effects of nonylphenols on soil microbial activity and water retention

7
Applied Soil Ecology 64 (2013) 77–83 Contents lists available at SciVerse ScienceDirect Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil Effects of nonylphenols on soil microbial activity and water retention G. Ojeda a,, J. Patrício a , H. Navajas b , L. Comellas b , J.M. Alca ˜ niz c,d , O. Ortiz c,d , E. Marks c , T. Natal-da-Luz a , J.P. Sousa a a IMAR – Institute of Marine Research, c/o Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, 3004-517 Coimbra, Portugal b IQS Technical College, Ramon Llull University, 08017 Barcelona, Spain c CREAF, Cerdanyola del Vallès 08193, Spain d Universitat Autònoma de Barcelona, Cerdanyola del Vallès 08193, Spain article info Article history: Received 30 March 2012 Received in revised form 24 October 2012 Accepted 26 October 2012 Keywords: Nonylphenol Peat Water retention Soil microbial activity abstract The main aim of this study is to analyze the influence of 4-nonylphenol (NP) on soil water retention and biological activity. Two doses of 4-nonylphenol (25 and 50 mg kg 1 ) were tested in a loam soil with and without peat amendment. In general, one week after the start of the experiment, the soil water content retained at 0.75 MPa of soil suction was 18% higher in the soil amended and its basal respiration (BR) was 15% higher than soil without peat. In contrast, the microbial activity indices (CM: coefficient of mineralization or BR:total organic carbon (TOC) ratio; Cmic:Corg: microbial biomass carbon (MBC):TOC ratio; qCO 2 : metabolic quotient or BR:MBC ratio) were higher in the soil without peat, compared to the soil amended with peat. On the other hand, the addition of NP to soil was able to modify soil biological but not physical (water retention, desorption) properties. When soil was amended with peat, MBC was reduced one week after applying NP. In contrast, no effects of NP on MBC were observed in the soil without peat. BR was reduced by 16% one week after applying 50 mg kg 1 of NP to soil with peat, and was increased by 46% one week after applying 25 mg kg 1 of NP to soil without peat. The effects of NP on MBC and BR could be associated more with the adsorption of NP by soil organic matter, while changes in CM or Cmic:Corg ratio were more closely related to changes in soil water retention. The potential toxic effects of NP (high qCO 2 values) were only observed in the absence of peat amendments. Peat addition reduced NP toxic effects on microorganisms. © 2012 Elsevier B.V. All rights reserved. 1. Introduction 4-Nonylphenol (NP) is the main degradation product of a group of non-ionic surfactants known as alkylphenol polyethoxylates, which have been a common component of domestic and indus- trial cleaning products (Roberts et al., 2006). The environmental occurrence of alkylphenols, such as NP, has been established since the late 1970s (Sheldon and Hites, 1978), with a worldwide pro- duction of about 500 Gg (Petrovic and Barceló, 2001). NP originates from the biodegradation of nonylphenol ethoxylates, which include other chemical forms of nonylphenol ethoxylates (nonylphe- nol, nonylphenol monoethoxylate, nonylphenol diethoxylate) and nonylphenol carboxy acids (4-nonylphenoxy acetic acid, nonylphe- noxy ethoxy acetic acid) (Domene et al., 2010). In general, NP could be adsorbed in soil particles by hydrophilic interaction with min- eral components and hydrophobic interaction with organic matter Corresponding author. Tel.: +351 239855760x415; fax: +351 239823603. E-mail address: g [email protected] (G. Ojeda). in sediments, as observed by John et al. (2000) or by closely binding to humic acids in soils (Höllrigl-Rosta et al., 2003). Estrogenic, toxic and carcinogenic effects of NP in various teleost fish species, birds and mammals have been reported by several researchers over the last decade, at doses as low as 0.05–0.1 mg L 1 in water (Soares et al., 2008; Sayed et al., 2012). Thus, in order to fight NP pollution, the European Union (EU) has published Direc- tives aimed at environmental protection. The Water Framework Directive (European Commission, 2000) includes limitations for nonylphenol and today most of their uses are regulated (European Commission, 2003) due to the fact that they are considered to be priority hazardous substances (PHS) (Soares et al., 2008). Although critical levels of nonylphenol ethoxylates have been proposed or fixed for sewage sludge (50 mg kg 1 )(European Communities, 2000), freshwater (28 gL 1 ) or saltwater (28 gL 1 ) ecosystems (EPA, 2005), soil ecosystems are still not regulated in terms of NP concentration in soil. NP has been detected in rain and snow, prob- ably as a result of NP evaporation from water surfaces, soil bodies and vegetation (Friesa and Puttman, 2004; Nelson et al., 1998). Surface waters, sediments, groundwater, air and soil are the environmental compartments where NP is commonly found 0929-1393/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsoil.2012.10.012

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Page 1: Effects of nonylphenols on soil microbial activity and water retention

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Applied Soil Ecology 64 (2013) 77–83

Contents lists available at SciVerse ScienceDirect

Applied Soil Ecology

journa l homepage: www.e lsev ier .com/ locate /apsoi l

ffects of nonylphenols on soil microbial activity and water retention

. Ojedaa,∗, J. Patrícioa, H. Navajasb, L. Comellasb, J.M. Alcanizc,d, O. Ortizc,d, E. Marksc, T. Natal-da-Luza,.P. Sousaa

