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Response of soil enzymes to Linear Alkylbenzene Sulfonate (LAS) addition in soil microcosms
Soil Biology & Biochemistry 41 (2009) 69–76
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Response of soil enzymes to Linear Alkylbenzene Sulfonate (LAS) addition in soil microcosms Marı´a del Mar Sa´nchez-Peinado a, Bele´n Rodelas a, b, Marı´a Victoria Martı´nez-Toledo a, b, Jesu´s Gonza´lez-Lo´pez a, b, *, Clementina Pozo a, b a b
Group of Environmental Microbiology, Institute of Water Research, University of Granada, Granada, Spain Group of Environmental Microbiology, Department of Microbiology, University of Granada, Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 May 2008 Received in revised form 17 September 2008 Accepted 23 September 2008 Available online 23 October 2008
Soil enzymatic activities (phosphatases, arylsulphatase and dehydrogenase) were measured in microcosm systems designed for the study of the impact of a commercial mixture of Linear Alkylbenzene Sulphonate (LAS) homologues on a xerofluvent agricultural soil. The soil microcosms consisted of glass columns filled with 800 g of dry soil which were fed with sterile commercial LAS solutions at concentrations of 10 or 50 mg l1 for periods of time up to 21 days. A soil microcosm fed with sterile distilled water was included in this study and considered as control. Our results showed that the continuous application of the anionic surfactant to soil increased the values of the enzymes acid and alkaline phosphatases and arylsulphatase. On the contrary, the dehydrogenase activity was decreased by the continuous application of 10 or 50 mg l1 LAS when compared with control microcosms. In addition, a statistically negative correlation was found between this enzymatic activity in the upper portion of the soil 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 bacteria populations and soil biological activities, phosphate concentration was regularly determined in the microcosm leachates. The phosphate concentration tested in the leachate of the microcosm continuously amended with 50 mg l1 LAS solution was significantly lower than the concentrations detected in the leachate of the microcosms continuously amended with 10 mg l1 LAS throughout the experiment. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: LAS Enzymatic activities Soil microcosm Heterotrophic bacteria P-PO3 4
1. Introduction Linear Alkylbenzene Sulfonate (LAS) is the most used anionic surfactant in detergents and cleaning agents. World-wide LAS consumption during 2005 was estimated as 2.5 million tons, and it is expected to reach values around 3.4 million tons in 2010 (Penteado et al., 2006). In Europe, the total production of LAS in the year 2004 was 487 ktons, corresponding to more than 80% to household detergents (CESIO, 2006). The consumption rate per habitant and per day is relatively high depending on the country. In this sense, Jensen et al. (2007) have recently reported that the consumption rate in Europe is in the range of 1.4–4 g person day1. The aerobic treatment of sewage in wastewater plants is sufficient to degrade more than 95% of the LAS input (Prats et al., 2006), but high LAS concentrations have been found in sewage sludge after anaerobic treatment. The use of raw wastewater as irrigation
* Corresponding author. Institute of Water Research, C/Ramo´n y Cajal No. 4, University of Granada, 18071 Granada, Spain. Tel.: þ34 958244170; fax: þ34 958246235. E-mail addresses: [email protected] (J. Gonza´lez-Lo´pez), [email protected] (C. Pozo). 0038-0717/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2008.09.019
water and the application of sewage sludge as soil fertilizer or soil conditioner are the most important introduction ways of LAS into natural ecosystems. LAS concentration in natural soils is typically low, ranging between 0.7 mg kg1 and 1.4 mg kg1 (Mortensen et al., 2001; Carlsen et al., 2002). Nevertheless, Solbe` et al. (2000) reviewed LAS concentrations in sludge-amended agricultural soils concluding that they were around 20 mg kg1, depending on the sludge application rate and the sampling time after the application. Moreover, Jensen et al. (2001) have reported that LAS concentrations ranging between 10 and 50 mg kg1 can be found in agricultural soils after the addition of sludge as a fertilizer. Several studies have focused on the effect of LAS derived from sludge 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 microbial activities (Sa´nchez-Peinado et al., 2008). Soil enzymes have been suggested as potential indicators of soil quality because of their essential role in different soil aspects such as nutrient mineralization and cycling, decomposition and synthesis of organic matter and degradation of xenobiotics (Bandick and Dick, 1999). Microorganisms are the main source of
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enzymes (extracellular and intracellular enzymes) in soils (Tabatabai, 1982), so their study is essential to understand the biological processes that occur in soils as well as the impact of several substances on their fertility. The main goal of this research was to investigate the effects of LAS exposure (as aqueous solution) on soil enzymes such as acid and alkaline phosphatases, arylsulphatase and dehydrogenase by using soil microcosms artificially polluted with different concentrations of detergent in order to understand the effects of this anionic surfactant on soil microbial activity.
Filtered air
6 cm Upper zone
Polyethylene tube
35 cm
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 (Southern Spain) which had no previous exposure to LAS contamination. After the removal of plant residues, samples were collected at a depth of 0–20 cm using a soil core sampler. Immediately after collection the soil was air dried and sifted with a sieve (diameter 2 mm) to remove 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 composition of the samples was as follows: organic matter, 13.9 g kg1; pH (water) 7.8; N total, 1.4 g kg1; phosphorous, 25 mg kg1; and potassium, 240 mg kg1. Soil texture was analyzed following Natural Resources Conservation Service (1999), while N total, phosphorus and potassium were determined by the techniques described 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. To avoid the soil loss a porous plate was located at the bottom of the glass cylinder and the leaching water was collected using a polyethylene tube connected to a sterile glass bottle. Sterile solutions of commercial LAS at concentrations of 10 or 50 mg l1 were continuously added through the experimental soil columns at 8.0 ml h1 using a peristaltic pump (Watson Marlow 505S, UK). These LAS concentrations are typically found in agricultural soils after the addition of sludge as a fertilizer (Jensen et al., 2001). All the soil columns and tubes used in the experiment were made of glass and polyethylene, respectively, in order to avoid the potential LAS adsorption (Fig. 1). The commercial LAS mixture used in the experiments contains 69% of water and 31% of active matter, with the following distribution of the linear alkyl chain homologues: 5-phenyl C10, 0.8%; phenyl C10, 9.8%; phenyl C11, 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 two sterile soil columns amended with sterile LAS solution at two concentrations (10 and 50 mg l1) and named as SLAS10 and SLAS50, respectively, was included in the study in order to establish the running time of each experiment by testing the appearance of the first LAS homologue (C10) in the leachate. The soil columns were sterilised by three successive autoclaving processes at 120 C during 60 min and the sterility of the systems was tested by inoculation of soil samples (1 g) into nutrient broth medium. To avoid contamination, the soil columns were upclosed with sterile rubber stoppers. For the experiments with sterile soil columns amended with sterile solutions containing 10 mg l1 of LAS, the running time of the study was established
Lower zone
Peristaltic pump
Porous plate
Polyethylene tube Sterile solution of LAS Leachate
Fig. 1. Diagram of the agricultural soil microcosm systems used in this study.
at 21 days, while for experiments with 50 mg l1 of LAS this time was only of 7 days, although these experiments were maintained over 21 days for comparison. The determination of LAS homologues in the leachate was made according to the methodology proposed by Nimer et al. (2007) as described in Section 2.3.3. After determination of running time the sterile microcosms were eliminated. Once the experiment running time was established, two new types of soil microcosms were built. The first type corresponded to non-sterile soil columns amended with sterilised distilled water (named as W). The second type corresponded to non-sterile soil columns amended with LAS solution at two different concentrations: 10 and 50 mg l1 of LAS (LAS10 and LAS50, respectively). The distilled water used was sterilised by autoclaving and the LAS solution was sterilised by filtration (0.22 mm, MilliporeÒ). For LAS10 microcosms the glass columns were broken down under sterile conditions after 7, 14 and 21 days, while for experiments using 50 mg l1 LAS, the soil columns were broken down after 3, 7 and 21 days of running. Once the columns were opened, the glass was removed and two soil portions were taken: upper and lower (see Fig. 1). These samples were homogenised and enclosed into sterile glass containers and remained at 4 C until analysis. All the microcosms W, LAS10 and LAS50 were replicated thrice for each sampling time.
