) content was obtained via the alkali reduction diﬀusion method; available phosphorus (AP) content was determined through the Mo-Sb colorimetric method, with a 0.05 mol L 1 HCl 0.025 mol L 1 H SO extraction; and available − − − 2 4 potassium (AK) content was determined via the photometric method. All procedures for soil chemical properties were carried out according to Chen [36]. Acid phosphatase activity was determined using disodium phenyl phosphate colorimetry [37,38], urease activity was measured by the indophenol-blue colorimetric method [39], dehydrogenase activity was found using TTC (2,3,5-triphenyltetrazolium chloride) colorimetry [37], and catalase activity was obtained using the methods of Johnson and Temple [40]. Each determination was replicated three times.

2.4. Data Analysis OriginPro 2019 (Originlab, Northampton, MA, USA) and SigmaPlot 10.0 (SYSTAT software, Chicago, IL, USA) were used to make figures. Tukey test was used to test for the treatment effects between different graphene concentrations for the same treatment time) (at p < 0.05) with SPSS 18.0 Forests 2020, 11, 258 5 of 16 Forests 2020, 11, 258 5 of 16 softwarecorrelations (IBM were Corporation, analyzed in Armonk, a randomized NY, USA). design For soilwith chemical repeated properties measures and using enzyme SPSS activities,18.0, and Pearsonfactor analysis correlations was conducted were analyzed using in OriginPro a randomized 2019 software design with (Originlab, repeated Northampton, measures using MA, SPSS USA). 18.0, and factor analysis was conducted using OriginPro 2019 software (Originlab, Northampton, MA, USA). 3. Results 3. Results

3.1. Plant Growth Attributes

3.1.1. Growth and Biomass Graphene influenced influenced the the growth growth and and biomass biomass of of L. L.olgensis olgensis seedlings,seedlings, and and the thedry drymasses masses of leaf, of leaf,stem stemand androot rootshowed showed basically basically the same the same trends trends (Figure (Figures 1, 2).1 andDuring2). During the whole the wholetreatment treatment period, period,root, stem root, and stem leaf and biomass leaf biomass generally generally increased increased at low at concentration low concentration of graphene of graphene (25–100 (25–100 mg L mg-1), 1 Lwith− ), withthe effects the eff ectsof all of treatments all treatments being being not not sign significant.ificant. However, However, these these biomass biomass parameters parameters often -11 significantlysignificantly decreaseddecreased atat highhigh concentrationconcentration ofof graphenegraphene (250–500(250–500 mgmg LL− ),), though though the the effects effects of few treatments were significantsignificant ((pp << 0.05),0.05), indicatingindicating thatthat highhigh concentrationconcentration of graphenegraphene inhibitedinhibited the growth ofof seedlings.seedlings. TheThe declinesdeclines grewgrew withwith increasingincreasing graphenegraphene concentrationconcentration (Figure (Figure2 ).2). Survival Survival rates of of the seedlings at 5050 daysdays werewere alsoalsodecreased decreased with with increasing increasing graphene graphene concentration concentration (Ck (Ck= = 98.3%, T1 = 90.3%,90.3%, T2 T2 == 81.7%,81.7%, T3 T3 == 76.7%,76.7%, T4 T4 = =60.0%60.0% and and T5 T5 = 50.0%= 50.0%).). The The increase increase in in biomass biomass at at low concentration ofof graphenegraphene followedfollowed thethe orderorder ofof 4040 >> 30 > 5050 days days (Figure (Figure 22).).

Figure 1.1. Growth vigorvigor ofofL. L. olgensisolgensisseedlings seedlings at at di differentﬀerent concentrations concentrations of of graphene graphene for for 30 30 (a) ( anda) and 50 b 1 (50) ( daysb) days (from (from left left to right,to right, treated treated with with 0 (Ck),0 (Ck), 25, 25, 50, 50, 100, 100, 250, 250, and and 500 500 mg mg L −L-1,, respectively;respectively; deaddead seedlings in (b) were removed). seedlings in (b) were removed).

Forests 2020, 11, 258 6 of 16

ForestsForests2020 2020, 11, ,11 258, 258 6 of6 16of 16

Figure 2. Dry masses of leaf (a), stem (b) and root (c) of L. olgensis seedlings at different concentrations of graphene. Values followed by the same letter for the same days are not significantly different at p < 0.05. Ck, T1, T2, T3, T4, and T5 represent 0, 25, 50, 100, 250, and 500 mg FigureFigure 2. Dry2. Dry masses masses of leaf of (a ),leaf stem ( (ab),) andstem root (b ()c )and of L. olgensisroot (c)seedlings of L. olgensis at different seedlings concentrations at different of L-1 graphene treatments, respectively. graphene.concentrations Values followedof graphene. by the Values same letter followed for the sameby the days same are notletter significantly for the disamefferent days at p