IMAR – Institute of Marine Research, c/o Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, 3004-517 Coimbra, PortugalIQS Technical College, Ramon Llull University, 08017 Barcelona, SpainCREAF, Cerdanyola del Vallès 08193, SpainUniversitat Autònoma de Barcelona, Cerdanyola del Vallès 08193, Spain

r t i c l e i n f o

rticle history:eceived 30 March 2012eceived in revised form 24 October 2012ccepted 26 October 2012

eywords:onylphenoleatater retention

oil microbial activity

a b s t r a c t

The main aim of this study is to analyze the influence of 4-nonylphenol (NP) on soil water retention andbiological activity. Two doses of 4-nonylphenol (25 and 50 mg kg−1) were tested in a loam soil with andwithout peat amendment. In general, one week after the start of the experiment, the soil water contentretained at −0.75 MPa of soil suction was 18% higher in the soil amended and its basal respiration (BR)was 15% higher than soil without peat. In contrast, the microbial activity indices (CM: coefficient ofmineralization or BR:total organic carbon (TOC) ratio; Cmic:Corg: microbial biomass carbon (MBC):TOCratio; qCO2: metabolic quotient or BR:MBC ratio) were higher in the soil without peat, compared to thesoil amended with peat. On the other hand, the addition of NP to soil was able to modify soil biologicalbut not physical (water retention, desorption) properties. When soil was amended with peat, MBC wasreduced one week after applying NP. In contrast, no effects of NP on MBC were observed in the soil

−1

without peat. BR was reduced by 16% one week after applying 50 mg kg of NP to soil with peat, andwas increased by 46% one week after applying 25 mg kg−1 of NP to soil without peat. The effects of NP onMBC and BR could be associated more with the adsorption of NP by soil organic matter, while changes inCM or Cmic:Corg ratio were more closely related to changes in soil water retention. The potential toxiceffects of NP (high qCO2 values) were only observed in the absence of peat amendments. Peat additionreduced NP toxic effects on microorganisms.

© 2012 Elsevier B.V. All rights reserved.

. Introduction

4-Nonylphenol (NP) is the main degradation product of a groupf non-ionic surfactants known as alkylphenol polyethoxylates,hich have been a common component of domestic and indus-

rial cleaning products (Roberts et al., 2006). The environmentalccurrence of alkylphenols, such as NP, has been established sincehe late 1970s (Sheldon and Hites, 1978), with a worldwide pro-uction of about 500 Gg (Petrovic and Barceló, 2001). NP originatesrom the biodegradation of nonylphenol ethoxylates, which includether chemical forms of nonylphenol ethoxylates (nonylphe-ol, nonylphenol monoethoxylate, nonylphenol diethoxylate) andonylphenol carboxy acids (4-nonylphenoxy acetic acid, nonylphe-oxy ethoxy acetic acid) (Domene et al., 2010). In general, NP could

e adsorbed in soil particles by hydrophilic interaction with min-ral components and hydrophobic interaction with organic matter

∗ Corresponding author. Tel.: +351 239855760x415; fax: +351 239823603.E-mail address: g [email protected] (G. Ojeda).

929-1393/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.apsoil.2012.10.012

in sediments, as observed by John et al. (2000) or by closely bindingto humic acids in soils (Höllrigl-Rosta et al., 2003).

Estrogenic, toxic and carcinogenic effects of NP in various teleostfish species, birds and mammals have been reported by severalresearchers over the last decade, at doses as low as 0.05–0.1 mg L−1

in water (Soares et al., 2008; Sayed et al., 2012). Thus, in order tofight NP pollution, the European Union (EU) has published Direc-tives aimed at environmental protection. The Water FrameworkDirective (European Commission, 2000) includes limitations fornonylphenol and today most of their uses are regulated (EuropeanCommission, 2003) due to the fact that they are considered to bepriority hazardous substances (PHS) (Soares et al., 2008). Althoughcritical levels of nonylphenol ethoxylates have been proposedor fixed for sewage sludge (50 mg kg−1) (European Communities,2000), freshwater (28 �g L−1) or saltwater (28 �g L−1) ecosystems(EPA, 2005), soil ecosystems are still not regulated in terms of NPconcentration in soil. NP has been detected in rain and snow, prob-

ably as a result of NP evaporation from water surfaces, soil bodiesand vegetation (Friesa and Puttman, 2004; Nelson et al., 1998).

Surface waters, sediments, groundwater, air and soil are theenvironmental compartments where NP is commonly found

Page 2: Effects of nonylphenols on soil microbial activity and water retention

7 Soil Ecology 64 (2013) 77–83

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Table 1Soil and peat physical–chemical properties.