2.3. Samples’ analysis 2.3.1. Counts of cultivable heterotrophic bacteria in samples from soil columns The number of cultivable aerobic heterotrophic bacteria in both soil portions (upper and lower) from the soil microcosm types (W, LAS10 and LAS50) at each incubation time was determined by the plate count method. For this, 1 g of soil from each homogenised sample (from upper and lower portions) was diluted in 9 ml of
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sterile saline solution (0.9% NaCl, w/v) and mixed thoroughly on a magnetic stirrer. Standard serial dilutions followed and 100 ml aliquots of each dilution were spread on plates of 1/10 diluted tripticase soy agar (TSA) medium (Oxoid, Basingstoke, Hampshire, England) (Avidano et al., 2005). Plates were incubated at 28 C in the dark for 3 days under aerobic conditions and then checked visually. All the counts were made in triplicate. 2.3.2. Enzymatic activities The determination of enzymatic activities in the homogenised soil samples taken from both upper and lower portions of each microcosm type (W, LAS10 and LAS50) was done in all cases according to the methods described by Tabatabai (1982). For acid phosphatase, 1 g of sieved soil (less than 2 mm) was combined with 2.0 ml of toluene, 4.0 ml pH 6.5 modified universal buffer and 1.0 ml of p-nitrophenol phosphate solution (0.025 M) for 1 h at 37 C. The p-nitrophenol released was determined colorimetrically at 410 nm using a Hitachi U-2000 (Tokyo, Japan) spectrophotometer, and compared with a standard. Alkaline phosphatase was analyzed in a similar manner, except that the modified universal buffer was adjusted to pH 11.0 using 0.1 N NaOH. For the analysis of arylsulphatase, 1 g of sieved soil (less than 2 mm), 0.25 ml toluene, 4.0 ml acetate buffer (0.5 M, pH 5.8) and 1.0 ml of p-nitrophenyl sulfate (0.025 M in acetate buffer) were mixed and incubated at 37 C. After 1 h, the reaction was stopped by the addition of 1 ml of 0.5 M CaCl2 and 4.0 ml of 0.5 M NaOH and filtered through a Whatman no. 42 filter paper. The p-nitrophenol released was measured colorimetrically at 410 nm. For the dehydrogenase activity, 20 g of dried sieved soil was combined with 0.2 g CaCO3. Three 6 g portions of mixture were incubated at 37 C with 1.0 ml 3% (w/v) 2,3,5-triphenyltetrazolium chloride and 2.5 ml distilled water. After 24 h, the soils were removed from incubation, mixed with 10 ml of reagent grade methanol and quantitatively filtered through absorbent cotton. The red color resulting from the production of triphenylformazan by soil dehydrogenase was washed from the cotton into a 100 ml volumetric flask using 10 ml portions of methanol, and the flask was brought to volume with additional methanol. The triphenylformazan was determined colorimetrically at 485 nm and compared with a standard curve. 2.3.3. Chemical determinations LAS 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 Nimer et al. (2007) and using an Agilent Technologies (Palo Alto, CA, USA) 1100 series HPLC equipment. Phosphorous (as P-PO3 4 ) concentration was determined in the leachate from soil microcosms W, LAS10 and LAS50 throughout the experiments following the spectrophotometric procedure described in Rodier (1989) and using a Hitachi U-2000 (Tokyo, Japan) spectrophotometer.
3. Results 3.1. LAS in soil samples Once removed from the glass columns, soil samples from each soil microcosms (two samples by column, corresponding to the upper and lower portions) were preserved by the immediate addition of 3% (v/v) formaldehyde until their analysis, following the methodology previously described in Nimer et al. (2007). Table 1 shows the concentration of LAS (mg kg1 soil) in each soil column portion at each sampling time. The surfactant concentrations were significantly higher (p < 0.05) in the sterile soil columns (SLAS10 and SLAS50) when compared with the non-sterile ones (LAS10 and LAS50). Moreover, in all cases, the highest LAS concentrations were always found in the upper soil column layer. 3.2. Cultivable heterotrophic bacteria in samples from soil columns Fig. 2 shows the cultivable aerobic heterotrophic bacterial counts (as log CFU g1) in soil samples from upper and lower portions of LAS10 (Fig. 2A) and LAS50 (Fig. 2B) microcosms. Bacterial counts from W microcosms (taking into account as control soil microcosms) were also included. Bacterial counts were significantly higher (p < 0.05) in the microcosms amended with LAS (LAS10 and LAS50) than in the W microcosms (Fig. 2), and the counts from LAS50 were significantly higher (p < 0.05) than those from LAS10 in the upper portion of the soil columns. Maximum bacterial counts in the LAS50 microcosms were recorded during the third experiment day (Fig. 2B), earlier than in the case of the LAS10 microcosms, where maximum bacterial counts were recorded during the 14th experiment day (Fig. 2A). Finally, bacterial counts were significantly higher (p < 0.05) in the upper portion than in the lower portion in all the studied microcosms. 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 dehydrogenase) in soil microcosms amended with sterile solutions containing 10 mg l1 (A) and 50 mg l1 (B) of LAS both in upper and lower portions of soil columns. The values of the soil enzymatic activities in soil microcosms amended with sterile distilled water (W) are included as controls. Table 1 LAS concentration (mg kg1 soil) in each soil microcosm and soil portion, at different sampling times. Soil microcosm type
Running time (days)
LAS10
7 14 21
1.4 9.2 0.7
0.3 3.1 0.6
SLAS10
7 14 21
13.3 22.1 30.0
0.5 0.8 1.2
LAS50
3 7 21
35.1 70.8 20.4
0.5 0.4 9.6
SLAS50
3 7 21
53.1 83.8 284.5
0.6 12.2 84.7
2.4. Statistical analysis One-way analysis of variance (ANOVA) was performed using the software package Statgraphics 3.0 Plus version (STSC Inc., Rockville, MD, USA) in order to identify the effect of different LAS concentrations on bacterial counts and soil enzyme activities. A significance level of 95% (p < 0.05) was selected. In addition, to evaluate the correlation among cultivable heterotrophic bacteria and enzymatic activities, a matrix of Pearson’s linear correlation coefficients was made using the same statistical package.
71
Upper portion (mg kg1 soil)
Lower portion (mg kg1 soil)
LAS10: soil microcosm amended with 10 mg l1 LAS solution. LAS50: soil microcosm amended with 50 mg l1 LAS solution. SLAS10: sterile soil microcosm amended with 10 mg l1 LAS solution. SLAS50: sterile soil microcosm amended with 50 mg l1 LAS solution.
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72
B
8
Upper portion
Log CFU/g dry soil
Log CFU/g dry soil
A
*
7.5 7 6.5
LSD (0.05)
6 5.5 0
7
14
8
*
*
*
7 6.5
LSD (0.05)
6 5.5
21
Upper portion
7.5
0
3
6
9
Days 8
Lower portion 7.5
*
7 LSD (0.05)
6 5.5 0
7
14
21
Log CFU/g dry soil
Log CFU/g dry soil
8
6.5
15
18
21
Lower portion
7.5
*
7 6.5
LSD (0.05)
6 5.5
0
3
6
9
Days LAS10
12
Days
12
15
18
21
Days W
LAS50
W
Fig. 2. Cultivable aerobic heterotrophic bacteria counts (as log CFU g1) in soil samples from upper and lower portions of LAS10 microcosms (A) and LAS50 microcosms (B). Bacterial counts 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 * are significantly different from control (p 0.05).
µg PNP-released/g soil/h
A
250 W
200
LAS10
150
100
50
0 0
7
14
21
B
In order to test the influence of LAS on nutrient availability and, as a consequence, on bacterial population and enzyme activities,
7
14
21
7
21
250 W
LAS50
200
150
100
50
0 0
3
7
21
0
3
Days upper portion
3.4. Phosphorous leachate analysis
0
Days
g PNP-released/g soil/h
As a general rule, the application of the anionic surfactant to soil at the different concentrations assayed significantly increased the mean values of extracellular enzymes (acid and alkaline phosphatases and arylsulphatase) both in the upper and lower portions of the soil columns with respect to the control microcosms, exception made for the acid phosphatase (lower portion) and the alkaline phosphatase (upper and lower portions) of LAS10 microcosm, where a positive but not significant trend was detected. By contrast, the dehydrogenase activity was always significantly decreased by the presence of LAS, when it was compared with control soil samples (W). Table 2 summarizes the results of an analysis of variance for each enzyme activity measured as affected by the different LAS treatment applied (LAS10 and LAS50). Significance level was generally higher for the LAS50 than for the LAS10 treatment. To evaluate the relationships between enzymatic soil activities and heterotrophic soil bacterial counts in each microcosm soil portion, a correlation matrix (Pearson’s linear correlation coefficients) was constructed (Table 3). A positive and statistically significant linear correlation (p < 0.05) existed between both phosphatase and arylsulphatase activities and aerobic heterotrophic soil bacterial counts in microcosms amended with sterile solutions containing 10 mg l1 (LAS10) and 50 mg l1 (LAS50) of LAS. These correlations showed always greater coefficient values in the upper portion of the microcosm than in the lower one. On the other hand, the correlation coefficients between the dehydrogenase activity and aerobic heterotrophic soil bacteria counts were always negative and higher in the upper portion of the microcosms than in the lower one. Finally, it is worth noting that in the microcosms without LAS (W) the counts of heterotrophic aerobic bacteria (as log CFU g1) were only positively and statistically correlated to the alkaline phosphatase activity.
lower portion
Fig. 3. Alkaline phosphatase activity (mg PNP released g1 wet soil h1 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 and standard deviations (SD) of three replicates.