Figure 3. Root morphology of L. olgensis ﬁne roots with diﬀerent concentrations of graphene (mean 1 ± S.D.,Figuren = 3.3). Root (a): morphology length; (b) surface of L. olgensis area;(c ):fine volume; roots with and (ddifferent): average conc diameter.entrations of graphene (mean ± 1 S.D., n = 3). (a): length; (b) surface area;(c): volume; and (d): average diameter. 3.1.3. Physiological Responses Figure 3. Root morphology of L. olgensis fine roots with different concentrations of graphene (mean 3.1.3. Physiological Responses 1 Graphene± 1 S.D., n generally = 3). (a): length; increased (b) surface the relative area;(c conductivity): volume; and (except (d): average for 250 diameter. and 500 mg L− at 40 days) 1 1 and MDAGraphene contents generally (except increased for 25, 50 the and relative 100 mg conductivity L− at 40 days (except and 50 for mg 250 L− andat 50500 days) mg L of-1 L.at olgensis40 days) leavesand3.1.3. MDA (FigurePhysiological contents4). The (except Responses increments for 25, 50 in theand relative100 mg L conductivity-1 at 40 days and 50 MDA mg L contents-1 at 50 days) both of grew L. olgensis with increasingleaves (Figure graphene 4). The concentration, increments in indicating the relative that conductivity graphene damaged and MDA the cellcontents membrane both grew of leaves, with Graphene generally increased the relative conductivity1 (except for 250 and 500 mg L-1 at 40 days) and the damage was the most serious at 500 mg L− . The order of maximum relative conductivity and increasing graphene concentration, indicating that graphene-1 damaged the cell-1 membrane of leaves, MDAand contentsMDA contents was generally (except for 50 >25,40 50> and30 days 100 mg (Figure L at4). 40 days and 50 mg L at 50 days) of L. olgensis leaves (Figure 4). The increments in the relative conductivity and MDA contents both grew with increasing graphene concentration, indicating that graphene damaged the cell membrane of leaves,

Forests 2020, 11, 258 7 of 16 Forests 2020, 11, 258 7 of 16 and the damage was the most serious at 500 mg L-1. The order of maximum relative conductivity and -1 andMDA the contents damage was generallythe most serious 50 > 40 at> 30500 days mg L(Figure. The order4). of maximum relative conductivity and Forests 2020, 11, 258 7 of 16 MDA contents was generally 50 > 40 > 30 days (Figure 4).

Figure 4. Relative conductivity (a) and malondialdehyde (MDA) contents (b) of L. olgensis leaves FiguretreatedFigure 4. with 4.RelativeRelative different conductivity conductivity concentratio (a) ( nsaand) andof malondialdehydegraphene malondialdehyde (mean ± (MDA)1 S.D., (MDA) n contents = contents3). Values (b) ( bfollowedof) ofL. L.olgensis olgensis by the leaves leavessame treatedtreated with with different different concentratio concentrationsns of of graphene graphene (mean (mean ± 1 S.D.,1 S.D., n =n 3).= 3). Values Values followed followed by by the the same same letter for the same days are not significantly different at p ±< 0.05. Ck, T1, T2, T3, T4 and T5 represent 0, letter25,letter 50, for for100, the the 250,same same and days days 500 are mg are not L not -1significantly graphene significantly treatments, different different at respectively.at p

0.05). For initially the three and treatment then increased, times, 250with or the 500 effects of some treatments being not significant (p > 0.05). For the three treatment times, 250 or 500 mg effects -1of some treatments being not significant (p > 0.05). For the three treatment times, 250 or 500 mg1 L graphene was generally the most toxic to the seedlings, meaning that at this concentration, a L -1graphene was generally the most toxic to the seedlings, meaning that at this concentration, a large mg− L graphene was·_ generally the most toxic to the seedlings, meaning that at this concentration, a large amount of_ O2 and H2O2 were induced in leaves. The toxic effects ranked in the order 30 > 50 > amount of O2· and·_ H2O2 were induced in leaves. The toxic effects ranked in the order 30 > 50 > 40 large40 days amount (Figure of O 5).2 and H2O2 were induced in leaves. The toxic effects ranked in the order 30 > 50 > days (Figure5). 40 days (Figure 5).

FigureFigure 5. 5. SuperoxideSuperoxide anion anion (a (a) )and and hydrogen hydrogen peroxide peroxide ( (bb)) contents contents of of L.L. olgensis olgensis leavesleaves treated treated with with Figuredifferentdifferent 5. Superoxide concentrations concentrations anion of of (graphea graphene.) and ne.hydrogen Values Values peroxide followed followed ( bby by) contentsthe the same same of letter letterL. olgensis for for the the leaves same same treated days days are arewith not not 1 differentsignificantlysignificantly concentrations different different at of pp

Forests 2020, 11, 258 8 of 16

Forestsdecreased. 2020, 11 ,Overall, 258 at 40 and 50 days, most of the treatments showed a decreasing in POD and8 ofSOD 16 activities, with a minimum at 250 or 500 mg L-1 at all treatment times (Figure 6). decreased. Overall, at 40 and 50 days, most of the treatments showed a decreasing in POD and SOD -1 activities,Forests 2020 ,with11, 258 a minimum at 250 or 500 mg L at all treatment times (Figure 6). 8 of 16

