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Chemosphere 185 (2017) 907e917

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Chemosphere

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Response of soil dissolved organic matter to microplastic addition inChinese loess soil

Hongfei Liu a, c, 1, Xiaomei Yang a, d, f, 1, Guobin Liu a, b, Chutao Liang a, b, Sha Xue a, b, *,Hao Chen d, Coen J. Ritsema d, Violette Geissen d, e

a State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Northwest A&F University, Yangling, 712100, PR Chinab Institute of Soil and Water Conservation, Chinese Academy of Sciences and Ministry Water Resources, Yangling, 712100, PR Chinac College of Forestry, Northwest A&F University, Yangling, Shaanxi, 712100, PR Chinad Soil Physics and Land Management, Wageningen University, P.O. Box 47, 6700 AA, Wageningen, The Netherlandse Institute of Crop Science and Resources Conservation (INRES), University of Bonn, 53115, Bonn, Germanyf College of Natural Resources and Environment, Northwest A&F University, Yangling, Shaanxi, 712100, PR China

h i g h l i g h t s

� Microplastic addition stimulated soil activity of fluorescein diacetate hydrolase (FDAse) in soil.� The lower level of microplastic addition had a negligible effect on the nutrient contents in DOM solution at day 30.� The higher level of microplastic addition significantly increased the nutrient contents in DOM solution.

a r t i c l e i n f o

Article history:Received 15 March 2017Received in revised form29 June 2017Accepted 13 July 2017Available online 18 July 2017

Handling Editor: Jian-Ying Hu

Keywords:MicroplasticDissolved organic carbon (DOC)Dissolved organic nitrogen (DON)Dissolved organic phosphorus (DOP)Excitation-emission matrix (EEM)

* Corresponding author. State Key Laboratory of Soion the Loess Plateau, Institute of Soil and Water ConsSciences and Ministry of Water Resources, Yangling, S

E-mail address: xuesha100@163.com (S. Xue).1 The equal author contribution.

http://dx.doi.org/10.1016/j.chemosphere.2017.07.0640045-6535/© 2017 Elsevier Ltd. All rights reserved.

a b s t r a c t

Plastic debris is accumulating in agricultural land due to the increased use of plastic mulches, which iscausing serious environmental problems, especially for biochemical and physical properties of the soil.Dissolved organic matter (DOM) plays a central role in driving soil biogeochemistry, but little informa-tion is available on the effects of plastic residues, especially microplastic, on soil DOM. We conducted asoil-incubation experiment in a climate-controlled chamber with three levels of microplastic added toloess soil collected from the Loess Plateau in China: 0% (control, CK), 7% (M1) and 28% (M2) (w/w). Weanalysed the soil contents of dissolved organic carbon (DOC), dissolved organic nitrogen (DON), NH4

þ,NO3

�, dissolved organic phosphorus (DOP), and PO43� and the activities of fluorescein diacetate hydrolase

(FDAse) and phenol oxidase. The higher level of microplastic addition significantly increased the nutrientcontents of the DOM solution. The lower level of addition had no significant effect on the DOM solutionduring the first seven days, but the rate of DOM decomposition decreased in M1 between days 7 and 30,which increased the nutrient contents. The microplastic facilitated the accumulation of high-molecular-weight humic-like material between days 7 and 30. The DOM solutions were mainly comprised of high-molecular-weight humic-like material in CK and M1 and of high-molecular-weight humic-like materialand tyrosine-like material in M2. The Microplastic stimulated the activities of both enzymes. Microplasticaddition thus stimulated enzymatic activity, activated pools of organic C, N, and P, and was beneficial forthe accumulation of dissolved organic C, N and P.

© 2017 Elsevier Ltd. All rights reserved.

l Erosion and Dryland armingervation, Chinese Academy ofhaanxi, 712100, PR China.

1. Introduction

The increasing use of plastic has increased the amount of plasticdebris in the environment, especially in the oceans. More than 240million tonnes of plastic are used every year (Thompson et al.,2009), contributing to 11.7e25% of the municipal waste in Europeand the USA. Only 1e2% of this plastic is recycled (Shent et al., 1999;