M. Sa´nchez-Peinado et al. / Soil Biology & Biochemistry 41 (2009) 69–76
A
100 90 80 70 60 50 40 30 20 10 0
W
0
7
LAS10
14
21
0
7
14
21
Days
50
W
LAS10
40 30 20 10 0
100 90 80 70 60 50 40 30 20 10 0
0
7
14
21
0
7
14
21
Days W
LAS50
B
70
60
0
3
7
21
0
3
7
21
Days upper portion
lower portion
Fig. 4. Acid phosphatase activity (mg PNP released g1 wet soil h1 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 and standard deviations (SD) of three replicates.
g PNP released/g soil/h
g PNP-released/g soil/h
B
70 60
g PNP released/g soil/h
g PNP-released/g soil/h
A
73
LAS50
W 50 40 30 20 10
phosphorous (as P-PO3 4 ) concentration was regularly determined in the microcosms leachates. Fig. 7 shows the phosphate concentrations detected in the leachate of the soil microcosms LAS10, LAS50 and W during the running experiment time. The dynamics of these 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 soil microcosm LAS10 was significantly higher than in LAS50 and W, and the minimum phosphates concentration was recorded in the LAS50 leachate throughout the whole experiment time.
0 0
3
7
21
0
3
7
21
Days upper portion
lower portion
Fig. 5. Arylsulphatase activity (mg PNP released g1 wet soil h1 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 value and standard deviations (SD) of three replicates.
4. Discussion Among the parameters revealing information on the quality of a soil, those relative to its biological composition and metabolic activity display a greater sensitivity to contamination processes (Garcı´a et al., 2003). Since the physical and chemical characteristics of a soil can be considered stable, any impact in a soil is detected initially by significant variations in its biological dynamics. The soil constitutes a biological system in which enzymatic activities play an important role in its biochemical status (Overbeck, 1991; Stryler, 1995). The measurement of the soil enzyme activities allows inferring the quality of the system, and is commonly used as an indicator of soil microbial activity (Sinsabaugh, 1994; Garcı´a et al., 2003). In addition, it acts as a good indicator of changes in the properties of the soil induced by the anthropogenic addition of compounds (Lobo et al., 2000). The lower concentrations of LAS detected in the non-sterile soil columns when compared with the sterile ones demonstrated the biotransformation process of the surfactant. This process reached its highest intensity after 14 days of experimentation in the case of
soil columns continuously amended with 10 mg l1 of surfactant, and after 7 days when the soil columns were amended with 50 mg l1 of LAS. On the other hand, the greatest proportion of the surfactant was retained in the upper portion of the columns (whether sterile or non-sterile). Previous studies (Jacobsen et al., 2004) revealed the same distribution pattern of the surfactant in experimental soil columns, with maximum concentrations registered in the first 15 cm, and showing a significant transport towards the lower layers. In our experiments, the lower concentrations of LAS detected in the deep layers of the microcosm may be due to the faster biotransformation process of the surfactant and to the higher retention of the surfactant in the upper layers of the soil column. The first LAS degradation homologue detected in the leachate was C10 (data not shown) because the longer chain homologues are more easily biodegradable when they are in the interstitial water, and once adsorbed to soil particles they are retained with greater force than the short chain homologues. Similar results were obtained by Jacobsen et al. (2004).
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Table 3 Pearson’s correlation coefficients between heterotrophic aerobic bacteria counts and enzymatic activities in each soil microcosm and portion sampled.
g TPF released/g soil/day
A 90 80
LAS10
W
Acid phosphatase
70 60 50 40 30 20 10 0 0
7
14
21
0
7
14
21
Days
Arylsulphatase
Dehydrogenase
LAS10 Upper portion Lower portion
0.82* 0.27
0.94* 0.61*
0.89* 0.63*
0.84* 0.73*
LAS50 Upper portion Lower portion
0.58* 0.58*
0.97* 0.75*
0.84* 0.75*
0.59* 0.27
0.83* 0.65*
0.54 0.47
0.18 0.31
W Upper portion Lower portion
0.05 0.06
*Significant at p 0.05. LAS10: soil microcosm amended with 10 mg l1 LAS solution. LAS50: soil microcosm amended with 50 mg l1 LAS solution. W: soil microcosms amended with sterilised distilled water (control).
B g TPF released/g soil/day
Alkaline phosphatase
90
LAS50
W
80 70 60 50 40 30 20 10 0
0
3
7
21
0
3
7
21
Days upper portion
lower portion
Fig. 6. Dehydrogenase activity (mg TPF released g1 dry weight day1 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 and standard deviations (SD) of three replicates.
The data obtained from agricultural soil microcosms have revealed that the enzymatic activities acid phosphatase, alkaline phosphatase and arylsulphatase were correlated positively with the bacterial counts under our experimental conditions. These results are in accordance with those previously reported by Nannipieri et al. (1979), Criquet et al. (2002) and Ne´ble et al. (2007). The values of phosphatase and arylsulphatase activities obtained in soil microcosms LAS10 were always higher than those obtained in control microcosms (W), although these differences were not statistically significant for the cases of acid phosphatase activity (lower portion) and alkaline phosphatase activity (upper and lower portions). On the contrary, the application of greater concentrations of LAS in the influent (50 mg l1) generated significantly higher values of phosphatase and arylsulphatase activities, with temporal distribution patterns characterized by earlier and more intense maximums of activity. While previous studies (Elsgaard et al., 2001a) suggested the absence of response of the arylsulphatase activity to LAS addition, our results have shown that the value of this enzymatic activity is increased by the presence of 10 or 50 mg l1 of this anionic surfactant. We suggest that a limitation of the bacterial growth by bio-available forms of sulphur in the continuously washed
agricultural soil microcosms assayed in our study could be the reason for this opposed behaviour. Thus, arylsulphatase activity could be involved in the breakage of the LAS benzene–S bond and in the release of S molecules to the medium, allowing the microorganisms not only to cover their requirements of a bio-available S form (Dodgson et al., 1982) but also to collaborate significantly in the biotransformation of the LAS molecule (Kertesz et al., 1994). In fact, several strains of Pseudomonas spp. (potentially capable to produce extracellular sulphatases, Gonza´lez et al., 2003) have been previously described as important members of LAS biotransformation consortia (Swisher, 1987; Jimenez et al., 1991). Indeed, several recent studies (Vong et al., 2002; Gonza´lez et al., 2003) have been carried out to investigate the possibility to use bacteria to increase the mobilisation of the different forms of S-organic in soils and to ‘‘in situ’’ accelerate the S biogeochemical cycle, in order to improve the fertility of agricultural soils. Under our experimental conditions (oligotrophic systems), the continuous leaching of nutrients from soil (either dissolved in the water and/or emulsified by the anionic surfactant), as well as the depletion of the nutritional resources in the soil as a consequence of microbial growth, could have affected the biological activities of the soil (Barber, 1995; Hartemink, 2005). In fact, our results show that the phosphate concentration detected in the leachate of the LAS50 microcosm was significantly lower than the concentrations detected in LAS10 leachates throughout the experiment. The counts of cultivable aerobic heterotrophic bacteria in soil samples from the LAS50 microcosms were significantly higher than those from LAS10, and consequently it could be suggested that the phosphate consumption was higher too. Thus, under these circumstances, the bacterial populations in this soil microcosm type may respond by increasing the extracellular phosphatase activity (mainly acid phosphatase) to obtain readily assimilable 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 phosphate concentration throughout the period of study was shown by the positive and statistically significant correlations found between the number of cultivable heterotrophic bacteria and the phosphatase activity (mainly alkaline phosphatase) in the W microcosm. No
Table 2 Summary of the results of an analysis of variance (ANOVA) for each of the enzyme activities measured, as affected by the different LAS treatment applied (LAS10 and LAS50). Acid phosphatase
LAS10 LAS50
Alkaline phosphatase
Arylsulphatase
Upper portion
Lower portion
Upper portion
Lower portion
Upper portion
Lower portion
Upper portion
Lower portion
p < 0.01 p < 0.05
NS p < 0.001
NS p < 0.05
NS p < 0.001
p < 0.001 p < 0.001
p < 0.01 p < 0.01
p < 0.05 p < 0.05
p < 0.01 p < 0.001
NS: not significant (p > 0.05).
Dehydrogenase
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75
approach constitutes a useful tool for preliminary studies on the effects of LAS on soils’ biological properties.