Figure 6. Peroxide (POD, a) and superoxide dismutase (SOD, b) activities of L. olgensis leaves treated with different concentrations of graphene at three sampling times (mean ± 1 S.D., n =3). FW = Figure 6. Peroxide (POD, a) and superoxide dismutase (SOD, b) activities of L. olgensis leaves treated Figurefresh mass.6. Peroxide Values (POD,followed a) byand the superoxide same letter dismutasefor the same (SOD, days bare) activitiesnot significantly of L. olgensis different leaves at p < with different concentrations of graphene at three sampling times (mean 1 S.D., n = 3). FW = fresh treated0.05. Ck, with T1, different T2, T3, concentrationsT4 and T5 represent of grap hene0, 25, at 50, three 100, sampling 250, and times500 mg (mean± L-1 graphene± 1 S.D., n treatments,=3). FW = mass. Values followed by the same letter for the same days are not significantly different at p < 0.05. Ck, freshrespectively. mass. Values followed by the same letter for the same days are not significantly different at p < T1, T2, T3, T4 and T5 represent 0, 25, 50, 100, 250, and 500 mg L 1 graphene treatments, respectively. 0.05. Ck, T1, T2, T3, T4 and T5 represent 0, 25, 50, 100, 250, and− 500 mg L-1 graphene treatments, Graphene generally reduced the contents of soluble protein and proline in L. olgensis leaves respectively.Graphene generally reduced the contents of soluble protein and proline in L. olgensis leaves (except -1 -1 (except for the soluble protein contents at 25, 50, 100 and 5001 mg L for 40 days and 501 mg L for 50 for the soluble protein contents at 25, 50, 100 and 500 mg L− for 40 days and 50 mg L− for 50 days, -1 -1 days,Graphene and the generallyproline contents reduced at the 25, contents 501 mg L of forsoluble 40 days protein and and501 mg proline L for in 50 L. days),olgensis and leaves their and the proline contents at 25, 50 mg L− for 40 days and 50 mg L− for 50 days), and their change (exceptchange for trends the soluble were similar protein (Figure contents 7). at At 25, 30 50, days, 100 andsoluble 500 mgprotein L-1 for and 40 prolinedays and contents 50 mg Lgradually-1 for 50 trends were similar (Figure7). At 30 days, soluble protein and proline contents gradually decreased days,decreased and the with proline increasing contents graphe at 25,ne 50 concentration; mg L-1 for 40 daysat 40 and days, 50 mgthey L -1first for 50increased days), andand their then with increasing graphene concentration; at 40 days, they first increased and then decreased; at 50 days, changedecreased; trends at 50were days, similar they first(Figure decreased, 7). At 30 increa days,sed soluble and then protein decreased, and proline usually contents being the gradually lowest at1 they first decreased, increased and then decreased, usually being the lowest at 250 or 500 mg L− , decreased250 or 500 with mg Lincreasing-1, and there graphe were significantne concentration; differences at 40between days, somethey treatmentsfirst increased (P < 0.05;and Figurethen and there were significant differences between some treatments (p < 0.05; Figure7). decreased;7). at 50 days, they first decreased, increased and then decreased, usually being the lowest at 250 or 500 mg L-1, and there were significant differences between some treatments (P < 0.05; Figure 7).

Figure 7. Soluble protein (a) and proline (b) contents of L. olgensis leaves with different graphene Figure 7. Soluble protein (a) and proline (b) contents of L. olgensis leaves with different graphene treatments (mean 1 S.D., n = 3). FW = fresh mass. Values followed by the same letter for the same treatments (mean ±± 1 S.D., n = 3). FW = fresh mass. Values followed by the same letter for the same days are not significantly different at p < 0.05. Ck, T1, T2, T3, T4, and T5 represent 0, 25, 50, 100, 250, Figuredays are7. Solublenot significantly protein ( adifferent) and proline at P < (0.05.b) contents Ck, T1, T2,of L. T3, olgensis T4, and leaves T5 represent with different 0, 25, 50, graphene 100, 250, and 500 mg L 1 graphene treatments, respectively. treatmentsand 500 mg (mean L−-1 graphene ± 1 S.D., treatments, n = 3). FW respectively.= fresh mass. Values followed by the same letter for the same daysGraphene are not asignificantlyffected photosynthetic different at P < pigment 0.05. Ck, contentsT1, T2, T3, with T4, and the T5 eff representects of some 0, 25, treatments50, 100, 250, being Graphene affected-1 photosynthetic pigment contents with the effects of some treatments being significantand 500 ( pmg< L0.05; graphene Figure 8treatments,). At 30 days, respectively. graphene reduced carotenoid and total chlorophyll contents; significant (p < 0.05; Figure 8). At 30 days, graphene reduced1 carotenoid and total chlorophyll however, at high graphene concentration (250–500 mg L− ), their contents increased slightly, but -1 contents;Graphene however, affected at high photosynthetic graphene concentration pigment conten (250–500ts with mg the L effects), their ofcontents some1 treatments increased slightly, being they were still lower than those of the control and were the lowest at 100 mg L− (Figure8). At 40 significantbut they were (p < still 0.05; lower Figure than 8). those At of30 thedays, control graphene and were reduced the lowest carotenoid at 100 andmg Ltotal-1 (Figure chlorophyll 8). At 40 and 50 days, graphene usually increased their contents, which first increased and then decreased contents;and 50 days,however, graphene at high usually graphene increased concentration their contents, (250–500 which mg L -1first), their increased contents and increased then decreased slightly, with increasing graphene concentration; the highest carotenoid and total chlorophyll contents were but they were still lower than those of the control and were the lowest at 100 mg L-1 (Figure 8). At 40 generally found at 50 or 100 mg L 1 (Figure8). and 50 days, graphene usually increased− their contents, which first increased and then decreased

