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Soil Biology & Biochemistry 41 (2009) 69–76

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Soil Biology & Biochemistry

journal homepage: www.elsevier .com/locate/soi lb io

Response of soil enzymes to Linear Alkylbenzene Sulfonate (LAS)addition in soil microcosms

Marıa del Mar Sanchez-Peinado a, Belen Rodelas a,b, Marıa Victoria Martınez-Toledo a,b,Jesus Gonzalez-Lopez a,b,*, Clementina Pozo a,b

a Group of Environmental Microbiology, Institute of Water Research, University of Granada, Granada, Spainb Group of Environmental Microbiology, Department of Microbiology, University of Granada, Granada, Spain

a r t i c l e i n f o

Article history:Received 19 May 2008Received in revised form17 September 2008Accepted 23 September 2008Available online 23 October 2008

Keywords:LASEnzymatic activitiesSoil microcosmHeterotrophic bacteriaP-PO4

3�

* Corresponding author. Institute of Water ReseaUniversity of Granada, 18071 Granada, Spain. Tel.:958246235.

E-mail addresses: jgl@ugr.es (J. Gonzalez-Lopez), c

0038-0717/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.soilbio.2008.09.019

a b s t r a c t

Soil enzymatic activities (phosphatases, arylsulphatase and dehydrogenase) were measured in micro-cosm systems designed for the study of the impact of a commercial mixture of Linear AlkylbenzeneSulphonate (LAS) homologues on a xerofluvent agricultural soil. The soil microcosms consisted of glasscolumns filled with 800 g of dry soil which were fed with sterile commercial LAS solutions at concen-trations of 10 or 50 mg l�1 for periods of time up to 21 days. A soil microcosm fed with sterile distilledwater was included in this study and considered as control. Our results showed that the continuousapplication of the anionic surfactant to soil increased the values of the enzymes acid and alkalinephosphatases and arylsulphatase. On the contrary, the dehydrogenase activity was decreased by thecontinuous application of 10 or 50 mg l�1 LAS when compared with control microcosms. In addition,a statistically negative correlation was found between this enzymatic activity in the upper portion of thesoil columns amended with LAS and the viable counts of heterotrophic aerobic microorganisms.Moreover, in order to test the influence of LAS on nutrient availability and, consequently, on bacteriapopulations and soil biological activities, phosphate concentration was regularly determined in themicrocosm leachates. The phosphate concentration tested in the leachate of the microcosm continuouslyamended with 50 mg l�1 LAS solution was significantly lower than the concentrations detected in theleachate of the microcosms continuously amended with 10 mg l�1 LAS throughout the experiment.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Linear Alkylbenzene Sulfonate (LAS) is the most used anionicsurfactant in detergents and cleaning agents. World-wide LASconsumption during 2005 was estimated as 2.5 million tons, and itis expected to reach values around 3.4 million tons in 2010 (Pen-teado et al., 2006). In Europe, the total production of LAS in the year2004 was 487 ktons, corresponding to more than 80% to householddetergents (CESIO, 2006). The consumption rate per habitant andper day is relatively high depending on the country. In this sense,Jensen et al. (2007) have recently reported that the consumptionrate in Europe is in the range of 1.4–4 g person day�1.

The aerobic treatment of sewage in wastewater plants is suffi-cient to degrade more than 95% of the LAS input (Prats et al., 2006),but high LAS concentrations have been found in sewage sludgeafter anaerobic treatment. The use of raw wastewater as irrigation

rch, C/Ramon y Cajal No. 4,þ34 958244170; fax: þ34

lpozo@ugr.es (C. Pozo).

All rights reserved.

water and the application of sewage sludge as soil fertilizer or soilconditioner are the most important introduction ways of LAS intonatural ecosystems.

LAS concentration in natural soils is typically low, rangingbetween 0.7 mg kg�1 and 1.4 mg kg�1 (Mortensen et al., 2001;Carlsen et al., 2002). Nevertheless, Solbe et al. (2000) reviewed LASconcentrations in sludge-amended agricultural soils concludingthat they were around 20 mg kg�1, depending on the sludgeapplication rate and the sampling time after the application.Moreover, Jensen et al. (2001) have reported that LAS concentra-tions ranging between 10 and 50 mg kg�1 can be found inagricultural soils after the addition of sludge as a fertilizer.