Parameter Unity Soil Peat

Dry matter % 98.4 97.4pH (Water, 1:5) – 8.3 7.8EC (25 ◦C, 1:5) dS/m 0.2 1.54Sand (2–0.2 mm) % 46.0 –Silt (0.2–0.05 mm) % 34.6 –Clay (<0.05 mm) % 19.4 –Textural class – Loam –Carbonates % 18 3.5Organic matter % 1.72 23.7Total N (Kjeldahl) % 0.08 0.83Degree of stability % – 67.2Cr mg kg−1 28 14Ni mg kg−1 18 7Pb mg kg−1 23 7Cu mg kg−1 25 16Zn mg kg−1 75 42Hg mg kg−1 52 0.02

soil amended with peat; P1, soil with peat and 25 mg of nonylphe-nol kg−1; and P2, soil with peat and 50 mg of nonylphenol kg−1.All treatments of the experiment were assayed in triplicate and asa result the number of samples were: 6 treatments × 3 replicates

Table 2Mean values of some biochemical properties with their statistical differences deter-mined by two-way variance analysis (n = 18) between treatments, one week afterNP application to soil.

Parameter Mean Parameter Mean Parameter Mean

NP (mg kg−1) p < 0.01b TOC (%) p < 0.001a,b MWD (mm) p < 0.05a,b

P0 0.00b P0 1.21a P0 1.52aP1 4.32a P1 0.90a P1 1.93aP2 7.89a P2 0.96a P2 2.16aS0 0.00b S0 0.63b S0 1.29bS1 3.25a S1 0.50b S1 1.43b

8 G. Ojeda et al. / Applied

Chang et al., 2007; Soares et al., 2008). In general, pollutant degra-ation is linked to its bioavailability, which could be defined as theraction that can be taken up or transformed by (micro)organismsde Weert et al., 2008). Since microbes are water-dependent orga-isms and require sufficient water to maintain their activity even inoil (Stotzky, 1986), studying how NP could modify soil water avail-bility may be important in order to explain its potential ecotoxicffects.

Soil hydraulic properties are influenced by certain soil prop-rties such as texture, bulk density and organic matter content,here soil sorptivity, the capacity to absorb/desorb water by cap-

llarity, could be altered by surfactants (Mingorance et al., 2007).on-ionic surfactants such as nonylphenols can decrease the liq-id surface tension and increase the solid–liquid contact angle,hich means that NP can reduce the wettability of solid surfaces

Luepakdeesakoon et al., 2006). Since soil water retention depends,mong other things, on the amount of organic carbon present in theoil (Rawls et al., 2003) and on soil wettability (Bachmann et al.,008), decreased wettability of soil aggregates would reduce slak-

ng and microcracking, increasing the pore space able to hold waterOjeda et al., 2008). Therefore, it is possible that a physical–chemicalnteraction among soil, organic matter and pollutants better defineshe pollutant’s bioavailability and its effects on soil.

Microbial mediated functions such as the turnover of C and, soil structure stability, and biodegradation of organic pollut-nts are of agronomic and environmental importance (Chenund Stotzky, 2002). Soil supports biogeochemical cycles becauseicroorganisms degrade virtually all organic compounds, includ-

ng xenobiotics and naturally occurring polyphenolic compoundsNannipieri et al., 2003). Thus, it is necessary to identify microbialarameters that allow the evaluation of the effects of pollutants onoil microorganisms. For this purpose, microbial basal respirationBR) and microbial biomass carbon (MBC) have been suggested asalid indicators of microbial activity (Lagomarsino et al., 2011).

Despite the fact that high NP concentrations can be toxic toicroorganisms (Corvini et al., 2006), studies on NP interactionith soil’s physical and microbial properties are scarce. The main

im of this study is to gain knowledge about: (i) the effects of NP onoil water retention, (ii) bioavailability of NP to soil microorganismss evaluated by microbial activity and (iii) the relationship betweenoil water retention and microbial activity in a soil amended witheat and polluted with NP.

. Materials and methods

.1. Experimental site, soil and peat properties

The experiment was carried out in the greenhouses of theutonomous University of Barcelona (Cerdanyola del Vallès, Spain).he soil used was collected from the B layer of a Typic CalcixereptSoil Survey Staff, 2010) at the same location. This soil had formerlyeen used for grain production and had been free of agrochemicalsor at least 10 years. After collection, the soil was air-dried for aeek and sieved (5 mm). Half of the soil collected was amendedith natural peat (also sieved to 5 mm) in order to increase the soil

rganic matter content by 1% adding a dose of 43 g of peat kg−1

f soil. It was intended to force the interaction of 4-nonylphenolNP) with a stable organic fraction, simulating a “clean” organicmendment. The eutrophic peat (Hemic Haplosaprist, Soil Surveytaff, 2010) selected was formed by intense anaerobic transfor-ation of vegetal remains in a coastal lagoon of the Ebro River

elta (Tarragona, NE, Spain) and contains a silty calcareous mineralatrix. Soil and peat properties are shown in Table 1. As additional

nformation, the temporal changes in NP concentration in soil, totalrganic carbon (TOC) and soil aggregate stability (evaluated by

mean weight diameter (MWD) values) during the experiment areshown in Table 2.

2.2. Experimental setup

The soil with and without peat was distributed in six trays(2 m × 10 m) in a thin layer of 3 cm to facilitate the homogeneousdistribution of 4-nonylphenol (NP) when it was applied. Two dosesof NP (25 mg kg−1 and 50 mg kg−1) of technical-grade NP of 95%purity (KAO Corporation, Barcelona, Spain) were used in this study.These doses were selected in order to evaluate the limit of NPconcentration in sewage sludge (50 mg kg−1) as regulated by work-ing document on sludge, 3rd draft (European Communities, 2000),when used as organic soil amendment, because this limit is the onlydirective that regulates NP concentration in soil.