1200
P-PO4 (µg/l)
1000
5. Conclusions
800 600 400 200
W
LAS10
LAS50
0 3
6
9
12
15
Days Fig. 7. Phosphorous concentrations (as P-PO4) in leachate samples from LAS10 and LAS50 microcosms during the running time. Data from W microcosms (control) were included too. Data are average values and standard deviations (SD) of three replicates.
other enzymatic activities were positively and significantly related to the numbers of cultivable heterotrophic bacteria in this microcosm. Nevertheless, it is worthy to note that these counts only represent a small part of total soil bacteria. Arylsulphatase and phosphatase activities are considered ‘‘specific parameters’’ since the enzymes catalyse concrete reactions and are mediated by certain bacterial groups which depend on specific substrates. On the contrary, the dehydrogenase activity is considered ‘‘a general’’ determination, as its measurement allows to evaluate the microbial metabolic processes that take place in the soil under a global view (Tabatabai, 1982; Garcı´a et al., 2003). Our results demonstrate that the dehydrogenase activity was progressively inhibited in the LAS10 and LAS50 soil microcosms, and that a statistically negative correlation existed between this enzymatic activity in the upper portion of the soil columns and the counts of aerobic heterotrophic microorganisms, while no significant correlations were detected between these parameters in control soil microcosms (W). Previous studies (Malkomes and Wo¨hler, 1983; Elsgaard et al., 2001b) demonstrated a similar inhibition pattern of the dehydrogenase activity in the presence of increasing concentrations of LAS. These results suggest a certain negative effect of LAS on soil microbial processes although the presence of this xenobiotic can favour other more specialised enzymatic processes, mediated by specific groups of microorganisms (Van Straalen and Van Gestel, 1993). Margesin and Schiner (1998) reported that the measurement of the dehydrogenase activity could be considered as a particularly appropriate method for the monitoring of soil contamination by anionic surfactants. In addition, this study demonstrated that the presence of another anionic surfactant (SDS) significantly inhibited the dehydrogenase activity of an agricultural soil, and this enzymatic activity only increased when 90% of degradation of surfactant was achieved. This could be explained by the fact that SDS may alter microorganism membrane structures within which dehydrogenases are located. Similarly, Schwuger and Bartnik (1999) described a number of diverse effects of LAS on cellular membrane structures as a consequence of its surface activity. In recent years, considerable interest has developed in the use of microcosm techniques for ecotoxicological assays (Edwards et al., 1997, 1998; Burrows and Edwards, 2000). Such microcosms have usually consisted of units containing intact or mixed soil supporting indigenous multiple biotic species, and have ranged in size from a few grams of soil to cores as large as a meter in diameter. Although field observations are needed for realistic calibration and validation of the information, our integrated in vitro microcosm
Soil enzymatic activities, in particular dehydrogenase activity, have been suggested as appropriate parameters for the monitoring of soil contamination. Using agricultural soil microcosm systems, we conclude that the continuous applications of different LAS solutions at 10 and 50 mg l1 concentrations for periods of time up to 21 days significantly decrease the dehydrogenase activity, while other enzymatic activities catalysed by specific bacterial groups (alkaline phosphatase, acid phosphatase and arylsulphatase) were significantly increased. Therefore, a statistically negative correlation between dehydrogenase activity and the number of heterotrophic aerobic microorganisms was observed, suggesting an inhibition of the soil dehydrogenase activity in response to the addition of increasing concentrations of LAS in agricultural soils. LASs also induce the alteration of the phosphorous turn-over by increasing its bioavailability. Further studies are needed in order to evaluate 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 Educacio´n y Ciencia (MEC), as part of Project Reference PPQ2003-07978V02-02 (Programa Nacional de IþD). B.R. was supported by Programa Ramo´n y Cajal (MEC, Spain). C. Pozo was supported by Programa Retorno de Doctores (Junta de Andalucı´a. Spain).
References Avidano, L., Gamalero, E., Cossa, G.P., Carrazo, E., 2005. Characterization of soil health in an Italian polluted site by using microorganisms as bioindicators. Applied Soil Ecology 30, 21–33. Bandick, A.K., Dick, R.P., 1999. Field management effects on soil enzyme activities. Soil Biology & Biochemistry 31, 1471–1479. Barber, S.A., 1995. Soil Nutrient Bioavailability. A Mechanistic Approach, second ed. Wiley-Interscience, New York, 384 pp. Bremmer, J.M., 1965. Inorganic forms of nitrogen. Agronomy 9, 1179–1237. Burrows, L., Edwards, C.A., 2000. The fate and environmental effects of the fungicide carbendazim in an innovative terrestrial microcosm system. In: Proceedings of the Brighton Crop Protection Conference on Pests and Diseases, 4C-2, pp. 365–370. Carlsen, L., Metzon, M.B., Kjelsmark, J., 2002. LAS in the terrestrial environment. Science of the Total Environment 290, 225–230. CESIO, Comisio´n europea de surfactantes y sus intermediarios orga´nicos, 2006. General Assembly. Santiago de Compostela, Spain. Criquet, S., Tagger, S., Vogt, G., Le Petit, J., 2002. Endoglucanase and beta-glycosidase activities in an evergreen oak litter: annual variation and regulating factors. Soil Biology & Biochemistry 34, 1111–1120. Criquet, S., Ferre, E., Farnet, A.M., Le Petit, J., 2004. Annual dynamics of phosphatase activities in an evergreen oak litter: influence of biotic and abiotic factors. Soil Biology & Biochemistry 36, 1111–1118. Del Olmo, M., Garballo, A., Nimer, M., Lo´pez, I., De Ferrer, J.A., Vı´lchez, J.L., 2004. Development of methodologies for different degrees of resolution of linear alkylbenzene sulfonates in groundwater and wastewater by liquid chromatography using sodium dodecyl sulphate. Chromatographia 60, 157–164. Dick, R.P., 1997. Soil enzyme activities as integrative indicators of soil health. In: Pankhurst, C.E., Doube, B.M., Gupta, V.V.S.R. (Eds.), Biological Indicators of Soil Health. CAB International, Wellingford, pp. 121–156. Dodgson, K.S., White, G.F., Fitzgerald, J.W., 1982. Sulfatases of Microbial Origin. CRC Press, Boca Rato´n, Florida, USA, 199 pp. Edwards, C.A., Knacker, T.T., Pokarzhevski, A.A., Subler, S., Parmelee, R., 1997. The use of soil microcosms in assessing the effects of pesticides on soil ecosystems. In: Proceedings of the International Symposium on Use of Nuclear and Related Techniques for Studying Environmental Behaviour of Crop Protection Chemicals. Vienna, Austria, pp. 1–23. Edwards, C.A., Knacker, T., Pokarzhevskii, A.A., 1998. The prediction of the fate and effects of pesticides in the environment using tiered laboratory terrestrial model ecosystems. In: Proceedings of the Brighton Crop Protection Conference on Pests and Diseases, 4C-1, pp. 267–272.