Several studies have focused on the effect of LAS derived fromsludge application on plants and soil fauna (Jensen et al., 2001,2007), but few studies exist about the effects of the addition of LAS(as aqueous solution) on soil microorganisms and their microbialactivities (Sanchez-Peinado et al., 2008).

Soil enzymes have been suggested as potential indicators of soilquality because of their essential role in different soil aspects suchas nutrient mineralization and cycling, decomposition andsynthesis of organic matter and degradation of xenobiotics(Bandick and Dick, 1999). Microorganisms are the main source of

Leachate

Peristaltic pump

Polyethylene tube

Porous plate

Filtered air

Polyethylene tube

Sterile solutionof LAS

Lowerzone

Upperzone

6 cm

35 cm

Fig. 1. Diagram of the agricultural soil microcosm systems used in this study.

M. Sanchez-Peinado et al. / Soil Biology & Biochemistry 41 (2009) 69–7670

enzymes (extracellular and intracellular enzymes) in soils (Taba-tabai, 1982), so their study is essential to understand the biologicalprocesses that occur in soils as well as the impact of severalsubstances on their fertility.

The main goal of this research was to investigate the effects ofLAS exposure (as aqueous solution) on soil enzymes such as acidand alkaline phosphatases, arylsulphatase and dehydrogenase byusing soil microcosms artificially polluted with different concen-trations of detergent in order to understand the effects of thisanionic surfactant on soil microbial activity.

2. Material and methods

2.1. Soil

The soil used for the column experiments (microcosm system)was collected from an agricultural soil near Granada city (SouthernSpain) which had no previous exposure to LAS contamination. Afterthe removal of plant residues, samples were collected at a depth of0–20 cm using a soil core sampler. Immediately after collection thesoil was air dried and sifted with a sieve (diameter 2 mm) toremove gravel and plant residues.

The soil used was a Typic Xerofluvent with silt loam texture,containing 14% clay, 20% sand and 65% silt. The chemical compo-sition of the samples was as follows: organic matter, 13.9 g kg�1; pH(water) 7.8; N total, 1.4 g kg�1; phosphorous, 25 mg kg�1; andpotassium, 240 mg kg�1. Soil texture was analyzed followingNatural Resources Conservation Service (1999), while N total,phosphorus and potassium were determined by the techniquesdescribed by Bremmer (1965), Olsen and Dean (1965) and Pratt(1954), respectively.

2.2. Microcosm experiments

Soil microcosms were prepared by filling glass columns(6.0 cm diameter � 35 cm length) containing 800 g of dry soil. Toavoid the soil loss a porous plate was located at the bottom of theglass cylinder and the leaching water was collected usinga polyethylene tube connected to a sterile glass bottle. Sterilesolutions of commercial LAS at concentrations of 10 or 50 mg l�1

were continuously added through the experimental soil columnsat 8.0 ml h�1 using a peristaltic pump (Watson Marlow 505S,UK). These LAS concentrations are typically found in agriculturalsoils after the addition of sludge as a fertilizer (Jensen et al.,2001). All the soil columns and tubes used in the experimentwere made of glass and polyethylene, respectively, in order toavoid the potential LAS adsorption (Fig. 1). The commercial LASmixture used in the experiments contains 69% of water and 31%of active matter, with the following distribution of the linear alkylchain homologues: 5-phenyl C10, 0.8%; phenyl C10, 9.8%; phenylC11, 33.9%; phenyl C12, 32.5%; phenyl C13, 22.6%; phenyl C14,0.3%. The commercial product also contained tetra-indol (0.10%)and paraffin (0.10%).

As a first step, a sterile microcosm system consisting of twosterile soil columns amended with sterile LAS solution at twoconcentrations (10 and 50 mg l�1) and named as SLAS10 andSLAS50, respectively, was included in the study in order toestablish the running time of each experiment by testing theappearance of the first LAS homologue (C10) in the leachate. Thesoil columns were sterilised by three successive autoclavingprocesses at 120 �C during 60 min and the sterility of the systemswas tested by inoculation of soil samples (1 g) into nutrient brothmedium. To avoid contamination, the soil columns were up-closed with sterile rubber stoppers. For the experiments withsterile soil columns amended with sterile solutions containing10 mg l�1 of LAS, the running time of the study was established

at 21 days, while for experiments with 50 mg l�1 of LAS this timewas only of 7 days, although these experiments were maintainedover 21 days for comparison. The determination of LAS homo-logues in the leachate was made according to the methodologyproposed by Nimer et al. (2007) as described in Section 2.3.3.After determination of running time the sterile microcosms wereeliminated.