Forests 2020, 11, 258 9 of 16 Forests 2020, 11, 258 9 of 16 with increasing graphene concentration; the highest carotenoid and total chlorophyll contents were generallywithForests increasing2020 ,found11, 258 grapheneat 50 or 100 concentr mg L-1 ation;(Figure the 8). highest carotenoid and total chlorophyll contents were9 of 16 generally found at 50 or 100 mg L-1 (Figure 8).

Figure 8. Pigment contents of L. olgensis leaves with different graphene treatments (mean ± 1 S.D., n = Figure 8. Pigment contents of L. olgensis leaves with different graphene treatments (mean 1 S.D., n = 3).Figure FW 8.= Pigmentfresh mass. contents Values of L.followed olgensis leavesby the with same different letter for graphene the same treatments days are (mean not significantly ± 1 S.D., n = 3). FW = fresh mass. Values followed by the same letter for the same days are not significantly different -1 different3). FW = atfresh p < 0.05.mass. Ck, Values T1, T2, followed T3, T4, and by T5the represent same letter 0, 25,for 50, the 100, same 250, days and 1 are500 notmg Lsignificantly graphene at p < 0.05. Ck, T1, T2, T3, T4, and T5 represent 0, 25, 50, 100, 250, and 500 mg L− graphene treatments, treatments,different at respectively.p < 0.05. Ck, T1,(a): T2,carotenoid T3, T4, and contents; T5 represent (b): total 0, chlorophyll 25, 50, 100, contents.250, and 500 mg L-1 graphene respectively. (a): carotenoid contents; (b): total chlorophyll contents. treatments, respectively. (a): carotenoid contents; (b): total chlorophyll contents. 3.2.3.2. Soil Soil Properties Properties 3.2. Soil Properties 3.2.1.3.2.1. Soil Soil Chemical Chemical Properties Properties 3.2.1. Soil Chemical Properties RegardingRegarding the the effects effects of of graphene graphene on on soil soil chemical chemical properties, properties, such such as as organic organic matter matter (OM), (OM), hydrolytichydrolyticRegarding nitrogen nitrogen the effects(HN), (HN), availableof available graphene phosphorus phosphorus on soil chemical (A (AP)P) and and properties, available available potassium potassiumsuch as organic (AK) (AK) contents, contents,matter (OM), at at 30 30 1 days,hydrolyticdays, promotion promotion nitrogen effects eff ects(HN), were were available observed phosphorus (exc (exceptept for (A APP) contentsand available at 25 andpotassium 100 mg (AK) L−-1 andand contents, AK AK contents contents at 30 1 1 -1 atdays,at 100 promotion mgmg LL−-1),), oftenoften effects reaching reaching were observed the the maximum ma (excximumept at 50forat or AP50 250 orcontents mg 250 L −mg ;at however, 25L-1 and; however, 100 at 40 mg and Lat 50 40and days,and AK inhibition50contents days, -1 1 -1 inhibitionatwas 100 often mg was found,L ), often often and found, thereaching minimums and the mima werenimumsximum observed wereat 50 atobserved or 250 250 mg mg Lat− 250L(except; mghowever, L for-1 (except AP at contents) 40for andAP contents) (Figure50 days,9). (FigureinhibitionAt different 9). wasAt treatment different often found, times, treatment and the organicthe times, minimums matter,the orga HN,werenic AP matter,observed and AKHN, at contents 250AP mgand had L AK-1 (except di contentsfferent for change hadAP contents)different patterns change(Figure(Figure patterns99).). At different (Figure treatment 9). times, the organic matter, HN, AP and AK contents had different change patterns (Figure 9). 160 Ck 800 140160 T1 a a b )

T2 -1 Ck a 800 ) T3 b

-1 a 140 T1 a a b 120 T4 b )

b -1 T2 a ac b b 600

) T5 T3 c b b b c -1 b c a 100120 T4 b a a a c c b a a b b 600 T5 a b b c b c a b b c b b c b b b b b c 10080 b b b a a a c c a 400 a b c a b b b b b b b 6080 b b 400

4060 200 Organic contentsmatter (gkg Hydrolytic nitrogenHydrolytic contents kg (mg 2040 200 Organic contentsmatter (gkg

200 0 nitrogenHydrolytic contents kg (mg 200 ) -1

) c a d 500 a 0 -1 a a a b 200

a ) a b b -1 ) c a a d 50 b a b b b -1 a a a b a b b a a a a b c 150 40 b b a b b b b b b a b b c b b a b b b b a 150 40 b b b a a c c b 30 b b b b c c b 100 c 30 c c 20 100 c d 20 50 Available potassium contents (mg contents kg Available (mg potassium