Nonylphenol was diluted in acetone (95%) due to its highhydrophobicity, prior to homogeneous spraying over the soil layer.The acetone was then left to evaporate for 24 h. Two days later, soiltreatments with/without peat and with/without NP were homog-enized mechanically in a cement mixer and then distributed in 5 Lmetallic lysimeters (each with 4.5 kg of soil and 0.5 kg of gravelin the bottom to facilitate drainage). The six treatments assayedwere: S0, soil without peat and nonylphenol; S1, soil with 25 mgof nonylphenol kg−1; S2, soil with 50 mg of nonylphenol kg−1; P0,

S2 4.63a S2 0.65b S2 1.88b

NP: 4-nonylphenol soil concentration. TOC: total organic carbon of soil aggregates<5 mm in diameter. MWD: mean weight diameter. Differences were significant forap-perMANOVA and bp-Monte Carlo.

Page 3: Effects of nonylphenols on soil microbial activity and water retention

Soil Ec

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er treatment × 1 sampling = 18 samples. Soil NP concentration wasnalyzed one week after the start of the experiment.

.3. Soil biochemical properties

Water-retention curves were estimated one week after the startf the experiment to describe the soil water content at −0.75 MPa,hich could be considered as appropriate moisture conditions foricrobial activity, as observed by Paradelo and Barral (2009). Soil

amples of 1.2 g (<5 mm) from each lysimeter were placed in a cupf known weight and saturated for ten minutes by using one filteraper strip in contact with a free water table, approximately 10 mmbove the soil surface, after which the water excess was carefullyemoved by a syringe. An electronic balance (with 0.0001 g preci-ion) and a WP4 Dewpoint PotentiaMeter (Decagon Devices, Inc.,ullman, WA, USA) were then used to record the gravimetric waterontent (w) and the soil suction ( ), within the range between −0.5nd −1.5 MPa as the sample dried by evaporation. The residenceime of water stored between −0.75 and −1.5 MPa (tdry) was also

easured and considered as a soil water retention property. TheP4 measures water potential by determining the relative humid-

ty of the air above a sample in a closed chamber. At equilibriumemperature, relative humidity is a direct measure of water poten-ial. Approximately 6 measurements of w and were obtainedor each soil sample at mean values of 25 ◦C and 50% relative airumidity recorded by a CR10X Datalogger (Campbell Scientific, Inc.,ogan, Utah, USA). Soil water content at −0.75 MPa, obtained fromater retention curves fitted by a power model (Ojeda et al., 2011),as used to determine differences in soil water retention between

reatments.Microbial biomass and basal respiration were measured in soil

amples sieved to <5 mm, one week after NP application to soil.BC was measured by the fumigation–extraction method (Vance

t al., 1987) using 0.5 M K2SO4 as the extraction solution (1:4oil:extraction solution, w/v) and an extractability factor kEC = 2.65Vance et al., 1987). Water content during fumigation was 45–55%f water holding capacity. Microbial BR was measured by the CO2volved from a fresh soil sample incubated at 21 ◦C and at 45% of itsater-holding capacity for 20 days (Anderson, 1982). During this

ime, the CO2 produced by the sample was collected by a sodiumydroxide solution of known concentration. After incubation, theoncentration of sodium hydroxide and the solution was comparedo a container without soil (blank). All sodium hydroxide lost dur-ng incubation time corresponded to the CO2 produced by the soilample.

Soil aggregate stability and TOC were determined in soil sam-les obtained one week after NP application to soil. Soil aggregatetability was measured by fast wetting test modified from Le Bis-onnais’s method (Le Bissonnais, 1996), without the use of ethanol.ne gram of soil aggregates (3–5 mm in diameter) was put inach of the six sieves of different mesh (2.0, 1.0, 0.5, 0.25, 0.125,.053 mm) on a Wet Sieving Apparatus (Eijkelkamp©) and wereapidly immersed in 90 mL of distilled water for 10 min. Next, alow, gentle mechanical up-and-down movement was applied formin. Finally, the aggregates retained on each sieve were driedt 40 ◦C for 24 h. The mean weight diameter (MWD) was calcu-ated as the sum of the mass fraction remaining in each range ofize between sieves (5.0–2.0, 2.0–1.0, 1.0–0.5, 0.5–0.25, 0.25–0.125,.125–0.053, <0.053 mm), multiplied by the mean aperture of thedjoining sieves. MWD value was considered a soil aggregate stabil-ty index. TOC content was measured by the wet oxidation methodNelson and Sommers, 1982) in whole soil samples (<5 mm diame-

er), and in soil aggregates (3–5 mm) selected for the soil aggregatetability test. The carbon mineralization coefficient and the micro-ial metabolic quotient (qCO2) were calculated, respectively, as theatio between basal respiration and organic carbon, and the ratio

ology 64 (2013) 77–83 79

between basal respiration and soil microbial biomass (Andersonand Domsch, 1985). Furthermore, the ratio of MBC to TOC was alsocalculated.