Once the experiment running time was established, two newtypes of soil microcosms were built. The first type corresponded tonon-sterile soil columns amended with sterilised distilled water(named as W). The second type corresponded to non-sterile soilcolumns amended with LAS solution at two different concentra-tions: 10 and 50 mg l�1 of LAS (LAS10 and LAS50, respectively). Thedistilled water used was sterilised by autoclaving and the LASsolution was sterilised by filtration (0.22 mm, Millipore�).

For LAS10 microcosms the glass columns were broken downunder sterile conditions after 7, 14 and 21 days, while for experi-ments using 50 mg l�1 LAS, the soil columns were broken downafter 3, 7 and 21 days of running. Once the columns were opened,the glass was removed and two soil portions were taken: upper andlower (see Fig. 1). These samples were homogenised and enclosedinto sterile glass containers and remained at 4 �C until analysis. Allthe microcosms W, LAS10 and LAS50 were replicated thrice for eachsampling time.

2.3. Samples’ analysis

2.3.1. Counts of cultivable heterotrophic bacteria in samplesfrom soil columns

The number of cultivable aerobic heterotrophic bacteria in bothsoil portions (upper and lower) from the soil microcosm types(W, LAS10 and LAS50) at each incubation time was determined bythe plate count method. For this, 1 g of soil from each homogenisedsample (from upper and lower portions) was diluted in 9 ml of

Table 1LAS concentration (mg kg�1 soil) in each soil microcosm and soil portion, at differentsampling times.

Soil microcosmtype

Running time(days)

Upper portion(mg kg�1 soil)

Lower portion(mg kg�1 soil)

LAS10 7 1.4 0.314 9.2 3.121 0.7 0.6

M. Sanchez-Peinado et al. / Soil Biology & Biochemistry 41 (2009) 69–76 71

sterile saline solution (0.9% NaCl, w/v) and mixed thoroughly ona magnetic stirrer. Standard serial dilutions followed and 100 mlaliquots of each dilution were spread on plates of 1/10 dilutedtripticase soy agar (TSA) medium (Oxoid, Basingstoke, Hampshire,England) (Avidano et al., 2005). Plates were incubated at 28 �C inthe dark for 3 days under aerobic conditions and then checkedvisually. All the counts were made in triplicate.

2.3.2. Enzymatic activitiesThe determination of enzymatic activities in the homogenised

soil samples taken from both upper and lower portions of eachmicrocosm type (W, LAS10 and LAS50) was done in all casesaccording to the methods described by Tabatabai (1982).

For acid phosphatase, 1 g of sieved soil (less than 2 mm) wascombined with 2.0 ml of toluene, 4.0 ml pH 6.5 modifieduniversal buffer and 1.0 ml of p-nitrophenol phosphate solution(0.025 M) for 1 h at 37 �C. The p-nitrophenol released wasdetermined colorimetrically at 410 nm using a Hitachi U-2000(Tokyo, Japan) spectrophotometer, and compared with a stan-dard. Alkaline phosphatase was analyzed in a similar manner,except that the modified universal buffer was adjusted to pH 11.0using 0.1 N NaOH.

For the analysis of arylsulphatase, 1 g of sieved soil (less than2 mm), 0.25 ml toluene, 4.0 ml acetate buffer (0.5 M, pH 5.8) and1.0 ml of p-nitrophenyl sulfate (0.025 M in acetate buffer) weremixed and incubated at 37 �C. After 1 h, the reaction was stoppedby the addition of 1 ml of 0.5 M CaCl2 and 4.0 ml of 0.5 M NaOH andfiltered through a Whatman no. 42 filter paper. The p-nitrophenolreleased was measured colorimetrically at 410 nm.