10 d

Available Available phosphoruscontents kg (mg 50 Available potassium contents (mg contents kg Available (mg potassium

Available Available phosphoruscontents kg (mg 0 0 30 40 50 30 40 50 0 Treatment times (days) Treatment times (days) 0 30 40 50 30 40 50 Treatment times (days) Treatment times (days) FigureFigure 9. 9. SoilSoil chemical chemical properties properties with with different different graphene graphene treatments treatments (mean (mean ± 11 S.D., S.D., nn == 3).3). Values Values ± followedFigurefollowed 9. bySoil by the thechemical same same letter letterproperties for for the the withsame same different days days are are graphenenot not significantly significantly treatments different di (meanfferent at ±p at <1 p0.05.S.D.,< 0.05. Ck,n = T1, Ck,3). ValuesT2, T1, T3, T2, -1 1 T4,followedT3, and T4, T5 and by represent the T5 representsame 0, letter 25, 0,50, for 25, 100, the 50, 250,same 100, and 250,days 500 andare mg not 500 L significantly mggraphene L− graphene treatments, different treatments, at respectively. p < 0.05. respectively. Ck, ( aT1,): organic T2, T3, (a): matterT4,organic and contents;T5 matter represent contents; (b): 0, hydrolytic 25, (b 50,): hydrolytic100, nitrogen250, and nitrogen 500contents; mg contents; L-1 (graphenec): available (c): available treatments, phosphorus phosphorus respectively. contents; contents; (a ):and organic and (d (d):): availablematteravailable contents; potassium potassium (b ):contents. contents. hydrolytic nitrogen contents; (c): available phosphorus contents; and (d): available potassium contents.

Forests 2020, 11, 258 10 of 16 Forests 2020, 11, 258 10 of 16

3.2.2.3.2.2. Soil Enzyme Activities GrapheneGraphene inhibitedinhibited soilsoil urease,urease, catalase, catalase, dehydrogenase dehydrogenase and and acid acid phosphatase phosphatase activities (except(except 1-1 1-1 forfor ureaseurease activityactivity atat 3030 days, days, catalase catalase activity activity at at 500 500 mg mg L −L forfor 3030 daysdays andand 100, 100, 250 250 and and 500 500 mg mg L L− 1-1 1-1 forfor 4040 days,days, dehydrogenasedehydrogenase activity activity at at 25 25 and and 50 50 mg mg L L− forfor 4040 daysdays andand25, 25, 50 50 and and 100 100 mg mg L L− forfor 5050 days),days), and most of the the differences differences in in enzyme enzyme activities activities were were significant significant (p ( p< <0.05;0.05; Figure Figure 10). 10 At). At30,30, 40 40and and 50 50days, days, the the activities activities of of different different enzymes enzymes showed showed a a certain certain change change trend due toto graphenegraphene concentrationconcentration (Figure(Figure 1010).).

Ck 8 T1 a b 1.6

) T2 a -1 T3 a a a T4 ) a a a -1 T5 a a a a 6 a a 1.2 a b b a b a b b a a b a a b b b 4 a 0.8 a b b b b c Urease activities (mg g 2 0.4 Acid phosphatase g activities (mg

0 0.0 0.40 c d a a a a ) a a a b a a -1 a a 15 a b b a a b ) b b b a b a a -1 b c b b b b b 0.35 b b b b 10

5 g (ml activities Catalase 0.30 Dehydrogenase g activities (mg

0.00 0 30 40 50 30 40 50 Treatment times (days) Treatment times (days)

Figure 10. Soil enzyme activities with different graphene treatments (mean 1 S.D., n = 3). Values Figure 10. Soil enzyme activities with different graphene treatments (mean ±± 1 S.D., n = 3). Values followedfollowed byby thethe samesame letterletter forfor thethe samesame daysdays areare notnot significantlysignificantly didifferentfferent atatp p< < 0.05.0.05. Ck, T1, T2, T3, 1 T4,T4, andand T5T5 representrepresent 0,0, 25,25, 50,50, 100, 100, 250, 250, and and 500 500 mg mg L −L-1 graphenegraphene treatments,treatments, respectively.respectively. ((aa):): acidacid phosphatasephosphatase activities;activities; ((bb):): ureaseurease activities;activities; ((cc):): dehydrogenasedehydrogenase activities;activities; andand ((dd):): catalase activities.activities. 3.2.3. Correlation Analysis of Soil Properties 3.2.3. Correlation Analysis of Soil Properties Under graphene treatment, different correlations were observed between soil chemical properties Under graphene treatment, different correlations were observed between soil chemical and enzyme activities, including notable positive correlations (p < 0.01; such as between OM contents properties and enzyme activities, including notable positive correlations (p < 0.01; such as between and HN contents and acid phosphatase activity, between AK and HN contents, and between acid OM contents and HN contents and acid phosphatase activity, between AK and HN contents, and phosphatase activity and HN contents) and significant positive correlations (p < 0.05; e.g., between AK between acid phosphatase activity and HN contents) and significant positive correlations (p < 0.05; contents and acid phosphatase activity, OM and AP contents between HN contents and urease activity, e.g., between AK contents and acid phosphatase activity, OM and AP contents between HN and between AP contents and dehydrogenase activity) (Table3). The many correlations found, for contents and urease activity, and between AP contents and dehydrogenase activity) (Table 3). The which some were much stronger than others, suggesting that ecological or environmental information many correlations found, for which some were much stronger than others, suggesting that overlapped among them From the factor analysis results (Figure 11), it can be further seen that, under ecological or environmental information overlapped among them From the factor analysis results different graphene concentrations and times, the changes in HN, organic matter and AK contents were (Figure 11), it can be further seen that, under different graphene concentrations and times, the most significant, followed by the changes in AP content, dehydrogenase and urease activities, and the changes in HN, organic matter and AK contents were most significant, followed by the changes in changes in catalase and acid phosphatase activities were the least significant. AP content, dehydrogenase and urease activities, and the changes in catalase and acid phosphatase activities were the least significant.