2.4. Nonylphenol analysis

Five grams of soil were placed in a 250 mL Erlemmeyer flask,adding 5 mL of a solution of 25 mg L−1 of internal standard of 4-n-heptilfenol in dichloromethane (DCM). Two ultrasonic extractionswith 50 mL of DCM were immediately carried out for 30 min. Theextract was decanted and placed in a 100 mL heart-shaped flaskusing a Pasteur pipette. The residue was washed twice with vol-umes of 5 mL of DCM to collect all of the extract. After that, theextract was concentrated in a Rotavapor at a bath temperatureof 40 ◦C until reaching a weight equivalent to 10 mL of DCM, thissolution being subsequently injected into a high resolution gaschromatography coupled with mass spectrometry (HRGC–MS). Allsamples were extracted in duplicate and were injected in duplicate.NP was quantified by HRGC–MS.

The chromatographic conditions developed for NP quantifi-cation in soil samples by HRGC–MS in SIM mode (Selected IonMonitoring) were: (i) equipment: Agilent Technologies 6890 N,(ii) mass spectrometer: Agilent Technologies 5973 inert MassSelective Detector, (iii) column: Agilent Technologies – 5 mmethylpolysiloxane 5% phenyl 95% (30 m, 0.25 mm, 0.25 �m), (iv)oven temperature: 100 ◦C (1 min) ramp to 20 ◦C/min to 280 ◦C(10 min), (v) chromatogram time: 21 min, (vi) injector tempera-ture: 250 ◦C 10:1 flow splitter mode: 10 mL/min, (vii) injectionvolume: 2 �L. Terms of the mass spectrometer: (i) acquisitionmode: SIM, (ii) mass (m/z): 135 and 192, (iii) ionization mode: E.I.,(iii) interface temperature: 150 ◦C, (iv) temperature of ion source:230 ◦C, (v) solvent delay: 5 min.

2.5. Statistical analysis

The main aim of this study was to analyze the influence of 4-nonylphenol (NP) on soil water retention and biological activity.Three doses of 4-nonylphenol (0, 25 and 50 mg kg−1) were testedin a loam soil (Typic Calcixerept) with and without peat amendment.We tested the effects of NP using univariate analysis methods avail-able in the PRIMER v6 statistical package (Clarke and Gorley, 2006)and the PERMANOVA + PRIMER add-on package (Anderson et al.,2008). Potentially significant differences in soil’s physical, chemi-cal and biological properties between “Dose” (3 levels: 0, 25 and50 mg kg−1) and “Substrate” (2 levels: Soil and Soil + Peat) weretested using a two-way PERMANOVA design (“Dose” and “Sub-strate” as fixed factors). Previous to the analyses, soil data werenormalized and a similarity matrix based on the Euclidean distancecoefficient was constructed. For the tests, the ‘Permutation of resid-uals under a reduced model’ was the permutation method chosen,and 9999 random permutations were used. The null hypothesiswas rejected when the significance level p was <0.05 (if the numberof permutation was lower than 150, the Monte Carlo permutationp was used). Significant terms and interactions were investigatedusing subsequent pair-wise comparisons with the PERMANOVA t-statistic and 9999 permutations. All regressions used for comparingthe impact of NP on soil properties were done with the StatView®(SAS Institute, 1998) statistical program.

3. Results

3.1. Effects on soil biochemical properties

Significant differences in water retention at −0.75 MPa(W−0.75 MPa) were observed between substrates (Table 3). Peatamendment increased soil water content at −0.75 MPa (Fig. 1a),

Page 4: Effects of nonylphenols on soil microbial activity and water retention

80 G. Ojeda et al. / Applied Soil Ecology 64 (2013) 77–83

(a)

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Fig. 1. Mean values and standard errors of (a) gravimetric water content at −0.75 MPa of soil suction (W−0.75 MPa) and (b) the residence time of water stored between −0.75a hout NS kg−1 ow

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nd −1.5 MPa (tdry), one week after nonylphenol application. P0: soil with peat wit0: soil without peat or NP, S1: soil + 25 mg of NP kg−1 of soil, S2: soil + 50 mg of NPithout peat) are not significantly different at p < 0.05.

ompared to soil without peat (p < 0.001), 18.4%. In contrast, differ-nces in the residence time of water stored between −0.75 and1.5 MPa (tdry) were not observed between substrates or doses

Table 3, Fig. 1b), although NP seemed to increase tdry values.On the other hand, with respect to MBC values, while no

ifferences between substrates were seen, significant differ-nces between NP doses and a significant interaction betweenubstrates × doses were observed (Table 3) one week after NP appli-ation. This means that the effect of the NP dose on MBC needs toe analyzed separately for each substrate. In general, without tak-

ng into account the type of substrate, MBC values of treatmentsithout NP or with an initial dose of 25 mg kg−1 of NP, were higher

han the MBC values in the treatments where an initial dose of0 mg kg−1 of NP was applied (p < 0.05). However, when each sub-trate was analyzed separately, MBC of soil amended with peatP0) was reduced significantly by both NP doses (P1: 38%, P2: 31%)p < 0.05), (Fig. 2a). In contrast, MBC in soil without peat (S0) wasot modified by NP doses, although the MBC values of soil with an

nitial dose of 25 mg kg−1 of NP (S1) were higher than the MBC val-es of soil with an initial dose of 50 mg kg−1 of NP (S2) (p < 0.001)

Fig. 2a).