For the dehydrogenase activity, 20 g of dried sieved soil wascombined with 0.2 g CaCO3. Three 6 g portions of mixture wereincubated at 37 �C with 1.0 ml 3% (w/v) 2,3,5-triphenyltetrazoliumchloride and 2.5 ml distilled water. After 24 h, the soils wereremoved from incubation, mixed with 10 ml of reagent grademethanol and quantitatively filtered through absorbent cotton. Thered color resulting from the production of triphenylformazan bysoil dehydrogenase was washed from the cotton into a 100 mlvolumetric flask using 10 ml portions of methanol, and the flaskwas brought to volume with additional methanol. The triphe-nylformazan was determined colorimetrically at 485 nm andcompared with a standard curve.

2.3.3. Chemical determinationsLAS content in samples from soil columns and leachates was

analyzed following the high-performance liquid chromatography(HPLC) technique proposed by Del Olmo et al. (2004) and Nimeret al. (2007) and using an Agilent Technologies (Palo Alto, CA, USA)1100 series HPLC equipment.

Phosphorous (as P-PO43�) concentration was determined in the

leachate from soil microcosms W, LAS10 and LAS50 throughoutthe experiments following the spectrophotometric proceduredescribed in Rodier (1989) and using a Hitachi U-2000 (Tokyo,Japan) spectrophotometer.

SLAS10 7 13.3 0.514 22.1 0.821 30.0 1.2

LAS50 3 35.1 0.57 70.8 0.4

21 20.4 9.6

SLAS50 3 53.1 0.67 83.8 12.2

21 284.5 84.7

LAS10: soil microcosm amended with 10 mg l�1 LAS solution. LAS50: soil microcosmamended with 50 mg l�1 LAS solution. SLAS10: sterile soil microcosm amended with10 mg l�1 LAS solution. SLAS50: sterile soil microcosm amended with 50 mg l�1 LASsolution.

2.4. Statistical analysis

One-way analysis of variance (ANOVA) was performed using thesoftware package Statgraphics 3.0 Plus version (STSC Inc., Rockville,MD, USA) in order to identify the effect of different LAS concen-trations on bacterial counts and soil enzyme activities. A signifi-cance level of 95% (p < 0.05) was selected.

In addition, to evaluate the correlation among cultivableheterotrophic bacteria and enzymatic activities, a matrix of Pear-son’s linear correlation coefficients was made using the samestatistical package.

3. Results

3.1. LAS in soil samples

Once removed from the glass columns, soil samples from eachsoil microcosms (two samples by column, corresponding to theupper and lower portions) were preserved by the immediateaddition of 3% (v/v) formaldehyde until their analysis, following themethodology previously described in Nimer et al. (2007). Table 1shows the concentration of LAS (mg kg�1 soil) in each soil columnportion at each sampling time. The surfactant concentrations weresignificantly higher (p < 0.05) in the sterile soil columns (SLAS10and SLAS50) when compared with the non-sterile ones (LAS10 andLAS50). Moreover, in all cases, the highest LAS concentrations werealways found in the upper soil column layer.

3.2. Cultivable heterotrophic bacteria in samples from soil columns

Fig. 2 shows the cultivable aerobic heterotrophic bacterialcounts (as log CFU g�1) in soil samples from upper and lowerportions of LAS10 (Fig. 2A) and LAS50 (Fig. 2B) microcosms.Bacterial counts from W microcosms (taking into account as controlsoil microcosms) were also included.

Bacterial counts were significantly higher (p < 0.05) in themicrocosms amended with LAS (LAS10 and LAS50) than in the Wmicrocosms (Fig. 2), and the counts from LAS50 were significantlyhigher (p < 0.05) than those from LAS10 in the upper portion of thesoil columns.

Maximum bacterial counts in the LAS50 microcosms wererecorded during the third experiment day (Fig. 2B), earlier than inthe case of the LAS10 microcosms, where maximum bacterialcounts were recorded during the 14th experiment day (Fig. 2A).

Finally, bacterial counts were significantly higher (p < 0.05) inthe upper portion than in the lower portion in all the studiedmicrocosms.

3.3. Enzymatic activities in samples from soil columns

Figs. 3–6 (A and B) show the evolution of soil enzyme activities(alkaline and acid phosphatases, arylsulphatase and dehydroge-nase) in soil microcosms amended with sterile solutions containing10 mg l�1 (A) and 50 mg l�1 (B) of LAS both in upper and lowerportions of soil columns. The values of the soil enzymatic activitiesin soil microcosms amended with sterile distilled water (W) areincluded as controls.