H. Liu et al. / Chemosphere 185 (2017) 907e917908

Panda et al., 2010). Plastic debris is accumulating in the environ-ment because of its durability and the limitation of recyclingtechnology (Barnes et al., 2009; Rillig, 2012). Microplastic, particles<5 mm in size (Claessens et al., 2011), have been studied in themarine environment and on shorelines, but few studies havefocused on the microplastic in soil and terrestrial systems (Rillig,2012; do Sul and Costa, 2014; Van Cauwenberghe et al., 2015).Microplastic can enter the soil either as primary microplastic fromindustrial abrasives and cosmetic products by sludge application(Cole et al., 2011) or secondary microplastic from the environ-mental degradation of plastic mulch. Agricultural sites and landfillsin Europe contain 1000-4000microplastic particles per kg of sludgedrymass (Zubris and Richards, 2005; Barnes et al., 2009). The use ofplastic films as agricultural mulches increased in China, especiallyin northern regions, nearly four-fold from 1991 to 2011, from0.32 to1.25 million tonnes which has contributed to the accumulation ofmicroplastic in farmland (Yearbook, 2012; Wang et al., 2013; Liuet al., 2014). Microplastic can be ingested by fauna due to theirsmall size and can thus accumulate in the food chain (Besselinget al., 2013; Huerta Lwanga et al., 2016b). Some studies haveidentified interactions between microplastic and the chemicalpollutants they absorb on their surfaces (Ivar do Sul and Costa,2014). Microplastic can also alter the physical properties of soiland can accumulate in soil, reaching levels that can affect soilfunction and biodiversity (Rillig, 2012).

Dissolved organic matter (DOM) plays an important role innumerous physical, chemical, and biological processes in soil(Kalbitz et al., 2000), including the cycling of soil organic carbon (C)and the transport of nutrients such as nitrogen and phosphorus(Kalbitz et al., 2003b). DOM represents <0.25% of the total soilorganic matter but facilitates the solubility and mobility of metalsand organic compounds (Kalbitz et al., 1997; Temminghoff et al.,1997) and thus increases the transport of pollutants (Kalbitzet al., 2000). DOM is a complex mixture of various labile andrecalcitrant organic substances (Michel et al., 2006) and is a sub-strate and the most important C source for microorganisms(Marschner and Kalbitz, 2003; DeForest et al., 2004a, 2004b). Soildissolved organic C (DOC) is amore sensitive indicator of changes insoil quality changes than total organic C (TOC), because short-termchanges in TOC are not easily detected (Purakayastha et al., 2008;Gong et al., 2009; Li et al., 2016).

The accumulation of microplastic in soil can affect microbialactivity, attributed to absorbed harmful contaminants (Rillig, 2012),earthworm activity (Huerta Lwanga et al., 2016b, 2017) and soilphysical properties, such as soil porosity and aggregate structure(Ladd et al., 1993; Rillig, 2012; Zhang et al., 2015). The activities ofsoil enzymes represent microbial activities and the availability ofsubstrates for microorganism uptake. Soil enzymes with high cat-alytic capacities are produced by soil microorganisms and are themain medium controlling the cycling of soil nutrients such as C, N,and P (Dick et al., 1994; Allison and Jastrow, 2006; Trasar-Cepedaet al., 2008). Phenol oxidase (PO) is involved in the degradationof phenolic compounds which are recalcitrant DOMs and decreasesthe DOM biodegradability (Marschner and Kalbitz, 2003). PO ac-tivity is negatively correlated with soil humification (Li et al., 2015).Fluorescein diacetate hydrolase (FDAse) can represent overall mi-crobial metabolic activity and is an effective indicator of short-termchanges of soil quality (Muscolo et al., 2014, 2015).

So far the effect of microplastic on soil fertility and microbialactivity were still not clear, although researches demonstrated thatplastic-film residues can decrease soil porosity, air circulation,microbial biomass and microbial activity and can probably affectsoil fertility (Moreno and Moreno, 2008; Kasirajan and Ngouajio,2012). A detailed study of the effects of microplastic on the dy-namics of soil DOM and enzymatic activities is needed for

minimizing environmental risks and determining the sustainabilityof farming practices. We used three-dimensional excitation-emis-sion matrix (EEM) fluorescence spectroscopy, a rapid, sensitive,selective, and reagent-free technique for fingerprinting organicmatter in terrestrial ecosystems, to study the composition of soilDOM (Chen et al., 2003; Matilainen et al., 2011).

Our specific aims were to: (1) determine the effect of micro-plastic on the quantities and composition of soil DOM, (2) deter-mine the effect of microplastic on the dynamics of soil enzymaticactivity, and (3) clarify the relationships between soil DOM andenzymatic activity.