76
´ nchez-Peinado et al. / Soil Biology & Biochemistry 41 (2009) 69–76 M. Sa
Elsgaard, L., Petersen, S.O., Debosz, K., Kristiansen, I.B., 2001a. Effects of linear alkylbenzene sulfonates (LAS) on soil microbiology. Tenside Surfactants Detergents 38, 94–97. Elsgaard, L., Petersen, S., Debozs, K., 2001b. Effects and risk assessment of linear alkylbenzene sulfonates in agricultural soil. 1. Short-term effects on soil microbiology. Environmental Toxicology and Chemistry 20, 1656–1663. Garcı´a, C., Gil, F., Herna´ndez, T., Trasar, C., 2003. Te´cnicas de ana´lisis de para´metros bioquı´micos en suelos: medida de actividades enzima´ticas y biomasa microbiana. Ediciones Mundi-Prensa, Madrid, 371 pp. Gonza´lez, S., Navarro, M., Lo´pez, J.I., Rad, C., 2003. Determinacio´n de la actividad arilsulfatasa del suelo. In: Garcı´a, C., Gil, F., Herna´ndez, T., Trasar, C. (Eds.), Te´cnicas de ana´lisis de para´metros bioquı´micos en suelos: medida de actividades enzima´ticas y biomasa microbiana. Ediciones Mundi-Prensa, Madrid, pp. 79–99. Hartemink, A.E., 2005. Nutrient stocks, nutrient cycling and soil changes in cocoa ecosystems – a review. Advances in Agronomy 86, 227–253. Jacobsen, A.M., Mortensen, G.K., Hansen, H.C.B., 2004. Degradation and mobility of linear alkylbenzene sulfonate and nonylphenol in sludge-amended soil. Journal of Environmental Quality 33, 232–240. Jensen, J., Løkke, H., Holmstrup, M., Krogh, P.H., Elsgaard, L., 2001. Effects and risk assessment of linear alkylbenzene sulfonates in agricultural soil. 5. Probabilistic risk assessment of linear alkylbenzene sulfonates in sludge-amended soils. Environmental Toxicology and Chemistry 20, 1690–1697. Jensen, J., Smith, S.R., Krogh, P.H., Versteeg, D.J., Temara, A., 2007. European risk assessment of LAS in agricultural soils revisited: species sensitivity distribution and risk estimates. Chemosphere 69, 880–892. Jimenez, L., Breen, A., Thomas, N., Federle, T.W., Sayler, G.S., 1991. Mineralization of linear alkylbenzene sulfonate by a 4-member aerobic bacterial consortium. Applied and Environmental Microbiology 57, 1566–1569. Kertesz, M.A., Cook, A.M., Leisinger, T., 1994. Microbial metabolism of sulphur- and phosphorous-containing xenobiotics. FEMS Microbiology Reviews 15, 195–215. Lobo, M.C., Sastre, I., Vicente, M.A., 2000. Las enzimas como medida del impacto ambiental en los suelos. In: Garcı´a, C., Herna´ndez, M.T. (Eds.), Investigacio´n y ˜ a. CEBAS-CSIC, Murcia, pp. perspectivas de la enzimologı´a de suelos en Espan 297–352. Malkomes, H.P., Wo¨hler, B., 1983. Testing and evaluating some methods to investigate side effects of environmental chemicals on soil microorganisms. Ecotoxicology and Environmental Safety 7, 284–294. Margesin, R., Schiner, F., 1998. Biodegradation of the anionic surfactant sodium dodecyl sulphate at low temperatures. International Biodeterioration and Biodegradation 41, 139–143. Mortensen, G.K., Elsgaard, H., Ambus, P., Jensen, E.S., Groen, C., 2001. Influence of plant growth on degradation of LAS in sludge-amended soil. Journal of Environmental Quality 30, 1266–1270. Nannipieri, P., Pedrazzini, F., Arcaa, P.G., Piovanelli, C., 1979. Changes in amino acids, enzyme activities, and biomass during soil microbial growth. Soil Science 127, 26–34. Natural Resources Conservation Service, 1999. Soil taxonomy, a basic system of soil classification for making and interpreting soil surveys. Agriculture Handbook, vol. 436, second ed. Washington, DC, USA, 869 pp.
Ne´ble, S., Calvert, V., Le Petit, J., Criquet, S., 2007. Dynamics of phosphatase activities in a cork oak litter (Quercus suber L.) following sewage sludge application. Soil Biology & Biochemistry 39, 2735–2742. Nimer, M., Ballesteros, O., Navalo´n, G., Crovetto, C., Verge, C., Lo´pez, I., Berna, J.L., 2007. New simple treatment for determination of linear alkylbenzene sulfonate (LAS) in agricultural soils by liquid chromatography with fluorescence detection. Analytical and Bioanalytical Chemistry 387, 2175–2184. Olsen, S.R., Dean, L.A., 1965. Phosphorous. In: Page, A.L., Miller, R.H., Keeney, D.R. (Eds.), Methods of Soil Analysis: Part 2. Chemical and Microbiological Properties. American Society of Agronomy, Soil Science Society of America, Madison, WI, USA, pp. 1035–1049. Overbeck, J., 1991. Early studies on ecto- and extracellular enzymes in aquatic environments. In: Chro´st, R.J. (Ed.), Microbial Enzymes in Aquatic Environments. Springer, New York, pp. 1–5. Penteado, J.C.P., Seoud, O.A.E., Carvalho, L.R.F., 2006. Linear alkylbenzene sulfonates: chemistry, environmental impact and analysis. Quimica Nova 29, 1038–1046. Pratt, P.F., 1954. Potassium release from exchangeable and non-exchangeable forms in soils. Ohio Agricultural Experiment Station Resource Bulletin 47, 747. Prats, D., Lopez, C., Vallejo, D., Varo, P., Leon, V.M., 2006. Effect of temperature on the biodegradation of linear alkylbenzene sulfonate and alcohol ethoxylate. Journal of Surfactants Detergents 9, 69–75. Rodier, J., 1989. Ana´lisis de Aguas. Ed. Omega, Barcelona, Spain, pp. 1059–1060. Sa´nchez-Peinado, M., Gonza´lez-Lo´pez, J., Rodelas, B., Galera, V., Pozo, C., Martı´nezToledo, M.V., 2008. Effect of linear alkylbenzene sulfonates on the growth of aerobic heterotrophic cultivable bacteria isolated from an agricultural soil. Ecotoxicology 17, 549–557. Schwuger, M.J., Bartnik, F.G., 1999. Interaction of anionic surfactants with proteins, enzymes and membranes. In: Gloxhuber, C. (Ed.), Anionic Surfactants – Biochemistry, Toxicology, Dermatology. Marcel Dekker Inc., New York, USA, pp. 1–49. Sinsabaugh, R.L., 1994. Enzymic analysis of microbial pattern and process. Biology and Fertility of Soils 17, 69–74. Solbe`, J., Berna, J.L., Cavalli, L., Feitjtel, T.C.J., Fox, K.K., Heinze, J., Marshall, S.J., de Wolf, W., 2000. Terrestrial risk assessment of LAS in sludge-amended soils. In: 5th World Cesio Congress, vol. 2, pp. 1433–1438. Speir, T.W., Ross, D.J., 1978. Soil phosphatase and sulphatase. In: Burns, R.G. (Ed.), Soil Enzymes. Academic Press, London, pp. 197–250. Stryler, L., 1995. Biochemistry, fourth ed. Freeman and Company, New York, 632 pp. Swisher, R.D., 1987. Surfactants Biodegradation, second ed. Marcel-Dekker Inc., New York, USA, 1085 pp. Tabatabai, M.A., 1982. Soil enzymes. In: Page, A.L., Miller, R.H., Keeney, D.R. (Eds.), Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties, second ed. Soil Science Society of America, Inc., Madison, WI, USA, pp. 902–947. Van Straalen, N.M., Van Gestel, C.A.M., 1993. Soil invertebrates and micro-organisms. In: Calow, P. (Ed.), Handbook of Ecotoxicology, vol. 1. Blackwell Scientific, Oxford, U.K., pp. 251–277. Vong, P.-C., Lasserre-Joulin, F., Guckert, A., 2002. Mobilization of labelled organic sulphur in rhizosphere of rape and barley and in non-rhizosphere soil. Journal of Plant Nutrition 25 (10), 2191–2204.
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Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Response of soil enzymes to Linear Alkylbenzene Sulfonate (LAS) addition in soil microcosms Marı´a del Mar Sa´nchez-Peinado a, Bele´n Rodelas a, b, Marı´a Victoria Martı´nez-Toledo a, b, Jesu´s Gonza´lez-Lo´pez a, b, *, Clementina Pozo a, b a b
Group of Environmental Microbiology, Institute of Water Research, University of Granada, Granada, Spain Group of Environmental Microbiology, Department of Microbiology, University of Granada, Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 May 2008 Received in revised form 17 September 2008 Accepted 23 September 2008 Available online 23 October 2008
Soil enzymatic activities (phosphatases, arylsulphatase and dehydrogenase) were measured in microcosm systems designed for the study of the impact of a commercial mixture of Linear Alkylbenzene Sulphonate (LAS) homologues on a xerofluvent agricultural soil. The soil microcosms consisted of glass columns filled with 800 g of dry soil which were fed with sterile commercial LAS solutions at concentrations of 10 or 50 mg l1 for periods of time up to 21 days. A soil microcosm fed with sterile distilled water was included in this study and considered as control. Our results showed that the continuous application of the anionic surfactant to soil increased the values of the enzymes acid and alkaline phosphatases and arylsulphatase. On the contrary, the dehydrogenase activity was decreased by the continuous application of 10 or 50 mg l1 LAS when compared with control microcosms. In addition, a statistically negative correlation was found between this enzymatic activity in the upper portion of the soil 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 bacteria populations and soil biological activities, phosphate concentration was regularly determined in the microcosm leachates. The phosphate concentration tested in the leachate of the microcosm continuously amended with 50 mg l1 LAS solution was significantly lower than the concentrations detected in the leachate of the microcosms continuously amended with 10 mg l1 LAS throughout the experiment. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: LAS Enzymatic activities Soil microcosm Heterotrophic bacteria P-PO3 4
1. Introduction Linear Alkylbenzene Sulfonate (LAS) is the most used anionic surfactant in detergents and cleaning agents. World-wide LAS consumption during 2005 was estimated as 2.5 million tons, and it is expected to reach values around 3.4 million tons in 2010 (Penteado et al., 2006). In Europe, the total production of LAS in the year 2004 was 487 ktons, corresponding to more than 80% to household detergents (CESIO, 2006). The consumption rate per habitant and per day is relatively high depending on the country. In this sense, Jensen et al. (2007) have recently reported that the consumption rate in Europe is in the range of 1.4–4 g person day1. The aerobic treatment of sewage in wastewater plants is sufficient to degrade more than 95% of the LAS input (Prats et al., 2006), but high LAS concentrations have been found in sewage sludge after anaerobic treatment. The use of raw wastewater as irrigation
* Corresponding author. Institute of Water Research, C/Ramo´n y Cajal No. 4, University of Granada, 18071 Granada, Spain. Tel.: þ34 958244170; fax: þ34 958246235. E-mail addresses: [email protected] (J. Gonza´lez-Lo´pez), [email protected] (C. Pozo). 0038-0717/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2008.09.019
water and the application of sewage sludge as soil fertilizer or soil conditioner are the most important introduction ways of LAS into natural ecosystems. LAS concentration in natural soils is typically low, ranging between 0.7 mg kg1 and 1.4 mg kg1 (Mortensen et al., 2001; Carlsen et al., 2002). Nevertheless, Solbe` et al. (2000) reviewed LAS concentrations in sludge-amended agricultural soils concluding that they were around 20 mg kg1, depending on the sludge application rate and the sampling time after the application. Moreover, Jensen et al. (2001) have reported that LAS concentrations ranging between 10 and 50 mg kg1 can be found in agricultural soils after the addition of sludge as a fertilizer. Several studies have focused on the effect of LAS derived from sludge 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 microbial activities (Sa´nchez-Peinado et al., 2008). Soil enzymes have been suggested as potential indicators of soil quality because of their essential role in different soil aspects such as nutrient mineralization and cycling, decomposition and synthesis of organic matter and degradation of xenobiotics (Bandick and Dick, 1999). Microorganisms are the main source of
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enzymes (extracellular and intracellular enzymes) in soils (Tabatabai, 1982), so their study is essential to understand the biological processes that occur in soils as well as the impact of several substances on their fertility. The main goal of this research was to investigate the effects of LAS exposure (as aqueous solution) on soil enzymes such as acid and alkaline phosphatases, arylsulphatase and dehydrogenase by using soil microcosms artificially polluted with different concentrations of detergent in order to understand the effects of this anionic surfactant on soil microbial activity.