5.5

6

6.5

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0 7 14 21Days

Lo

g C

FU

/g

d

ry so

il

LSD (0.05)

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g C

FU

/g

d

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il

LSD (0.05)

5.5

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g C

FU

/g

d

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il

LSD (0.05)

5.5

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0 3 6 9 12 15 18 21

0 7 14 21 0 3 6 9 12 15 18 21

DaysL

og

C

FU

/g

d

ry so

il

LSD (0.05)

A B

Upper portionUpper portion

Lower portion Lower portion

*

**

*

*

LAS10 W LAS50 W

*

Fig. 2. Cultivable aerobic heterotrophic bacteria counts (as log CFU g�1) in soil samples from upper and lower portions of LAS10 microcosms (A) and LAS50 microcosms (B). Bacterialcounts from W microcosms (control) were also included. Values are an average of three experiments. Bars: LSD between means (Student’s t-test, p � 0.05). Means marked with * aresignificantly different from control (p � 0.05).

0

50

100

150

200

250

0 3 7 21 0 3 7 21Days

0 7 14 21 0 7 14 21Days

B

A

0

50

100

150

200

250

g P

NP

-released

/g

so

il/h

µg

P

NP

-released

/g

so

il/h

upper portion lower portion

W LAS10

W LAS50

Fig. 3. Alkaline phosphatase activity (mg PNP released g�1 wet soil h�1 of incubation)of soil samples from upper and lower portions of LAS10 (A) and LAS50 (B) microcosms.Data from W microcosms (control) were included too. Data are average values andstandard deviations (SD) of three replicates.

M. Sanchez-Peinado et al. / Soil Biology & Biochemistry 41 (2009) 69–7672

As a general rule, the application of the anionic surfactant to soilat the different concentrations assayed significantly increased themean values of extracellular enzymes (acid and alkaline phospha-tases and arylsulphatase) both in the upper and lower portions of thesoil columns with respect to the control microcosms, exceptionmade for the acid phosphatase (lower portion) and the alkalinephosphatase (upper and lower portions) of LAS10 microcosm, wherea positive but not significant trend was detected. By contrast, thedehydrogenase activity was always significantly decreased by thepresence of LAS, when it was compared with control soil samples(W). Table 2 summarizes the results of an analysis of variance foreach enzyme activity measured as affected by the different LAStreatment applied (LAS10 and LAS50). Significance level wasgenerally higher for the LAS50 than for the LAS10 treatment.

To evaluate the relationships between enzymatic soil activitiesand heterotrophic soil bacterial counts in each microcosm soilportion, a correlation matrix (Pearson’s linear correlation coeffi-cients) was constructed (Table 3). A positive and statisticallysignificant linear correlation (p < 0.05) existed between bothphosphatase and arylsulphatase activities and aerobic heterotro-phic soil bacterial counts in microcosms amended with sterilesolutions containing 10 mg l�1 (LAS10) and 50 mg l�1 (LAS50) ofLAS. These correlations showed always greater coefficient values inthe upper portion of the microcosm than in the lower one. On theother hand, the correlation coefficients between the dehydroge-nase activity and aerobic heterotrophic soil bacteria counts werealways negative and higher in the upper portion of the microcosmsthan in the lower one. Finally, it is worth noting that in themicrocosms without LAS (W) the counts of heterotrophic aerobicbacteria (as log CFU g�1) were only positively and statisticallycorrelated to the alkaline phosphatase activity.

3.4. Phosphorous leachate analysis

In order to test the influence of LAS on nutrient availability and,as a consequence, on bacterial population and enzyme activities,

0102030405060708090

100

0 3 7 21 0 3 7 21

B

0102030405060708090

100

0 7 14 21 0 7 14 21Days

Days

g P

NP

-released

/g

so

il/h

g P

NP

-relea

sed

/g

so

il/h

upper portion lower portion

A

W LAS10

WLAS50

Fig. 4. Acid phosphatase activity (mg PNP released g�1 wet soil h�1 of incubation) ofsoil samples from upper and lower portions of LAS10 (A) and LAS50 (B) microcosms.Data from W microcosms (control) were included too. Data are average values andstandard deviations (SD) of three replicates.