2. Materials and methods

2.1. Experimental design

This experiment was conducted in the climate-controlledchamber (AGC-Doo3N, Hangzhou, China) at the Institute of Soiland Water Conservation, Chinese Academy of Sciences, Yangling,China. The soil used in this experiment was collected in Ansaicounty on the Loess Plateau, and soil properties can be seen inTable 1. The soil is mainly composed of Cultivated loessial soils(calcaric cambisols, FAO) developed on wind-deposited loessparental material and is characterised by the parental material, theabsence of bedding, a loose silty texture, macroporosity, andwetness-induced collapsibility. Two hundred grams were incu-bated in PVC pots 10 cm in diameter and 9 cm in height. Ourexperiment contained three treatments: 1) CK, no microplasticadded to the soil; 2) M1,14 g of microplastic added to the soil (7%w/w); 3) M2, 56 g microplastic added to the soil (28% w/w). Themicroplastic used in this experiment is made of polypropylene(Youngling-TECH company, Beijing, China). The density of Micro-plastic is 0.91 g/cm3, and its bending strength is 200 kg/cm2. Theparticle size of microplastic is below 180 mm. The soils were slightlycompacted using a small manual soil compactor. The soilcompactor was set to fall 10 times by gravity at the pot height toguarantee the same compaction for all samples. The microplasticcontents were chosen based on the research of Huerta Lwanga et al.(2016a) but simulating the hotspots of plastic debris in the field.Soil moisture was maintained at 60% of field capacity throughoutthe experiment. Each treatment had three replicates. The pots wereincubated in the light at 28 �C (relative humidity of 80%, 300 m(photons) m�2 s�1). The soil was sampled from each pot after 0, 1, 3,7, 14, and 30 days, so the experiment had a total of 54 pots: 3treatments � 3 replicates � 6 days. The soil samples were passedthrough a 2 mm sieve, and then one subsample was storedat �80 �C for analysing soil enzymatic activities, and another sub-sample was stored at 4 �C for measuring DOM chemical properties.

2.2. Analysis of soil DOM concentration and composition

DOM was extracted by adding 120 mL of distilled water to 40 gsubsamples of homogenised soil (1:3 soil:water, w/w) as describedby Kalbitz et al. (2003a, 2013b). All extracts were centrifuged at4000 rpm for 10 min, and the supernatants were filtered throughpre-rinsed 0.45 mm cellulose-acetate membranes (Schleicher &Schuell). The filtered solutions were stored frozen until analysis.Total dissolved N (TDN), DOC, NH4

þ, NO3�, total dissolved P (TDP),

and PO43� contents were measured in all samples using standard

soil test procedures of the Chinese Ecosystem Research Network(CERN Editorial Committee, 1996). DOC contents were determinedusing a TOC analyser (liquid TOC II, Elementar, Germany). TDNcontents were determined using alkaline persulfate digestion-UVspectrophotometric method (Doyle et al., 2004). TDP contentswere measured using the ammonium molybdate

Table 1Principle properties of selected soil.

Properties Value

Particle size distribution:<0.002 mm (clay) (%) 18.390.002e0.02 mm (%) 24.950.02e2 mm (%) 55.85>0.2 (%) 0.81pH 8.45Total organic carbon (g*Kg�1) 8.68Total nitrogen (g*Kg�1) 0.22Total phosphorus (g*Kg�1) 0.55Nitrate nitrogen (mg*Kg�1) 6.71Ammonium nitrogen (mg*Kg�1) 3.37Available phosphorus (mg*Kg�1) 3.04

H. Liu et al. / Chemosphere 185 (2017) 907e917 909

spectrophotometric method (Galhardo and Masini, 2000). PO43�

contents were determined using phospho-molybdenum bluemethod (Jarvie et al., 2002). NH4

þ content was measured by an AA3continuous flow autoanalyzer (AutoAnalyzer3-aa3, Bran＋Luebbe,Germany). NO3

� content was determined by ultraviolet colorimetrywith an ultraviolet spectrophotometer (UV2300, Shanghai, China).Dissolved organic N (DON) and dissolved organic P (DOP) contentswere calculated as TDN- (NH4

þþNO3�) and TDP- PO4

3�, respectively.UVeVis absorption from 200 to 500 nm (1 nm steps) was

measured in a 10-mm quartz cuvette, with Milli-Q-water used as ablank. The specific UV absorbance at 254, 280 and 365 nm weremeasured for all samples. EEM spectrograms of the subsampleswere measured using an F-4600 fluorescence spectrometer(HITACHI, Japan). The voltage of the photomultiplier tubes was setat 700 V, and the slits for both excitation and emissionwere 5 nm ata scanning speed of 1200 nm min�1. EEM were recorded in a rangeof excitation wavelengths of 200e450 nm and in a range of emis-sion wavelengths of 250e450 nm. Before measurement, thewavelength-dependent light intensity of the light source and thelight sensitivity of the detector were corrected. Excitation andemission intensities for instrument-specific biases were correctedbased on correction factors supplied by manufacturer. Each EEMwas corrected for inner filtering effects by multiplying it by acorrection matrix, which was calculated for each wavelength pairfrom the sample absorbance by assuming excitation (Ex) andemission (Em) pathlengths of 5 mm in a 10 mm cuvette (Ohno,2002). The EEM spectrograms were combined into a three-dimensional data array: 54 samples � 51 excitations � 41 emis-sions. The Raman scatter effects were removed from the data setbefore analysis by subtracting the spectrogram for Milli-Q-waterfrom the sample spectrogram. Rayleigh scatter effects wereremoved from the dataset by excluding emission measurementsmade at wavelengths less than or equal to the excitationwavelength þ20 nm.