Filtered air
6 cm Upper zone
Polyethylene tube
35 cm
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 (Southern Spain) which had no previous exposure to LAS contamination. After the removal of plant residues, samples were collected at a depth of 0–20 cm using a soil core sampler. Immediately after collection the soil was air dried and sifted with a sieve (diameter 2 mm) to remove 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 composition of the samples was as follows: organic matter, 13.9 g kg1; pH (water) 7.8; N total, 1.4 g kg1; phosphorous, 25 mg kg1; and potassium, 240 mg kg1. Soil texture was analyzed following Natural Resources Conservation Service (1999), while N total, phosphorus and potassium were determined by the techniques described 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. To avoid the soil loss a porous plate was located at the bottom of the glass cylinder and the leaching water was collected using a polyethylene tube connected to a sterile glass bottle. Sterile solutions of commercial LAS at concentrations of 10 or 50 mg l1 were continuously added through the experimental soil columns at 8.0 ml h1 using a peristaltic pump (Watson Marlow 505S, UK). These LAS concentrations are typically found in agricultural soils after the addition of sludge as a fertilizer (Jensen et al., 2001). All the soil columns and tubes used in the experiment were made of glass and polyethylene, respectively, in order to avoid the potential LAS adsorption (Fig. 1). The commercial LAS mixture used in the experiments contains 69% of water and 31% of active matter, with the following distribution of the linear alkyl chain homologues: 5-phenyl C10, 0.8%; phenyl C10, 9.8%; phenyl C11, 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 two sterile soil columns amended with sterile LAS solution at two concentrations (10 and 50 mg l1) and named as SLAS10 and SLAS50, respectively, was included in the study in order to establish the running time of each experiment by testing the appearance of the first LAS homologue (C10) in the leachate. The soil columns were sterilised by three successive autoclaving processes at 120 C during 60 min and the sterility of the systems was tested by inoculation of soil samples (1 g) into nutrient broth medium. To avoid contamination, the soil columns were upclosed with sterile rubber stoppers. For the experiments with sterile soil columns amended with sterile solutions containing 10 mg l1 of LAS, the running time of the study was established
Lower zone
Peristaltic pump
Porous plate
Polyethylene tube Sterile solution of LAS Leachate
Fig. 1. Diagram of the agricultural soil microcosm systems used in this study.
at 21 days, while for experiments with 50 mg l1 of LAS this time was only of 7 days, although these experiments were maintained over 21 days for comparison. The determination of LAS homologues in the leachate was made according to the methodology proposed by Nimer et al. (2007) as described in Section 2.3.3. After determination of running time the sterile microcosms were eliminated. Once the experiment running time was established, two new types of soil microcosms were built. The first type corresponded to non-sterile soil columns amended with sterilised distilled water (named as W). The second type corresponded to non-sterile soil columns amended with LAS solution at two different concentrations: 10 and 50 mg l1 of LAS (LAS10 and LAS50, respectively). The distilled water used was sterilised by autoclaving and the LAS solution was sterilised by filtration (0.22 mm, MilliporeÒ). For LAS10 microcosms the glass columns were broken down under sterile conditions after 7, 14 and 21 days, while for experiments using 50 mg l1 LAS, the soil columns were broken down after 3, 7 and 21 days of running. Once the columns were opened, the glass was removed and two soil portions were taken: upper and lower (see Fig. 1). These samples were homogenised and enclosed into sterile glass containers and remained at 4 C until analysis. All the microcosms W, LAS10 and LAS50 were replicated thrice for each sampling time.
2.3. Samples’ analysis 2.3.1. Counts of cultivable heterotrophic bacteria in samples from soil columns The number of cultivable aerobic heterotrophic bacteria in both soil portions (upper and lower) from the soil microcosm types (W, LAS10 and LAS50) at each incubation time was determined by the plate count method. For this, 1 g of soil from each homogenised sample (from upper and lower portions) was diluted in 9 ml of
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sterile saline solution (0.9% NaCl, w/v) and mixed thoroughly on a magnetic stirrer. Standard serial dilutions followed and 100 ml aliquots of each dilution were spread on plates of 1/10 diluted tripticase soy agar (TSA) medium (Oxoid, Basingstoke, Hampshire, England) (Avidano et al., 2005). Plates were incubated at 28 C in the dark for 3 days under aerobic conditions and then checked visually. All the counts were made in triplicate. 2.3.2. Enzymatic activities The determination of enzymatic activities in the homogenised soil samples taken from both upper and lower portions of each microcosm type (W, LAS10 and LAS50) was done in all cases according to the methods described by Tabatabai (1982). For acid phosphatase, 1 g of sieved soil (less than 2 mm) was combined with 2.0 ml of toluene, 4.0 ml pH 6.5 modified universal buffer and 1.0 ml of p-nitrophenol phosphate solution (0.025 M) for 1 h at 37 C. The p-nitrophenol released was determined colorimetrically at 410 nm using a Hitachi U-2000 (Tokyo, Japan) spectrophotometer, and compared with a standard. Alkaline phosphatase was analyzed in a similar manner, except that the modified universal buffer was adjusted to pH 11.0 using 0.1 N NaOH. For the analysis of arylsulphatase, 1 g of sieved soil (less than 2 mm), 0.25 ml toluene, 4.0 ml acetate buffer (0.5 M, pH 5.8) and 1.0 ml of p-nitrophenyl sulfate (0.025 M in acetate buffer) were mixed and incubated at 37 C. After 1 h, the reaction was stopped by the addition of 1 ml of 0.5 M CaCl2 and 4.0 ml of 0.5 M NaOH and filtered through a Whatman no. 42 filter paper. The p-nitrophenol released was measured colorimetrically at 410 nm. For the dehydrogenase activity, 20 g of dried sieved soil was combined with 0.2 g CaCO3. Three 6 g portions of mixture were incubated at 37 C with 1.0 ml 3% (w/v) 2,3,5-triphenyltetrazolium chloride and 2.5 ml distilled water. After 24 h, the soils were removed from incubation, mixed with 10 ml of reagent grade methanol and quantitatively filtered through absorbent cotton. The red color resulting from the production of triphenylformazan by soil dehydrogenase was washed from the cotton into a 100 ml volumetric flask using 10 ml portions of methanol, and the flask was brought to volume with additional methanol. The triphenylformazan was determined colorimetrically at 485 nm and compared with a standard curve. 2.3.3. Chemical determinations LAS 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 Nimer et al. (2007) and using an Agilent Technologies (Palo Alto, CA, USA) 1100 series HPLC equipment. Phosphorous (as P-PO3 4 ) concentration was determined in the leachate from soil microcosms W, LAS10 and LAS50 throughout the experiments following the spectrophotometric procedure described in Rodier (1989) and using a Hitachi U-2000 (Tokyo, Japan) spectrophotometer.