Forests 2020, 11, 258 11 of 16

Forests 2020, 11, 258 11 of 16 Table 3. Pearson correlations of soil chemical properties with different graphene treatments. Organic matter content (OM), hydrolytic nitrogen content (HN), available phosphorus content (AP), available Tablepotassium 3. Pearson content correlations (AK), dehydrogenase of soil chemical activity properties (Deh withy), catalase diﬀerent activity graphene (Cata), treatments. urease Organicactivity matter(Urea), content and acid (OM), phosph hydrolyticatase activity nitrogen (Phos). content (HN), available phosphorus content (AP), available potassium content (AK), dehydrogenase activity (Dehy), catalase activity (Cata), urease activity (Urea), and acid phosphataseOM activityHN (Phos). AP AK Urea Dehy Cata HN 0.651 ** AP 0.138OM 0.243 HN AP AK Urea Dehy Cata HNAK 0.320 0.651 *** 0.355 ** 0.377 * AP 0.138 0.243 Urea 0.115 0.216 * －0.023 －0.036 AK 0.320 * 0.355 ** 0.377 * * － UreaDehy 0.122 0.115 0.2120.216 * 0.3070.023 0.1520.036 0.035 − − DehyCata － 0.1220.197 － 0.2120.193 － 0.3070.255 * － 0.1520.207 0.0210.035 －0.118 − Cata 0.197** 0.193** 0.255 0.207* 0.021 0.118 － Phos 0.563− 0.550− 0.181− 0.347− 0.112 0.100− 0.107 Phos 0.563 ** 0.550 ** 0.181 0.347 * 0.112 0.100 0.107 * p < 0.05；** p < 0.01. − * p < 0.05; ** p < 0.01.

Figure 11. Factor analyses of soil chemical properties and enzyme activities with different grapheneFigure 11. treatments. Factor analyses of soil chemical properties and enzyme activities with different graphene treatments. 4. Discussion 4. DiscussionThis study found that graphene influenced the growth of L. olgensis seedlings, especially survival ratesThis of the study seedlings found at that 50 days graphene were decreased, influenced and the the growth declines of grewL. olgensis with increasingseedlings, grapheneespecially concentrationsurvival rates (Figure of the1 ).seedlings The seedling at 50 biomass days were declined decreased, at high and graphene the declines concentration grew with (the inhibitionincreasing rosegraphene with graphene concentration concentration) (Figure 1). but The was seedling promoted bi atomass low concentrationdeclined at high (except graphene for 50 days),concentration and the 1 most(the inhibition positive e roseffects with were graphene generally concentration) found at 25or but 50 was mg promoted L− (Figure at2 low). As concentration a new type of(except carbon for nanomaterials,50 days), and the graphene most positive is widely effects applied were and generally may enter found soil environments at 25 or 50 mg in many L-1 (Figure ways. 2). Thus, As ita maynew posetype potentialof carbon impacts nanomaterials, to ecosystems, graphene including is widely their applied soils and may plants. enter Similarly, soil environments some other in studies many alsoways. have Thus, shown it may that pose graphene potential influences impacts to plant ecosystems, growth andincluding biomass their accumulation. soils and plants. For Similarly, example, 1 thesome growth other of pakstudies choi, also tomato, have and shown spinach that was severelygraphene inhibited influences under plant 500–2000 growth mg Land− graphene biomass treatmentsaccumulation. for 20For days example, [41]; graphene the growth quantum of pak dots choi, inhibited tomato, theand seedling spinach height was severely of coriander inhibited [20]; afterunder exposing 500–2000 wheat mg L to-1 graphene fortreatments 30 days, for its growth20 days was[41]; inhibited, graphene the quantum levels of dots several inhibited nutrient the elementsseedling (N,height K, Ca,of coriander Mg, Fe, Zn,[20]; and after Cu) exposing in the plant wheat decreased, to graphene and for its 30 nutritional days, its balancegrowth waswas brokeninhibited, [23 ].Therefore,the levels theof several effectsof nutrient graphene elemen on plantts (N, growth K, Ca, varied Mg, with Fe, grapheneZn, and concentrationCu) in the plant and treatmentdecreased, time. and its nutritional balance was broken [23].Therefore, the effects of graphene on plant growthAt present,varied with the impactgraphene of grapheneconcentration on plant and rootstreatment and leavestime. is still controversial, with positive, negativeAt present, or no obvious the impact effects. of Ongraphene the one on hand, plant graphene roots and [19 leaves] and iron is still nanoparticles controversial, [18 ]with can elongatepositive, root cells of wheat and promote root elongation; under treatment with graphene quantum dots, the