The EEM contours of four components identified by the PAR-AFAC analysis are shown in Fig. 5. Component 1 had two protein-like fluorescence peaks centered at excitation/emission (Ex/Em)wavelength pairs of 215/280 and 260/280 nm. This component wasassociated with tyrosine-like substances which indicated a moredegraded peptide material (Coble et al., 1998; Parlanti et al., 2000).The Ex/Emwavelength pairs of component 2 were centered at 225/450 nm, and this component was identified as a UVC humic-likecomponents associated with high-molecular-weight and aromatichumus (Stedmon and Markager, 2005; Murphy et al., 2006).Component 3 had a humic-like fluorescence peak centered at theEx/Em wavelength pairs at 235/420 nm. This component wasassociated with high-molecular-weight humus (Stedmon et al.,2003; Murphy et al., 2006). The Ex/Em wavelength pairs ofcomponent 4 were centered at 220/440 nm. This component

originated from a UVA humic-like substance associated with fluo-rescence resembling that of fulvic acid (Cory and McKnight, 2005;Stedmon and Markager, 2005).

The activities of two enzymes were measured: PO and FDAse.FDAse activity was measured by the method adapted from (Greenet al., 2006; Daou et al., 2016). One gramme of soil was added to9 mL of 0.1 M phosphate buffer, pH 7, and the suspension wasshaken for 30 min. After shaking, 180 mL of the sample suspensionwere dispensed into wells of a black 96-well microplate, 20 mL of20 mM fluorescein diacetate (FDA) were dispensed to columns toserve as an assay, and 20 mL of phosphate buffer were dispensedinto columns to serve as controls. Reference standard wellsreceived 20 mL of substrate plus 180 mL phosphate buffer. Each assaymicroplate also contained one column of phosphate buffer blanksfor measuring background fluorescence in the substrate. Phosphatebuffer blank wells received 200 mL phosphate buffer. The micro-plates were covered and incubated in the dark at 30 �C for 1 h. TheFDAse activities were expressed in units of mg Kg�1 h�1.

PO activity was determined spectrophotometrically in clear 96-well microplates using L-3, 4-dihydroxyphenylalanine (L-DOPA) asa substrate as described by DeForest (2009). One gramme dry massof fresh soil was mixed with 125 mL of 50 mM acetate buffer (pH5.0) made bymixing sodium acetate trihydrate with distilled water.The samples were shaken for 30min. After shaking, 50 mL of the soilsuspensions were placed in microplate wells containing 150 mL ofbuffer that served as controls or containing 150 mL of 25 mM L-DOPA for sample analysis. Reference standard wells received 150 mLof substrate plus 50 mL acetate buffer. Each assay microplate alsocontained one column of phosphate buffer blanks for measuringbackground fluorescence in the substrate. Phosphate buffer blankwells received 200 mL acetate buffer. The microplates were coveredand incubated in the dark for 24 h. PO activity was quantified bymeasuring absorbance at 450 nm and expressed in units of mmolh�1 g�1.

2.3. Data analysis

One-way analysis of variance was used to determine the effectsof microplastic addition and time on chemical properties of soilDOM and enzyme activities. The means of significant effects atP < 0.05 were then compared using the Duncan’s multiple-rangetest. All statistical analyses were performed using SPSS 21.0. TheEEM data were analysed using MatLab 2010a (MathWorks Inc.,USA). Parallel factor analysis (PARAFAC) modelling of the fluores-cence EEMs was conducted with MATLAB using the DOMFluortoolbox (Stedmon and Bro, 2008) following the proceduresdescribed by (Stedmon and Bro, 2008). Figures were drawn usingSigmaPlot 10.0 software.

3. Results

3.1. Impact of microplastic addition on the dynamics of soil WEOM

The microplastic significantly affected DOC contents (Fig. 1).DOC content was higher in M2 and significantly lower in M1 thanCK on days 0 and 1. DOC content increased significantly in M1 andM2 during the first three days after microplastic addition,decreased between days 3 and 7, and then remained stable until theend of the experiment. DOC content was similar in CK and M1 butwas significantly higher in M2 between days 3 and 30. At day 30,DOC content in M2 increased by 35% relative to CK.

TDN contents ranged from 21.4 to 42.1 mg kg�1. TDN contentwas significantly lower in M1 than CK and M2 on days 0, 1 (Fig. 2)but was significantly higher in M1 and M2 than CK between days 7and 30, and was highest in M2. DON contents ranged from 3.4 to

Fig. 1. DOC, TDP, DOP and inorganic phosphorus concentration after microplastic addition. Error bars are standard deviations. Capital letters and Lowercase letters designatesignificant differences between sampling time and treatments, separately (P < 0.05).

Fig. 2. TDN, DON, NH4þ and NO3- concentration after microplastic addition. Error bars are standard deviations. Capital letters and Lowercase letters designate significant dif-ferences between sampling time and treatments, separately (P < 0.05).