0

10

20

30

40

50

60

70

0 3 7 21 0 3 7 21

B

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g P

NP

released

/g

so

il/h

g P

NP

released

/g

so

il/h

upper portion lower portion

A

W LAS10

W LAS50

Fig. 5. Arylsulphatase activity (mg PNP released g�1 wet soil h�1 of incubation) of soilsamples from upper and lower portions of LAS10 (A) and LAS50 (B) microcosms. Datafrom W microcosms (control) were included too. Data are average value and standarddeviations (SD) of three replicates.

M. Sanchez-Peinado et al. / Soil Biology & Biochemistry 41 (2009) 69–76 73

phosphorous (as P-PO43�) concentration was regularly determined

in the microcosms leachates. Fig. 7 shows the phosphate concen-trations detected in the leachate of the soil microcosms LAS10,LAS50 and W during the running experiment time. The dynamics ofthese values in the soil column amended with sterile water (W)showed the carrying away of the phosphates across the soil column.The phosphates’ concentration detected in the leachate of soilmicrocosm LAS10 was significantly higher than in LAS50 and W,and the minimum phosphates concentration was recorded in theLAS50 leachate throughout the whole experiment time.

4. Discussion

Among the parameters revealing information on the quality ofa soil, those relative to its biological composition and metabolicactivity display a greater sensitivity to contamination processes(Garcıa et al., 2003). Since the physical and chemical characteristicsof a soil can be considered stable, any impact in a soil is detectedinitially by significant variations in its biological dynamics. The soilconstitutes a biological system in which enzymatic activities playan important role in its biochemical status (Overbeck, 1991; Stryler,1995). The measurement of the soil enzyme activities allowsinferring the quality of the system, and is commonly used as anindicator of soil microbial activity (Sinsabaugh, 1994; Garcıa et al.,2003). In addition, it acts as a good indicator of changes in theproperties of the soil induced by the anthropogenic addition ofcompounds (Lobo et al., 2000).

The lower concentrations of LAS detected in the non-sterile soilcolumns when compared with the sterile ones demonstrated thebiotransformation process of the surfactant. This process reachedits highest intensity after 14 days of experimentation in the case of

soil columns continuously amended with 10 mg l�1 of surfactant,and after 7 days when the soil columns were amended with50 mg l�1 of LAS.

On the other hand, the greatest proportion of the surfactant wasretained in the upper portion of the columns (whether sterile ornon-sterile). Previous studies (Jacobsen et al., 2004) revealed thesame distribution pattern of the surfactant in experimental soilcolumns, with maximum concentrations registered in the first15 cm, and showing a significant transport towards the lowerlayers. In our experiments, the lower concentrations of LAS detec-ted in the deep layers of the microcosm may be due to the fasterbiotransformation process of the surfactant and to the higherretention of the surfactant in the upper layers of the soil column.The first LAS degradation homologue detected in the leachate wasC10 (data not shown) because the longer chain homologues aremore easily biodegradable when they are in the interstitial water,and once adsorbed to soil particles they are retained with greaterforce than the short chain homologues. Similar results wereobtained by Jacobsen et al. (2004).

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g T

PF

released

/g

so

il/d

ay

g T

PF

release

d/g

so

il/d

ay

upper portion lower portion

A

W LAS10

W LAS50

Fig. 6. Dehydrogenase activity (mg TPF released g�1 dry weight day�1 of incubation) ofsoil samples from upper and lower portions of LAS10 (A) and LAS50 (B) microcosms.Data from W microcosms (control) were included too. Data are average values andstandard deviations (SD) of three replicates.

Table 3Pearson’s correlation coefficients between heterotrophic aerobic bacteria countsand enzymatic activities in each soil microcosm and portion sampled.

Acidphosphatase

Alkalinephosphatase

Arylsulphatase Dehydrogenase

LAS10Upper portion 0.82* 0.94* 0.89* �0.84*Lower portion 0.27 0.61* 0.63* �0.73*

LAS50Upper portion 0.58* 0.97* 0.84* �0.59*Lower portion 0.58* 0.75* 0.75* �0.27

WUpper portion 0.05 0.83* 0.54 �0.18Lower portion �0.06 0.65* 0.47 �0.31

*Significant at p � 0.05. LAS10: soil microcosm amended with 10 mg l�1 LAS solu-tion. LAS50: soil microcosm amended with 50 mg l�1 LAS solution. W: soil micro-cosms amended with sterilised distilled water (control).