With respect to soil respiration (BR), significant differences of BRalues between substrates, no differences between NP doses and aignificant interaction between substrates × doses were observed

able 3ummary of two-way variance analysis of some soil physical and biochemical prop-rties between substrates (S: soil, P: soil + peat) and NP doses (D0: without NP, D1:ith 25 mg of nonylphenol kg−1 soil, D2: with 50 m nonylphenol kg−1 soil), one week

fter nonylphenol application to soil.

Parameters Pseudo-F

S: substrate D: dose S × D: interaction

W−0.75 MPa (g g−1) 49.5***a,***b NS NStdry (min) NS NS NSMBC (�Cmic g−1) NS 9.7**a**b 13.0**a***b

BR (�C-CO2 kg−1 d−1) 12.3**a**b NS 10.1**a**b

CM (�g C-CO2 g−1 C d−1) 15.5**a**b 6.1*a*b NSCmic:Corg (�g Cmic g−1 C g−1) 9.3*a*b 10.1**a**b 10.2**a**b

qCO2 (�g C-CO2 �g C d−1) 8.0*a*b 25.4***a***b 28.6***a***b

−0.75 MPa: gravimetric water content at −0.75 MPa of soil suction. tdry: the residenceime of water stored between −0.75 and −1.5 MPa. MBC: microbial biomass car-on. BR: basal or soil respiration. CM: coefficient of mineralization or ratio betweenR: TOC. Cmic:Corg: ratio between MBC and TOC. qCO2: metabolic quotient or ratioetween BR: MBC. NS: not significant. Differences were significant at p < 0.05 (*),.01 (**) or 0.001 (***) for ap-perMANOVA and bp-Monte Carlo.

P, P1: soil + peat + 25 mg of NP kg−1 of soil, P2: soil + peat + 50 mg of NP kg−1 of soil,f soil. Bars with the same letter at each soil property and substrate (soil with and

(Table 3), one week after the start of the experiment. In general,BR in soil with peat was higher than in soil without peat (p < 0.01),15%. Analysing each substrate separately, when soil was amendedwith peat (P0), BR was reduced but only when an initial dose of50 mg kg−1 of NP (P2) was applied to soil (p < 0.05), 16% (Fig. 2b). Incontrast, BR in soil without peat (S0) was increased only when aninitial dose of 25 mg kg−1 of NP (S1) was applied to soil (p < 0.05),46% (Fig. 2b).

Regarding microbial activity indices, clear differences in thecoefficient of mineralization (CM) values between substrates andbetween NP doses, without a significant interaction between sub-strates × doses (Table 3), were observed one week after the startof the experiment. Globally, when soil was amended with peat itsCM was lower than the CM of soil without peat (p < 0.01), a 32.1%(Fig. 3a). On the other hand, without taking into account the type ofsubstrate (soil with or without peat), the CM of treatments withoutNP was increased only when an initial dose of 25 mg kg−1 of NP wasapplied (p < 0.01).

When the ratio between MBC and total organic carbon(Cmic:Corg) was analyzed, differences in Cmic:Corg values betweensubstrates or between NP doses, and a significant interactionbetween substrates × doses were observed (Table 3), one weekafter NP application. Thus, the mean values of Cmic:Corg ratio in soilwith peat were lower than in soil without peat (p < 0.05), a 35.7%.Without taking into account the type of substrate, the Cmic:Corg

ratio of the treatments without NP was not modified when NP wasapplied, although the Cmic:Corg ratio of treatments with an initialdose of 25 mg kg−1 of NP was higher than the treatments with aninitial dose of 50 mg kg−1 of NP (p < 0.01). Analysing each substrateseparately, the Cmic:Corg ratio of soil amended with peat withoutNP (P0) was not modified by any NP dose (P1 or P2) (Fig. 3b). Incontrast, despite the fact that the Cmic:Corg ratio in the soil withoutpeat or NP (S0) was not modified when NP was applied to soil, theCmic:Corg ratio in the soil with an initial dose of 25 mg kg−1 of NP(S1) was higher than in the soil with an initial dose of 50 mg kg−1

of NP (S2) (p < 0.01) (Fig. 3b).Finally, like the Cmic:Corg ratio, differences in the metabolic quo-

tient (qCO2 = BR: MBC ratio) values between substrates or betweenNP doses, and a significant interaction between substrates × doseswere observed (Table 3) one week after NP application. Globally,

the qCO2 in the soil with peat was lower than in the soil withoutpeat (p < 0.05), a 22%. Without taking into account the type of sub-strate, the qCO2 of the treatments without NP or with an initial doseof 25 mg kg−1 of NP were was lower than the treatments with an
Page 5: Effects of nonylphenols on soil microbial activity and water retention

G. Ojeda et al. / Applied Soil Ecology 64 (2013) 77–83 81

0

100

200

300

400

500

MB

C (µ

Cm

icg

-1)

0

5

10

15

BR (µ

C-C

O2

Kg- 1

d-1)