H. Liu et al. / Chemosphere 185 (2017) 907e917910

24.1 mg kg�1. Microplastic addition significantly increased DONcontent, especially between days 14 and 30. Microplastic addition,however, significantly decreased NO3

� content during the first threedays, but NO3

� content did not differ significantly among thetreatments between days 7 and 30. Microplastic addition had nosignificant effect on NH4

þ content. TDN, DOC, and NO3� contents

decreased quickly in CK between days 1 and 7, but Microplasticaddition notably slowed the decrease.

TDP and DOP contents ranged from 0.2 to 1.68mg kg�1 and from

0.05 to 1.57 mg kg�1, respectively. Microplastic addition signifi-cantly increased TDP and DOP contents, especially by day 30, andthe contents were highest in M2 (Fig. 1). TDP and DOP contentstended to increase in M1 and M2 relative to CK. In contrast, PO4

3�

contents decreased quickly in M1 and M2 between days 14 and 30and were similar in all treatments by 30 day. PO4

3� content washighest in M2 between days 0 and 15 but did not notably betweenCK and M1.

Fig. 3. FDAse and phenol oxidase activities after microplastic addition. Error bars are standard deviations. Capital letters and Lowercase letters designate significant differencesbetween sampling time and treatments, separately (P < 0.05).

Fig. 4. Changes in SUVA254, SUVA280 and SUVA 365 indices after microplastic addition. Error bars are standard deviations. Capital letters and Lowercase letters designate sig-nificant differences between sampling time and treatments, separately (P < 0.05).

H. Liu et al. / Chemosphere 185 (2017) 907e917 911

3.2. Impact of microplastic addition on the dynamics of soil FDAseand PO activities

FDAse activities ranged from 5.7 to 24.2 mg kg�1 h�1. FDAseactivity decreased quickly in CK and M1 in the first three days andincreased between days 3e7. FDAse activity decreased inM2withinthe first day, increased between days 1 and 3, and then decreasedbetween days 3 and 7 (Fig. 3). FDAse activity in CK and M2remained stable between days 7 and 30, but it in M1 decreasedbetween days 7 and 30. Microplastic addition significantlyincreased FDAse activity, which was highest in M2. PO activitiesranged from 5.2 to 9.8mg kg�1 h�1. PO activity decreased inM1 andM2 in the first three days, subsequently increased between days 3and 7, and then decreased between days 7 and 30. PO activityvaried oppositely in CK.

3.3. Changes in the UV/Vis absorption spectrum of DOM

Specific UV absorbance (SUVA) at 254 nm is associated with

natural organic matter content in natural waters (Najm et al., 1994).SUVA 280 was introduced to represent total aromaticity (Sarathyand Mohseni, 2007), and SUVA 365 has been demonstrated to in-crease with molecular size (Peuravuori and Pihlaja, 2004). SUVA254, SUVA 280 and SUVA 365 tended to vary similarly over timeafter microplastic addition (Fig. 4). SUVA 254, SUVA 280 and SUVA365 increased in M2 during the first seven days and then sharplydecreased between days 7 and 14, which indicated the formation ofhigh-molecular-weight aromatic compounds during the first sevendays and their decomposition between days 7 and 14. SUVA 254,SUVA 280 and SUVA 365 remained stable in M2 between days 14and 30. SUVA 254, SUVA 280, and SUVA 365 sharply increased in CKduring the first day, decreased between days 1 and 3, thenincreased until day 14, and finally decreased until the end of theexperiment. Similarly, SUVA 254, SUVA 280, and SUVA 365immediately increased in M1 during the first day, decreased be-tween days 1 and 3, increased until day 7, and finally decreaseduntil the end of the experiment. SUVA 254, SUVA 280, and SUVA365 were highest in M2 and lowest in M1, indicating that a

a b

c d

e f

Fig. 5. Contour plots (c, d, e, f) and emission (a) and excitation (b) loadings of the four fluorescent components obtained by PARAFAC.

H. Liu et al. / Chemosphere 185 (2017) 907e917912

moderate level of Microplastic inhibited, and that a high level ofMicroplastic facilitated the formation of high-molecular-weightaromatic compounds.

Clear differences in EEM appearance at the start and end of theincubations were apparent (Fig. 6). Component 1 accumulated inCK between days 0 and 7 and subsequently decomposed until theend of the experiment. Component 1 increased in M1 during thefirst 14 days and subsequently decomposed until the end of theexperiment. Component 1 increased in M2 during the first threedays, decreased until day 7, and then increased between days 7 and30 (Fig. 7). Component 1 content was higher in CK than M1 and M2between days 1 and 14. However, component 1 content was sig-nificant higher inM2 than CK andM1 at day 30while it in CKwas aslow as M1. Component 2 varied similarly over time in CK, M1 andM2 after Microplastic addition. It accumulated rapidly during the

first day after microplastic addition, decreased until day 7, thenincreased between days 7 and 14, and finally decomposed until theend of the experiment. The component 2 content was significantlyhigher in M2 than CK and M1 throughout the experiment, espe-cially at days 1 and 30. However, component 2 content inM1was aslowas CK at day 1,14 and 30. Component 3 also increased quickly inM1 and M2 during the first day after Microplastic addition,decomposed sharply between days 1 and 7, then accumulatedquickly until day 14, and finally remained stable until the end of theexperiment. In contrast, component 3 decomposed immediately inCK until day 7 and then remained constant until the end of theexperiment. Component 3 was higher in CK thanM1 and M2 at day0 but was higher in M1 and M2 than CK between days 14 and 30.Component 3 was higher in M2 than M1 between days 1 and 3.Component 4 immediately increased in CK and M2 during the first