3. Results 3.1. LAS in soil samples Once removed from the glass columns, soil samples from each soil microcosms (two samples by column, corresponding to the upper and lower portions) were preserved by the immediate addition of 3% (v/v) formaldehyde until their analysis, following the methodology previously described in Nimer et al. (2007). Table 1 shows the concentration of LAS (mg kg1 soil) in each soil column portion at each sampling time. The surfactant concentrations were significantly higher (p < 0.05) in the sterile soil columns (SLAS10 and SLAS50) when compared with the non-sterile ones (LAS10 and LAS50). Moreover, in all cases, the highest LAS concentrations were always found in the upper soil column layer. 3.2. Cultivable heterotrophic bacteria in samples from soil columns Fig. 2 shows the cultivable aerobic heterotrophic bacterial counts (as log CFU g1) in soil samples from upper and lower portions of LAS10 (Fig. 2A) and LAS50 (Fig. 2B) microcosms. Bacterial counts from W microcosms (taking into account as control soil microcosms) were also included. Bacterial counts were significantly higher (p < 0.05) in the microcosms amended with LAS (LAS10 and LAS50) than in the W microcosms (Fig. 2), and the counts from LAS50 were significantly higher (p < 0.05) than those from LAS10 in the upper portion of the soil columns. Maximum bacterial counts in the LAS50 microcosms were recorded during the third experiment day (Fig. 2B), earlier than in the case of the LAS10 microcosms, where maximum bacterial counts were recorded during the 14th experiment day (Fig. 2A). Finally, bacterial counts were significantly higher (p < 0.05) in the upper portion than in the lower portion in all the studied microcosms. 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 dehydrogenase) in soil microcosms amended with sterile solutions containing 10 mg l1 (A) and 50 mg l1 (B) of LAS both in upper and lower portions of soil columns. The values of the soil enzymatic activities in soil microcosms amended with sterile distilled water (W) are included as controls. Table 1 LAS concentration (mg kg1 soil) in each soil microcosm and soil portion, at different sampling times. Soil microcosm type
Running time (days)
LAS10
7 14 21
1.4 9.2 0.7
0.3 3.1 0.6
SLAS10
7 14 21
13.3 22.1 30.0
0.5 0.8 1.2
LAS50
3 7 21
35.1 70.8 20.4
0.5 0.4 9.6
SLAS50
3 7 21
53.1 83.8 284.5
0.6 12.2 84.7
2.4. Statistical analysis One-way analysis of variance (ANOVA) was performed using the software package Statgraphics 3.0 Plus version (STSC Inc., Rockville, MD, USA) in order to identify the effect of different LAS concentrations on bacterial counts and soil enzyme activities. A significance level of 95% (p < 0.05) was selected. In addition, to evaluate the correlation among cultivable heterotrophic bacteria and enzymatic activities, a matrix of Pearson’s linear correlation coefficients was made using the same statistical package.
71
Upper portion (mg kg1 soil)
Lower portion (mg kg1 soil)
LAS10: soil microcosm amended with 10 mg l1 LAS solution. LAS50: soil microcosm amended with 50 mg l1 LAS solution. SLAS10: sterile soil microcosm amended with 10 mg l1 LAS solution. SLAS50: sterile soil microcosm amended with 50 mg l1 LAS solution.
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72
B
8
Upper portion
Log CFU/g dry soil
Log CFU/g dry soil
A
*
7.5 7 6.5
LSD (0.05)
6 5.5 0
7
14
8
*
*
*
7 6.5
LSD (0.05)
6 5.5
21
Upper portion
7.5
0
3
6
9
Days 8
Lower portion 7.5
*
7 LSD (0.05)
6 5.5 0
7
14
21
Log CFU/g dry soil
Log CFU/g dry soil
8
6.5
15
18
21
Lower portion
7.5
*
7 6.5
LSD (0.05)
6 5.5
0
3
6
9
Days LAS10
12
Days
12
15
18
21
Days W
LAS50
W
Fig. 2. Cultivable aerobic heterotrophic bacteria counts (as log CFU g1) in soil samples from upper and lower portions of LAS10 microcosms (A) and LAS50 microcosms (B). Bacterial counts 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 * are significantly different from control (p 0.05).
µg PNP-released/g soil/h
A
250 W
200
LAS10
150
100
50
0 0
7
14
21
B
In order to test the influence of LAS on nutrient availability and, as a consequence, on bacterial population and enzyme activities,
7
14
21
7
21
250 W
LAS50
200
150
100
50
0 0
3
7
21
0
3
Days upper portion
3.4. Phosphorous leachate analysis
0
Days
g PNP-released/g soil/h
As a general rule, the application of the anionic surfactant to soil at the different concentrations assayed significantly increased the mean values of extracellular enzymes (acid and alkaline phosphatases and arylsulphatase) both in the upper and lower portions of the soil columns with respect to the control microcosms, exception made for the acid phosphatase (lower portion) and the alkaline phosphatase (upper and lower portions) of LAS10 microcosm, where a positive but not significant trend was detected. By contrast, the dehydrogenase activity was always significantly decreased by the presence of LAS, when it was compared with control soil samples (W). Table 2 summarizes the results of an analysis of variance for each enzyme activity measured as affected by the different LAS treatment applied (LAS10 and LAS50). Significance level was generally higher for the LAS50 than for the LAS10 treatment. To evaluate the relationships between enzymatic soil activities and heterotrophic soil bacterial counts in each microcosm soil portion, a correlation matrix (Pearson’s linear correlation coefficients) was constructed (Table 3). A positive and statistically significant linear correlation (p < 0.05) existed between both phosphatase and arylsulphatase activities and aerobic heterotrophic soil bacterial counts in microcosms amended with sterile solutions containing 10 mg l1 (LAS10) and 50 mg l1 (LAS50) of LAS. These correlations showed always greater coefficient values in the upper portion of the microcosm than in the lower one. On the other hand, the correlation coefficients between the dehydrogenase activity and aerobic heterotrophic soil bacteria counts were always negative and higher in the upper portion of the microcosms than in the lower one. Finally, it is worth noting that in the microcosms without LAS (W) the counts of heterotrophic aerobic bacteria (as log CFU g1) were only positively and statistically correlated to the alkaline phosphatase activity.
lower portion
Fig. 3. Alkaline phosphatase activity (mg PNP released g1 wet soil h1 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 and standard deviations (SD) of three replicates.
M. Sa´nchez-Peinado et al. / Soil Biology & Biochemistry 41 (2009) 69–76
A
100 90 80 70 60 50 40 30 20 10 0
W
0
7
LAS10
14
21
0
7
14
21
Days
50
W
LAS10
40 30 20 10 0
100 90 80 70 60 50 40 30 20 10 0
0
7
14
21
0
7
14
21
Days W
LAS50
B
70
60
0
3
7
21
0
3
7
21
Days upper portion
lower portion
Fig. 4. Acid phosphatase activity (mg PNP released g1 wet soil h1 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 and standard deviations (SD) of three replicates.
g PNP released/g soil/h
g PNP-released/g soil/h
B
70 60
g PNP released/g soil/h
g PNP-released/g soil/h
A
73
LAS50
W 50 40 30 20 10
phosphorous (as P-PO3 4 ) concentration was regularly determined in the microcosms leachates. Fig. 7 shows the phosphate concentrations detected in the leachate of the soil microcosms LAS10, LAS50 and W during the running experiment time. The dynamics of these 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 soil microcosm LAS10 was significantly higher than in LAS50 and W, and the minimum phosphates concentration was recorded in the LAS50 leachate throughout the whole experiment time.
0 0
3
7
21
0
3
7
21
Days upper portion
lower portion
Fig. 5. Arylsulphatase activity (mg PNP released g1 wet soil h1 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 value and standard deviations (SD) of three replicates.