TreatmentsP0 P1 P2

a

bb

S0 S1 S2

a

ab

b

P0 P1 P2

aab

b

S0 S1 S2

a

ab

b

Treatments

(a) (b)

F (b) sow 1 of sN ith an

iswwpN

3

irabwio(

toit(nw

F(pn

ig. 2. Mean values and standard errors of (a) microbial biomass carbon (MBC) andithout NP, P1: soil + peat + 25 mg of NP kg−1 of soil, P2: soil + peat + 50 mg of NP kg−

P kg−1 of soil. Bars with the same letter at each soil property and substrate (soil w

nitial dose of 50 mg kg−1 of NP (p < 0.05). Analysing each substrateeparately, when soil was amended with peat (P0) its qCO2 valuesere increased only when an initial dose of 25 mg kg−1 of NP (P1)as applied (Fig. 3c), a 33%. In contrast, the qCO2 of soil withouteat (S0) was increased only when an initial dose of 50 mg kg−1 ofP (S2) was applied (Fig. 3c), a 167%.

.2. Relationships between soil properties

Since soil water is a main factor affecting soil microbial activ-ty, the interaction between different soil properties and soil wateretention was analyzed. The gravimetric water content of soilt −0.75 MPa, which corresponds to the middle suction valueetween −0.033 MPa (field capacity) and −1.5 MPa (permanentilting point), was not dependent on nonylphenol concentration

n soil (NP) (Fig. 4a), but it was significantly related to the totalrganic carbon (TOC) of soil (Fig. 4b) and mean weight diameterMWD) of water-stable soil aggregates (Fig. 4c).

On the other hand, decreases in the coefficient of mineraliza-ion (CM) values (Fig. 5a) and the microbial biomass carbon and soilrganic carbon ratio (Cmic:Corg) (Fig. 5b) corresponded to increasesn water retention at −0.75 MPa of soil suction (W−0.75 MPa). In con-

rast, no significant relationship between the metabolic quotientqCO2) and W−0.75 MPa (Fig. 5c) was observed. Similarly, no sig-ificant relationships between soil basal respiration (BR) or MBCith W−0.75 MPa were observed (data not shown), and no significant

0

5

10

15

20

25

CM

(µg

C-CO

2g-1

C d-1

)

S0 S1 S20

200

400

600

800

1000

1200

Cm

ic:C

org

(µg

Cm

icg

- 1C

g-1

)

P0 P1 P2Treatments

a

P0 P1 P2

b b

a

(a) (b)

a a

b

a a

ig. 3. Mean values and standard errors of (a) coefficient of mineralization (CM), (b) microqCO2), one week after nonylphenol application. P0: soil with peat without NP, P1: soil + peeat or NP, S1: soil + 25 mg of NP kg−1 of soil, S2: soil + 50 mg of NP kg−1 of soil. Bars with tot significantly different at p < 0.05.

il basal respiration (BR), one week after nonylphenol application. P0: soil with peatoil, S0: soil without peat or NP, S1: soil + 25 mg of NP kg−1 of soil, S2: soil + 50 mg ofd without peat) are not significantly different at p < 0.05.

relationships between microbial activity (BR, MBC, CM, Cmic:Corg orqCO2) and NP values were observed (data not shown).

4. Discussion

Soil water retention, as evaluated by the soil water content at−0.75 MPa, was not significantly influenced by NP (Figs. 1a and4a). Instead, peat amendment was able to increase water reten-tion as expected. Rawls et al. (2004) observed that the sensitivityof a soil’s water retention properties depends on the initial organiccarbon content. In this case, the peat dose used signifies an effectiveincrease in soil organic carbon content (Table 2), able to incrementsoil water retention.

Another important factor in soil water retention processes isrelated to the soil’s ability to resist the movement of air bubblesduring the entry of water without a generalized collapse of soilaggregates (slaking). One week after the start of the experiment,it was observed that peat increased soil organic carbon and aggre-gate stability (Table 2) and that these increments corresponded toincreases in soil water retention (Fig. 4b and c). Probably, improve-ments in soil aggregate stability mediated by increments in soilorganic carbon helps to preserve soil pores able to hold water(Ojeda et al., 2008). Moreover, the potential hydrophobicity of NP

(Soares et al., 2008; Xia and Jeong, 2004) and dry peat (Michelet al., 2001), may also help to increase soil aggregate stability dur-ing soil wetting processes. On the other hand, peat is an organicmaterial, with low density in its solid phase (Sławinski et al., 2002),

S0 S1 S20

0.02

0.04

0.06

0.08

0.10

qC

O2

(µg

C-C

O2µ

gC

d-1

)

P0 P1 P2 S0 S1 S2TreatmentsTreatments

a

ab

b

a

a

bbb b

(c)

bial biomass carbon:total organic carbon ratio (Cmic:Corg) and (c) metabolic quotientat + 25 mg of NP kg−1 of soil, P2: soil + peat + 50 mg of NP kg−1 of soil, S0: soil withouthe same letter at each soil property and substrate (soil with and without peat) are

Page 6: Effects of nonylphenols on soil microbial activity and water retention

82 G. Ojeda et al. / Applied Soil Ecology 64 (2013) 77–83

Fig. 4. Interactions between gravimetric water content at −0.75 MPa of soil suction (W−075 MPa) vs. (a) nonylphenol concentration on soil (NP), (b) total organic carbon of soil(TOC) and (c) mean weight diameter (MWD), one week after nonylphenol addition. P0: soil with peat without NP, P1: soil + peat + 25 mg of NP kg−1 of soil, P2: soil + peat + 50 mgof NP kg−1 of soil, S0: soil without peat or NP, S1: soil + 25 mg of NP kg−1 of soil, S2: soil + 50 mg of NP kg−1 of soil.