Forests 2020, 11, 258 12 of 16 average length and dry mass of coriander root and the root dry mass and length and leaf length of garlic all significantly increased, but the number of garlic leaves did not change [20]. On the other hand, the appropriate concentration of graphene promoted adventitious root elongation and main root formation in Populus tremula Linn, increasing its root number and making its root system strong and deeply colored. However, a high concentration of graphene obviously inhibited the growth of Populus tremula Linn; the plants had few fibrous roots, their leaves were small, and the aboveground part was light green and slightly yellow [21]. In this study, at 30 and 40 days, low concentration of graphene thickened and expanded the root system of L. olgensis (Figure3), which may be because graphene influenced the division and differentiation of root tip cells. However, at higher concentration 1 of graphene (>50 mg L− ), the L. olgensis roots became shorter, and both the root volume and surface area decreased (Figure3). It is possible that high concentration of graphene can inhibit the cell division and destroy the cell structure; thus, normal root physiology is affected, and root growth is inhibited. Relative conductivity and MDA are significant indicators of physiological plant stress and can be used to reflect the permeability of plant cell membrane. Their contents are in direct proportion to the damage to plants caused by adverse conditions. Under adverse conditions, the plant cell membrane is damaged, the relative conductivity increases, and a large amount of superoxide anion and hydrogen peroxide (H2O2), which are the main active oxygen radicals, are also produced and accumulated in plant cells [42]. Under the graphene treatment, the contents of ROS and H2O2 in cabbage, tomato, and spinach increased to various degrees, indicating that graphene induced oxidative stress in these plants [41]. In this study, all concentrations of graphene treatments for 30 days and high concentration 1 of graphene (>50 mg L− ) for 40 and 50 days also increased the relative conductivity, MDA, superoxide anion and hydrogen peroxide contents of L. olgensis leaves, and these parameters increased with increasing graphene concentration at 30 days (Figures4 and5). Similarly, Liu et al. [ 43] also found that 1 high concentration of graphene (50–200 mg L− ) significantly increased MDA contents in the root tips of rice seedlings and then the degree of peroxidation increased. Under adverse conditions, plant cells will produce a certain self-regulatory response to prevent toxicity [44]. POD and SOD can effectively remove excessive free radicals and peroxide in plants and improve the adaptability of plants to adverse conditions. When nanoparticles contact with organisms, a large amount of ROS will be produced in plant cells. SOD and POD were the enzymes to firstly act on ROS, and the increase of SOD and POD activities played a role as an acute detoxification measure [44]. At the same time, the induction of the two enzyme activities also suggested that the plant was in a state of oxidative stress. In this study, after the treatment of L. olgensis seedlings with graphene, especially 1 at 40 and 50 days, the lower concentration of graphene (<100 mg L− ) caused some oxidative stress to these seedlings, and the activities of SOD and POD increased to remove the excess active oxygen produced by graphene. However, under the higher-concentration graphene treatments, SOD and POD activities were reduced, meaning that the active oxygen produced by graphene in the seedlings may have exceeded the scavenging ability of antioxidant systems (SOD and POD). The excessive ROS then caused oxidative damage to the cell membrane, which is also reflected on the increase in relative conductivity and MDA, superoxide anion and hydrogen peroxide contents (Figures4 and5). 1 Our results also suggested that the lower concentration of graphene (<100 mg L− ) caused some oxidative stress to L. olgensis seedlings at 40 and 50 days. The toxicity mechanism of graphene to L. olgensis is closely related to oxidative stress, which is consistent with the results of Begum et al. [22]. The short-term toxicity of graphene to L. olgensis seedlings is mainly oxidative stress to roots and root growth inhibition, but after long-term treatment, the antioxidant system in seedlings will be seriously damaged. It is generally believed that when plants are stressed by pollutants, they begin to show strong osmotic regulation ability, which decreases or loses with the increase in stress level [42]. Proline and soluble proteins are important osmotic regulators in the plant cytoplasm and can maintain the osmotic pressure of plants, improve their resistance to external stress and prevent cell dehydration. At present, changes in proline and soluble protein contents have not been mentioned in studies on the effect of Forests 2020, 11, 258 13 of 16 graphene on plants [41,45]. In this study, after graphene treatment, the soluble protein and proline contents of L. olgensis leaves generally decreased with increasing graphene concentration (Figure7), suggesting that a high concentration of graphene significantly reduced the osmotic adjustment ability of the seedlings. Chlorophyll directly affects plant photosynthesis and is an important indicator of plant growth and health as well as environmental change. Studies have shown that the chlorophyll content and PSII 1 activity of wheat decreased after being exposed to graphene for 30 days [23], and 500 mg L− graphene also significantly reduced the chlorophyll contents of wheat leaves [43]. The same is true in this study (Figure8), indicating that graphene may a ffect the photosynthetic process of L. olgensis seedlings. In addition to plant growth and physiological parameters, changes in soil characteristics, such as enzyme activities and chemical properties, are also closely related to the effects of pollutants or foreign substances on the environment [24,25]. In particular, soil enzyme activity can be used to evaluate the impact of foreign pollutants on soil biological activity [23] and effectively reflect the state of material circulation and energy flow in the soil [46]. Organic matter is an important carrier of soil enzymes and an energy source for microorganisms [47]. Soil urease and catalase are closely associated to organic matter contents and microbial quantity, especially urease, which can promote the hydrolysis of urea to ammonia and carbonic acid [46,48]. Dehydrogenase catalyzes dehydrogenation reactions and acts as an intermediate transmitter of hydrogen. When graphene was in contact with Haplic Cambisols, the activities of acid phosphatase, urease, dehydrogenase, and catalase all decreased (Figure 10), which was consistent with the results of Ahmed [49] and Wang et al. [50]. This result indicates that graphene may inhibit the activity and viability of some microorganisms in this soil [48]. However, with the increase in graphene treatment time, especially at 50 days, the decrease in enzyme activities in Haplic Cambisols was no longer significant (except for acid phosphatase activity) (Figure 10), which indicates that the toxicity of graphene in this soil is limited, as found by Li et al. [25]. In particular, catalase is closely related to the size and activity of the bacterial community and can also reflect the intensity of soil microbial process to a certain extent. In this study, a high concentration of graphene 1 1 (500 mg L− at 30 days and 100–500 mg L− at 40 days) often increased catalase activity at 30 and 40 days, though the effects of the treatments were not all significant (p < 0.05) (Figure 10), suggesting that soil microorganisms may produce some oxidative stress in this period, but the change of catalase activity was not significant when the treatment time was prolonged (at 40 and 50 days), which also showed that the toxicity of graphene in this soil was limited. When the treatment time was shorter (30 days), graphene increased the soil HN contents and urease activity, but for a longer time (40 and 50 days), these parameters were inhibited (Figures9 and 10), which suggested that graphene may also affect the nitrogen cycle in Haplic Cambisols. For urease and dehydrogenase activities, their declines in some graphene treatments at 50 days were higher than those at 40 days, which may be because graphene undergoes dispersion; with the increase of contact time, its toxicity to some microorganisms, such as bacteria, increases. In addition, graphene usually decreased the HN, AP, and AK contents in Haplic Cambisols (Figure9), which may influence the absorption of the above nutrients (N, P, and K) by seedlings and lead to changes in their root morphological characteristics. Acid phosphatase is an important index to evaluate soil phosphorus, and all concentrations of graphene significantly inhibited its activity (Figure 10), suggesting that graphene in Haplic Cambisols may affect the phosphorus cycle.