Day30

A B

C D

E F

Fig. 6. Representative fluorescence emissioneexcitation matrices (EEMs) at the start and end of 30-day incubations with or without microplastic addition. Panels show EEMs fromCK (A,B), M1 (C,D), an M2 (E,F). Panel columns represent EEM at the start of the incubation experiment, left, and at the end of the incubation, right.

H. Liu et al. / Chemosphere 185 (2017) 907e917 913

day, decreased in M2 until day 3 and in CK until day 7, then accu-mulated until day 14, and finally decomposed until day 30.Component 4 accumulated in M1 between days 0 and 7 anddecomposed sharply between days 7 and 30. Component 4throughout the experiment was lowest in CK. Component 4 washigher in M2 than CK and M1 at days 0, 1, 14 and 30. However,component 4 content in M1 was as low as CK at day 0 and 30.

4. Discussion

4.1. Effects of microplastic addition on soil DOM quantity

The soil microbial community plays a vital role in nutrient

cycling, including mineralization, biodegradation (Firestone et al.,2002; Murugan et al., 2014). Plastic residues in soil decrease soilsaturated hydraulic conductivity (Wang et al., 2015) and influencesoil microbial communities (Jiang et al., 2014). Soil microbial car-bon, nitrogen, soil fluorescein diacetate hydrolysis, soil dehydro-genase and Simpson indices declined by about 28.9e73.5%,38.2e76.2%, 1.6e30.7%, 14.9e59.0%, 1.8e18.7%, respectively (Wanget al., 2016) after the addition of plastic residues. However, theseresults only concern about the effect of the addition of larger par-ticle size debris (20 mm � 20 mm pieces) instead of microplasticson soil microbial activities. Our study initially presented the effectsof microplastic addition on soil enzyme activities. Microplasticaddition significantly promoted FDAse between day 7 and 30, and

Fig. 7. Changes of the Fmax values of the four components identified by PARAFAC after microplastic addition. Error bars are standard deviations. Capital letters and Lowercaseletters designate significant differences between sampling time and treatments, separately (P < 0.05).

H. Liu et al. / Chemosphere 185 (2017) 907e917914

increased PO activities between day 7 and 14, and these beneficialeffects were positively correlated with the content of microplastic(Fig. 3). Microplastic can sorb harmful contaminants from the soilsolution and alter soil physical properties, such as increasingporosity and changing aggregate structure, so microplastic may beincorporated into the dynamic structure of aggregates (Ladd et al.,1993; Rillig, 2012; Zhang et al., 2015; Huerta Lwanga et al., 2016b,2017). Previous studies have shown that porosity, specific surfacearea, and aggregate structure are positively correlated with soilmicrobial activities (Girvan et al., 2003; Arthur et al., 2012; Naveedet al., 2016). An increase in porosity may increase the flow of air insoil, which would enrich aerobic microorganisms (Rubol et al.,2013).

TDN, DON, TDP, and DOP contents in the DOM solution weresignificantly higher in M2 than M1 and CK, indicating that the highlevel of microplastic addition facilitated the release of soil nutrientsinto the soil solution and the accumulation of DOM. TDN, DON, TDP,and DOP contents were also higher in M1 than CK between days 14and 30 but did not differ significantly between CK and M1 duringthe first seven days, so that the beneficial effect of low contentmicroplastic addition on accumulation of DOM is a slow process.DOM dynamics depend on the imbalance between production andin situ mineralization (Schimel and Weintraub, 2003; Gogo et al.,2014). In our experiment, no external organic matter was addedto the soil, so the soil microorganisms likely played an importantrole in DOM dynamics. The activities of soil enzymes can be in-dicators of microbial activity and the availability of substrates formicrobial uptake. Soil enzymes also decompose organic matter andcatalyse important transformations in C, N, and P cycles(Wallenstein and Burns, 2011). PO activity is involved in thedegradation of recalcitrant (phenolic) compounds such as tanninand lignin (Keuskamp et al., 2015). PO activity increased aftermicroplastic addition, especially between days 7 and 14, leading tomore poorly dissolved high-molecular-weight compounds in soildecomposed into easily dissolved low-molecular-weight com-pounds, thus increasing dissolved organic matter. FDAse activity

can represent general metabolic activity and is a good indicator ofthe intensity of soil life and microbial activity (Perucci, 1992).Microplastic addition significantly stimulated the activity of FDAse,resulting in the enhancement of microbial hydrolytic activity onsoil organic matter or DOM, which would lead to the accumulationof DOM (Fig. 3). An increase in DOM hydrolysis would not alter theapparent DOC content, because dissolved compounds remain sol-uble after hydrolysis (Gogo et al., 2014).