F or MB− l withN l + 50 m

ww

t(abbSwclNNe

thwsttgplopbq

ig. 5. Interactions between (a) CM or coefficient of mineralization, (b) Cmic:Corg

0.75 MPa of soil suction (W−075 MPa), one week after nonylphenol addition. P0: soiP kg−1 of soil, S0: soil without peat or NP, S1: soil + 25 mg of NP kg−1 of soil, S2: soi

hich, once wet, has high water retention capacity, as observed inetlands.

Regarding the biological response of soil to peat and NP addi-ions, the results suggest that stimulation of soil microbial activityrepresented by microbial biomass and soil respiration) by peatmendment could be inhibited by NP (Fig. 2), which tended toe more retained in the soil with peat (Table 2). In this case, NPioavailability was not limited by organic matter as reported byoares et al. (2008), when microbial biomass and soil respirationas tested at approximately 50% of its water holding capacity. In

ontrast, in the absence of peat, soil respiration tended to be stimu-ated by NP in similar soil water contents (Fig. 2b). Biodegradation ofP is probably easier without peat, because adsorption processes ofP in soil particles are mainly controlled by organic matter (Soarest al., 2008).

In general, the mineralization of soil organic matter (CM) andhe microbial biomass – soil organic carbon ratio (Cmic:Corg) wereigher in the soil without peat, in comparison with soil amendedith peat. When the relationship between CM or Cmic:Corg and

oil water content was analyzed (Fig. 5a and b), it was possibleo observe that higher water content in peat treatment tendedo reduce CM and Cmic:Corg. Probably, a higher limitation of oxy-en diffusion, due to higher water content in soil amended witheat, could reduce microbial activity, decreasing CO2 emissions and

imiting the growth of microbial biomass and the mineralization

f soil organic matter. Since aerobic soil microbial activity takeslace within a specific water content range, where the balanceetween the rates of substrate and gas diffusion is optimal or ade-uate (Schjønning et al., 2011), the soil water content at −0.75 MPa,

C:TOC ratio and (c) qCO2 or metabolic quotient vs. gravimetric water content atpeat without NP, P1: soil + peat + 25 mg of NP kg−1 of soil, P2: soil + peat + 50 mg ofg of NP kg−1 of soil.

could be considered an appropriate value for evaluating soil micro-bial activity, as observed by Paradelo and Barral (2009). However,it should be pointed out that the soil water content of BR and MBCexperiments were performed at 50% of field capacity and it does notcorrespond exactly to the soil water content at −0.75 MPa, althoughboth are directly related by characteristic water retention curve.

At least, high values of metabolic quotient were observed whena dose of 50 mg kg−1 was added to soil without peat, and on aminor scale, when a dose of 25 mg kg−1 was added to soil with peat(Fig. 3c). Due to high metabolic quotients being indicative of stressin the microbial pool (Anderson and Domsch, 1990, 2010) causedby soil pollution (Fließbach et al., 1994), it is possible to considerthat only a high dose of NP (50 mg kg−1 of NP) was toxic to soilmicroorganisms.

5. Conclusions

The short term effects of 4-nonylphenol (NP) and peat addi-tions to soil water retention and microbial activity were studied.It was observed that soil water retention was increased mainlyby peat amendments rather than by NP additions, while soil des-orptivity (the capacity of soil to retain water during the dryingprocess) was not modified by peat or NP additions. Peat was ableto increase NP retention in the soil, although NP bioavailabilitywas not limited by NP sorption onto peat. Moreover, an increase in

soil water retention, mediated by peat amendment rather than NPdoses applied, was probably the key factor able to regulate the min-eralization of organic matter and the proportion between microbialbiomass and soil organic carbon. Since NP applied to soil contained
Page 7: Effects of nonylphenols on soil microbial activity and water retention

Soil Ec

5coao

A

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R

A

A

A

A

A

B

C

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E

E

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G. Ojeda et al. / Applied

% impurities, further research is needed to assess its role on soil NPoncentration. Finally, due to sewage sludge is not the only sourcef NP in soils, the present study is useful to illustrate how NP isble to modify soil microbial activity, in the presence or absence ofrganic amendments.

cknowledgements

This research was carried out as part of the TOXIFENOL projectcontract CTM2006-14163-CO2-01/TECNO of the Spanish Ministryf Environment). We wish to thank the Fundacão para a Ciência eTecnologia – FCT (Portugal) for Gerardo Ojeda’s postdoctoral fel-

owship. It was also subsidized by the European Social Fund andortuguese National Funds (POPH & QREN Human Potential Oper-tional Program), through a contract of J. Patrício (Ciência 2008).

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