5. Conclusions This study is the first to provide experimental evidence revealing the interrelations between graphene and L. olgensis seedlings and Haplic Cambisols in Northeast China. After graphene treatments for 30, 40, and 50 days, the dry masses of the seedlings all increased first and then decreased, generally 1 being highest at 25 or 50 mg L− . At 30 and 40 days, the root morphology increased first and then 1 decreased, reaching maximum at 50 or 100 mg L− ; however, at 50 days, it was inhibited at all concentrations of graphene. As to the relative conductivity, malondialdehyde (MDA), superoxide Forests 2020, 11, 258 14 of 16 anion and hydrogen peroxide contents of L. olgensis leaves, they increased at 30 days, but usually decreased first and then increased at 40 and 50 days, generally reaching the minimum at 50 mg 1 L− . For the peroxidase (POD) and superoxide dismutase (SOD) activities and chlorophyll, soluble protein and proline contents, they decreased at 30 days, and the effects increased with increasing graphene concentration; however, they increased first and then decreased at 40 and 50 days, reaching 1 the maximum at 50 mg L− . At 30 days, graphene increased the organic matter, hydrolytic nitrogen, available phosphorus, and available potassium contents of Haplic Cambisols, but at 40 and 50 days, they often decreased. Graphene usually had an inhibitory effect on the activities of acid phosphatase, urease, dehydrogenase and catalase. Therefore, graphene caused different degrees of oxidative stress in L. olgensis seedlings, and their antioxidant systems might be seriously damaged by long-term treatments. When graphene concentration reaches a certain level, it may also affect nitrogen and phosphorus cycling in Haplic Cambisols. Although the exact mechanisms of the graphene response are difficult to infer, this study clearly demonstrates the beneficial value or harmful effect of graphene at different concentrations and treatment time on these seedlings in this soil.

Author Contributions: Conceptualization, J.S. and X.C.; investigation, K.C., C.D. and N.L.; data curation, K.C. and C.D.; writing—original draft, J.S. and C.D.; writing—review and editing, J.S., K.C., C.D., N.L. and X.C.; funding acquisition, J.S. All authors have read and agreed to the published version of the manuscript. Funding: This work was financially supported by the National Natural Science Foundation of China (31370613), the Fundamental Research Funds for the Central Universities (2572019CP15). Conflicts of Interest: The authors declare no conflict of interest.

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