The beneficial effect of microplastic addition on DOM nutrientavailability tended to increase over time aftermicroplastic addition.Some of the changes in concentrations of DOM observed during thefirst few days of the incubation might be partly related to thesample disturbance during mixing. As the influence of sampledisturbance on DOM nutrient availability decreases over time, theresults of the later sampling are more reliable. Similarly, thebeneficial effect of microplastic addition on microbial activity alsoincreased over time, further promoting the cycling of soil C, N and P,and increasing the bioavailability of soil C, N and P. DOC and DONare concentrated in different fractions of DOM (hydrophobic andhydrophilic, respectively) (Kaiser and Zech, 2000; Petrone et al.,2009). The hydrophobic acid fraction is considered to be lessdegradable than hydrophilic neutral fraction (Jandl and Sollins,1997; Kalbitz et al., 2003a). The labile fractions of DOM, such ascarbohydrates, are preferentially degraded by microorganisms, sorefractory DOM primarily accumulates during biodegradation(Ogawa et al., 2001; Kalbitz et al., 2003a, 2003b). In this study,microplastic addition had a more beneficial effect on DOC accu-mulation than DON within the first 3 days, as the DON contents inM1 and M2 were the same as CK at day 3. The lowerer decline ratesof DON and TDN contents in M1 and M2 between days 3 and 14relative to CK contributed to the accumulation of DON and TDN. Themain DOM components were likely recalcitrant materials such ashigh-molecular-weight humic-like material and fulvic acid, indi-cating more recalcitrant material in M1 and M2 at day 7 relative toCK (Fig. 7). PO, which can degrade insoluble high-molecular-weightcompounds l, such as Lignin, likely played a crucial role in DOM

H. Liu et al. / Chemosphere 185 (2017) 907e917 915

mineralization between days 7 and 30 (Yamashita and Tanoue,2003, 2004; Fellman et al., 2008). The higher PO activities in M1and M2 relative to CK between days 7 and 14 contributed to thedecomposition of insoluble high-molecular-weight materials, thuspromoting the accumulation of DON.

Our results suggested that microplastic addition immediatelycontributed to the suppression of the accumulation of NO3

�, but thiseffect was transient. Although Microplastic increases soil porosity,specific surface area, and oxygen content which can enhance thenitrification process, the low content of NH4

þ in soil is the limit forthe nitrification process. Microplastic addition stimulated soil mi-crobial activity which urged microbes to fiercely compete forlimited NH4

þ in soil, therefore limiting the nitrification process. Thereduction of NO3

� content after microplastic addition was notmainly on account of microbial degradation because microbespreferentially utilized the DON component of the DOM solution asN source relative to NO3

� and NH4þ (Ghani et al., 2013). Microplastic

addition increased microbial activities and favored DOC accumu-lation, whereas increased C-substrates exists to enable heterotro-phic microbial growth (NH4

þ immobilization) to dominate overautotrophic growth (NH4

þ oxidation) (Jones et al., 2004), thusinhibiting the NO3

� accumulation. The NH4þ and inorganic P con-

tents in the DOM solution were very low relative to the NO3� con-

tent. Microplastic addition had a negligible effect on NH4þ content in

DOM solution. The high level of microplastic addition couldpotentially increase inorganic P contents. However, Microplasticaddition had no significant effect on long-term inorganic P con-tents. The dynamics of inorganic N and P depend on the balancebetween mineralization of organic matter and in situ immobiliza-tion by chemoautotrophic bacteria. FDAse activity was much higherat day 0 than days 14 and 30, indicating a slower rate of soil mi-crobial mineralization between days 14e30, which would decreasethe contents of inorganic N and P.

4.2. Effects of microplastic addition on soil DOM quality

High level of microplastic addition facilitated the formation ofhigh-molecular-weight aromatic compounds in the DOM solutionduring the first seven days. However, SUVA 254, SUVA 280, andSUVA 365 subsequently decreased sharply in M2 between days 7and 14, suggesting that the high-molecular-weight aromatic com-pounds in the DOM solution were decomposed into smaller com-pounds, as supported by the EEM spectroscopic characteristics ofDOM (Fig. 7). High level of microplastic addition tended to facilitatethe long-term accumulation of high-molecular-weight aromaticcompounds relative to CK, while lower level of microplastic addi-tion initially promoted the degradation of high-molecular-weightaromatic compounds, but finally had a negligible effect on high-molecular-weight aromatic compounds contents and the molecu-lar size of humic substance.