M. Sanchez-Peinado et al. / Soil Biology & Biochemistry 41 (2009) 69–7674

The data obtained from agricultural soil microcosms haverevealed that the enzymatic activities acid phosphatase, alkalinephosphatase and arylsulphatase were correlated positively withthe bacterial counts under our experimental conditions. Theseresults are in accordance with those previously reported by Nan-nipieri et al. (1979), Criquet et al. (2002) and Neble et al. (2007). Thevalues of phosphatase and arylsulphatase activities obtained in soilmicrocosms LAS10 were always higher than those obtained incontrol microcosms (W), although these differences were notstatistically significant for the cases of acid phosphatase activity(lower portion) and alkaline phosphatase activity (upper and lowerportions). On the contrary, the application of greater concentrationsof LAS in the influent (50 mg l�1) generated significantly highervalues of phosphatase and arylsulphatase activities, with temporaldistribution patterns characterized by earlier and more intensemaximums of activity.

While previous studies (Elsgaard et al., 2001a) suggested theabsence of response of the arylsulphatase activity to LAS addition,our results have shown that the value of this enzymatic activity isincreased by the presence of 10 or 50 mg l�1 of this anionicsurfactant. We suggest that a limitation of the bacterial growth bybio-available forms of sulphur in the continuously washed

Table 2Summary of the results of an analysis of variance (ANOVA) for each of the enzyme activit

Acid phosphatase Alkaline phosphatase

Upper portion Lower portion Upper portion Lower portion

LAS10 p < 0.01 NS NS NSLAS50 p < 0.05 p < 0.001 p < 0.05 p < 0.001

NS: not significant (p > 0.05).

agricultural soil microcosms assayed in our study could be thereason for this opposed behaviour. Thus, arylsulphatase activitycould be involved in the breakage of the LAS benzene–S bond and inthe release of S molecules to the medium, allowing the microor-ganisms not only to cover their requirements of a bio-available Sform (Dodgson et al., 1982) but also to collaborate significantly inthe biotransformation of the LAS molecule (Kertesz et al., 1994). Infact, several strains of Pseudomonas spp. (potentially capable toproduce extracellular sulphatases, Gonzalez et al., 2003) have beenpreviously described as important members of LAS biotransfor-mation consortia (Swisher, 1987; Jimenez et al., 1991). Indeed,several recent studies (Vong et al., 2002; Gonzalez et al., 2003) havebeen carried out to investigate the possibility to use bacteria toincrease the mobilisation of the different forms of S-organic in soilsand to ‘‘in situ’’ accelerate the S biogeochemical cycle, in order toimprove the fertility of agricultural soils.

Under our experimental conditions (oligotrophic systems), thecontinuous leaching of nutrients from soil (either dissolved inthe water and/or emulsified by the anionic surfactant), as well asthe depletion of the nutritional resources in the soil as a conse-quence of microbial growth, could have affected the biologicalactivities of the soil (Barber, 1995; Hartemink, 2005). In fact, ourresults show that the phosphate concentration detected in theleachate of the LAS50 microcosm was significantly lower thanthe concentrations detected in LAS10 leachates throughout theexperiment. The counts of cultivable aerobic heterotrophic bacteriain soil samples from the LAS50 microcosms were significantlyhigher than those from LAS10, and consequently it could be sug-gested that the phosphate consumption was higher too. Thus,under these circumstances, the bacterial populations in this soilmicrocosm type may respond by increasing the extracellularphosphatase activity (mainly acid phosphatase) to obtain readilyassimilable inorganic P (as ion phosphate) from organic P forms(Speir and Ross, 1978; Dick, 1997; Criquet et al., 2004).

The effect of the soil column washing on the phosphateconcentration throughout the period of study was shown by thepositive and statistically significant correlations found between thenumber of cultivable heterotrophic bacteria and the phosphataseactivity (mainly alkaline phosphatase) in the W microcosm. No

ies measured, as affected by the different LAS treatment applied (LAS10 and LAS50).

Arylsulphatase Dehydrogenase

Upper portion Lower portion Upper portion Lower portion

p < 0.001 p < 0.01 p < 0.05 p < 0.01p < 0.001 p < 0.01 p < 0.05 p < 0.001

0

200

400

600

800

1000

1200

3 6 9 12 15Days

P-P

O4 (µg

/l)

LAS50LAS10 W

Fig. 7. Phosphorous concentrations (as P-PO4) in leachate samples from LAS10 andLAS50 microcosms during the running time. Data from W microcosms (control) wereincluded too. Data are average values and standard deviations (SD) of three replicates.