A large proportion of the DOM in soil is humic acid (HA) andfulvic acid (FA) (Thurman, 1985). HA, with a molecular weight>1200 Da, is more readily degraded than FA, with a molecularweight of 500e1200 Da, because of its highly aromatic structure(Kisand et al., 2008; Rocker et al., 2012). Component 2, representinghigh-molecular-weight and aromatic humic materials, weresignificantly higher in M2 than CK throughout the experiment,especially at days 1 and 30, but there were no significant differ-ences of component 2 between CK and M1 throughout the exper-iment, suggesting that high level of microplastic addition promotedthe accumulation of humic substances while low level of micro-plastic addition had a negligible effect on humic substances.However, component 3, representing high-molecular-weight hu-mic material, accumulated rapidly between days 7 and 30 in M1and M2 relative to CK. This accumulation can be attributed to the

different variations of PO activities among CK, M1, and M2. PO ac-tivities between days 7 and 30 decreased in M1 and M2, butincreased in CK, which slowed the decomposition of humic-likematerial after microplastic addition, thus facilitating the accumu-lation of humic-like material. HA-rich materials are usefulamendments for soil remediation because they improve soilstructure, stability, water-holding capacity, nutrient availability,and sorption of metal ions (Schnitzer, 2000; Clemente and Bernal,2006; Hayes and Malcom, 2001a, 2001b). Microplastic additionpromoted the accumulation of high-molecular-weight humic-likematerial which improved the quality of soil. FA is the most mobilefraction and a major component of DOM in the environment(Stevenson, 1994; Maie et al., 2004). The content of FA in soil posesa large environmental risk, because it affects the transport andbioavailability of environmental contaminants, including heavymetals (Chirenje et al., 2002; Plaza et al., 2005), polycyclic aromatichydrocarbons (PAHs) (Perminova et al., 2001; Gunasekara and Xing,2003), and other chemicals (Stevenson, 1994) as carrying agentsand complexing media. Component 4, representing FA, wassignificantly higher in M2 than CK throughout the experiment,especially at days 1 and 30, and was significantly higher in M1 thanCK between days 1 and 14, but were the same between CK and M1at days 0 and 30, implying that the beneficial effect of microplasticaddition on fulvic acid content is positively correlated with thecontent of microplastic. The increase in FA-like material aftermicroplastic addition therefore indicates a higher risk of environ-mental pollution (Fig. 7).

Microbial metabolites such as peptides generally accumulatewhen carbohydrate content is low so that other compounds such aslignin-derived moieties and lipids can serve as energy and C sour-ces for microorganisms. Tyrosine-like fluorescence indicates morehighly degraded peptides, and tryptophan-like fluorescence mayindicate the presence of intact proteins or less degraded peptides(Mayer et al., 1999; Yamashita and Tanoue, 2003, 2004). Previousstudy showed that protein-like component is a useful predictor ofbiodegradable DOC compared to other fluorescent indicators(Fellman et al., 2008). In this study, microplastic addition initiallydecreased the tyrosine-like material in the DOM solution, whichmay have been due to the higher microbial activity in M1 and M2,contributing to enhanced the decomposition of tyrosine-like ma-terial. The tyrosine-like material, however, decreased in CK and M1between days 7 and 30 but increased sharply in M2 between days14 and 30, implying that the low level of micorplastic addition hadno significant influence on the long-term content of tyrosine-likematerial and that the beneficial effect of high level of microplasticaddition on the accumulation of tyrosine-like material in the DOMsolution could persist for a long time. Finally, the DOM in CK andM1 was mainly composed of high-molecular-weight humic mate-rial by the end of the experiment, indicating that this material wasless biodegradable. However, the DOM solution in M2 wascomposed of high content tyrosine-like material, high-molecular-weight humic material and fulvic acid, which was related to thehigh microbial enzyme activity in M2.

5. Conclusions

The occurrence of microplastic in soil affected soil DOM and theactivities of soil enzymes. Micropalstic addition stimulated soilactivity of fluorescein diacetate hydrolase (FDAse) in soil. The lowerlevel of microplastic addition had a negligible effect on the contentsof organic carbon, inorganic nitrogen, total phosphorus, high-molecular-weight humic-like material and fulvic acid in DOM so-lution at day 30 after microplastic added. The higher level ofaddition significantly increased the nutrient contents of the DOM,including those of DOC, DON, NO3

�, DOP, and PO43� and high-

H. Liu et al. / Chemosphere 185 (2017) 907e917916

molecular-weight humic-like material and fulvic acid. These implythat the accumulation of microplastic probably stimulate soilenzymatic activity which is useful for organic C, N, and P dissolvedin soil and increasing the available source for plant. Thus, furtherstudy needs to be considered on the interaction between micro-plastic accumulation and plant growth, as well as the risk assess-ment of particle uptake by plant. .

Acknowledgements

This work was supported by the Natural Science Foundation ofChina (41371510), West Young Scholars Project of the ChineseAcademy of Sciences (XAB2015A05), EU Horizon 2020 project(ISQAPER: 635750).

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