M. Sanchez-Peinado et al. / Soil Biology & Biochemistry 41 (2009) 69–76 75

other enzymatic activities were positively and significantly relatedto the numbers of cultivable heterotrophic bacteria in this micro-cosm. Nevertheless, it is worthy to note that these counts onlyrepresent a small part of total soil bacteria.

Arylsulphatase and phosphatase activities are considered‘‘specific parameters’’ since the enzymes catalyse concrete reac-tions and are mediated by certain bacterial groups which dependon specific substrates. On the contrary, the dehydrogenase activityis considered ‘‘a general’’ determination, as its measurement allowsto evaluate the microbial metabolic processes that take place in thesoil under a global view (Tabatabai, 1982; Garcıa et al., 2003). Ourresults demonstrate that the dehydrogenase activity was progres-sively inhibited in the LAS10 and LAS50 soil microcosms, and thata statistically negative correlation existed between this enzymaticactivity in the upper portion of the soil columns and the countsof aerobic heterotrophic microorganisms, while no significantcorrelations were detected between these parameters in controlsoil microcosms (W). Previous studies (Malkomes and Wohler,1983; Elsgaard et al., 2001b) demonstrated a similar inhibitionpattern of the dehydrogenase activity in the presence of increasingconcentrations of LAS. These results suggest a certain negativeeffect of LAS on soil microbial processes although the presence ofthis xenobiotic can favour other more specialised enzymaticprocesses, mediated by specific groups of microorganisms (VanStraalen and Van Gestel, 1993).

Margesin and Schiner (1998) reported that the measurement ofthe dehydrogenase activity could be considered as a particularlyappropriate method for the monitoring of soil contamination byanionic surfactants. In addition, this study demonstrated that thepresence of another anionic surfactant (SDS) significantly inhibitedthe dehydrogenase activity of an agricultural soil, and this enzy-matic activity only increased when 90% of degradation of surfactantwas achieved. This could be explained by the fact that SDS may altermicroorganism membrane structures within which dehydroge-nases are located. Similarly, Schwuger and Bartnik (1999) describeda number of diverse effects of LAS on cellular membrane structuresas a consequence of its surface activity.

In recent years, considerable interest has developed in the use ofmicrocosm techniques for ecotoxicological assays (Edwards et al.,1997, 1998; Burrows and Edwards, 2000). Such microcosms haveusually consisted of units containing intact or mixed soil supportingindigenous multiple biotic species, and have ranged in size froma few grams of soil to cores as large as a meter in diameter.Although field observations are needed for realistic calibration andvalidation of the information, our integrated in vitro microcosm

approach constitutes a useful tool for preliminary studies on theeffects of LAS on soils’ biological properties.

5. Conclusions

Soil enzymatic activities, in particular dehydrogenase activity,have been suggested as appropriate parameters for the monitoringof soil contamination. Using agricultural soil microcosm systems,we conclude that the continuous applications of different LASsolutions at 10 and 50 mg l�1 concentrations for periods of time upto 21 days significantly decrease the dehydrogenase activity, whileother enzymatic activities catalysed by specific bacterial groups(alkaline phosphatase, acid phosphatase and arylsulphatase) weresignificantly increased. Therefore, a statistically negative correla-tion between dehydrogenase activity and the number of hetero-trophic aerobic microorganisms was observed, suggesting aninhibition of the soil dehydrogenase activity in response to theaddition of increasing concentrations of LAS in agricultural soils.LASs also induce the alteration of the phosphorous turn-over byincreasing its bioavailability. Further studies are needed in order toevaluate the effects of this anionic surfactant on the soil quality,particularly in aspects such as nutrient mineralization and cycling.

Acknowledgments

This research was funded by the Spanish Ministerio de Educa-cion y Ciencia (MEC), as part of Project Reference PPQ2003-07978-V02-02 (Programa Nacional de IþD). B.R. was supported byPrograma Ramon y Cajal (MEC, Spain). C. Pozo was supported byPrograma Retorno de Doctores (Junta de Andalucıa. Spain).

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