4. Discussion Among the parameters revealing information on the quality of a soil, those relative to its biological composition and metabolic activity display a greater sensitivity to contamination processes (Garcı´a et al., 2003). Since the physical and chemical characteristics of a soil can be considered stable, any impact in a soil is detected initially by significant variations in its biological dynamics. The soil constitutes a biological system in which enzymatic activities play an important role in its biochemical status (Overbeck, 1991; Stryler, 1995). The measurement of the soil enzyme activities allows inferring the quality of the system, and is commonly used as an indicator of soil microbial activity (Sinsabaugh, 1994; Garcı´a et al., 2003). In addition, it acts as a good indicator of changes in the properties of the soil induced by the anthropogenic addition of compounds (Lobo et al., 2000). The lower concentrations of LAS detected in the non-sterile soil columns when compared with the sterile ones demonstrated the biotransformation process of the surfactant. This process reached its highest intensity after 14 days of experimentation in the case of
soil columns continuously amended with 10 mg l1 of surfactant, and after 7 days when the soil columns were amended with 50 mg l1 of LAS. On the other hand, the greatest proportion of the surfactant was retained in the upper portion of the columns (whether sterile or non-sterile). Previous studies (Jacobsen et al., 2004) revealed the same distribution pattern of the surfactant in experimental soil columns, with maximum concentrations registered in the first 15 cm, and showing a significant transport towards the lower layers. In our experiments, the lower concentrations of LAS detected in the deep layers of the microcosm may be due to the faster biotransformation process of the surfactant and to the higher retention of the surfactant in the upper layers of the soil column. The first LAS degradation homologue detected in the leachate was C10 (data not shown) because the longer chain homologues are more easily biodegradable when they are in the interstitial water, and once adsorbed to soil particles they are retained with greater force than the short chain homologues. Similar results were obtained by Jacobsen et al. (2004).
´ nchez-Peinado et al. / Soil Biology & Biochemistry 41 (2009) 69–76 M. Sa
74
Table 3 Pearson’s correlation coefficients between heterotrophic aerobic bacteria counts and enzymatic activities in each soil microcosm and portion sampled.
g TPF released/g soil/day
A 90 80
LAS10
W
Acid phosphatase
70 60 50 40 30 20 10 0 0
7
14
21
0
7
14
21
Days
Arylsulphatase
Dehydrogenase
LAS10 Upper portion Lower portion
0.82* 0.27
0.94* 0.61*
0.89* 0.63*
0.84* 0.73*
LAS50 Upper portion Lower portion
0.58* 0.58*
0.97* 0.75*
0.84* 0.75*
0.59* 0.27
0.83* 0.65*
0.54 0.47
0.18 0.31
W Upper portion Lower portion
0.05 0.06
*Significant at p 0.05. LAS10: soil microcosm amended with 10 mg l1 LAS solution. LAS50: soil microcosm amended with 50 mg l1 LAS solution. W: soil microcosms amended with sterilised distilled water (control).
B g TPF released/g soil/day
Alkaline phosphatase
90
LAS50
W
80 70 60 50 40 30 20 10 0
0
3
7
21
0
3
7
21
Days upper portion
lower portion
Fig. 6. Dehydrogenase activity (mg TPF released g1 dry weight day1 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 and standard deviations (SD) of three replicates.
The data obtained from agricultural soil microcosms have revealed that the enzymatic activities acid phosphatase, alkaline phosphatase and arylsulphatase were correlated positively with the bacterial counts under our experimental conditions. These results are in accordance with those previously reported by Nannipieri et al. (1979), Criquet et al. (2002) and Ne´ble et al. (2007). The values of phosphatase and arylsulphatase activities obtained in soil microcosms LAS10 were always higher than those obtained in control microcosms (W), although these differences were not statistically significant for the cases of acid phosphatase activity (lower portion) and alkaline phosphatase activity (upper and lower portions). On the contrary, the application of greater concentrations of LAS in the influent (50 mg l1) generated significantly higher values of phosphatase and arylsulphatase activities, with temporal distribution patterns characterized by earlier and more intense maximums of activity. While previous studies (Elsgaard et al., 2001a) suggested the absence of response of the arylsulphatase activity to LAS addition, our results have shown that the value of this enzymatic activity is increased by the presence of 10 or 50 mg l1 of this anionic surfactant. We suggest that a limitation of the bacterial growth by bio-available forms of sulphur in the continuously washed
agricultural soil microcosms assayed in our study could be the reason for this opposed behaviour. Thus, arylsulphatase activity could be involved in the breakage of the LAS benzene–S bond and in the release of S molecules to the medium, allowing the microorganisms not only to cover their requirements of a bio-available S form (Dodgson et al., 1982) but also to collaborate significantly in the biotransformation of the LAS molecule (Kertesz et al., 1994). In fact, several strains of Pseudomonas spp. (potentially capable to produce extracellular sulphatases, Gonza´lez et al., 2003) have been previously described as important members of LAS biotransformation consortia (Swisher, 1987; Jimenez et al., 1991). Indeed, several recent studies (Vong et al., 2002; Gonza´lez et al., 2003) have been carried out to investigate the possibility to use bacteria to increase the mobilisation of the different forms of S-organic in soils and to ‘‘in situ’’ accelerate the S biogeochemical cycle, in order to improve the fertility of agricultural soils. Under our experimental conditions (oligotrophic systems), the continuous leaching of nutrients from soil (either dissolved in the water and/or emulsified by the anionic surfactant), as well as the depletion of the nutritional resources in the soil as a consequence of microbial growth, could have affected the biological activities of the soil (Barber, 1995; Hartemink, 2005). In fact, our results show that the phosphate concentration detected in the leachate of the LAS50 microcosm was significantly lower than the concentrations detected in LAS10 leachates throughout the experiment. The counts of cultivable aerobic heterotrophic bacteria in soil samples from the LAS50 microcosms were significantly higher than those from LAS10, and consequently it could be suggested that the phosphate consumption was higher too. Thus, under these circumstances, the bacterial populations in this soil microcosm type may respond by increasing the extracellular phosphatase activity (mainly acid phosphatase) to obtain readily assimilable 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 phosphate concentration throughout the period of study was shown by the positive and statistically significant correlations found between the number of cultivable heterotrophic bacteria and the phosphatase activity (mainly alkaline phosphatase) in the W microcosm. No
Table 2 Summary of the results of an analysis of variance (ANOVA) for each of the enzyme activities measured, as affected by the different LAS treatment applied (LAS10 and LAS50). Acid phosphatase
LAS10 LAS50
Alkaline phosphatase
Arylsulphatase
Upper portion
Lower portion
Upper portion
Lower portion
Upper portion
Lower portion
Upper portion
Lower portion
p < 0.01 p < 0.05
NS p < 0.001
NS p < 0.05
NS p < 0.001
p < 0.001 p < 0.001
p < 0.01 p < 0.01
p < 0.05 p < 0.05
p < 0.01 p < 0.001
NS: not significant (p > 0.05).
Dehydrogenase
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75
approach constitutes a useful tool for preliminary studies on the effects of LAS on soils’ biological properties.
1200
P-PO4 (µg/l)
1000
5. Conclusions
800 600 400 200
W
LAS10
LAS50
0 3
6
9
12
15
Days Fig. 7. Phosphorous concentrations (as P-PO4) in leachate samples from LAS10 and LAS50 microcosms during the running time. Data from W microcosms (control) were included too. Data are average values and standard deviations (SD) of three replicates.
other enzymatic activities were positively and significantly related to the numbers of cultivable heterotrophic bacteria in this microcosm. Nevertheless, it is worthy to note that these counts only represent a small part of total soil bacteria. Arylsulphatase and phosphatase activities are considered ‘‘specific parameters’’ since the enzymes catalyse concrete reactions and are mediated by certain bacterial groups which depend on specific substrates. On the contrary, the dehydrogenase activity is considered ‘‘a general’’ determination, as its measurement allows to evaluate the microbial metabolic processes that take place in the soil under a global view (Tabatabai, 1982; Garcı´a et al., 2003). Our results demonstrate that the dehydrogenase activity was progressively inhibited in the LAS10 and LAS50 soil microcosms, and that a statistically negative correlation existed between this enzymatic activity in the upper portion of the soil columns and the counts of aerobic heterotrophic microorganisms, while no significant correlations were detected between these parameters in control soil microcosms (W). Previous studies (Malkomes and Wo¨hler, 1983; Elsgaard et al., 2001b) demonstrated a similar inhibition pattern of the dehydrogenase activity in the presence of increasing concentrations of LAS. These results suggest a certain negative effect of LAS on soil microbial processes although the presence of this xenobiotic can favour other more specialised enzymatic processes, mediated by specific groups of microorganisms (Van Straalen and Van Gestel, 1993). Margesin and Schiner (1998) reported that the measurement of the dehydrogenase activity could be considered as a particularly appropriate method for the monitoring of soil contamination by anionic surfactants. In addition, this study demonstrated that the presence of another anionic surfactant (SDS) significantly inhibited the dehydrogenase activity of an agricultural soil, and this enzymatic activity only increased when 90% of degradation of surfactant was achieved. This could be explained by the fact that SDS may alter microorganism membrane structures within which dehydrogenases are located. Similarly, Schwuger and Bartnik (1999) described a number of diverse effects of LAS on cellular membrane structures as a consequence of its surface activity. In recent years, considerable interest has developed in the use of microcosm techniques for ecotoxicological assays (Edwards et al., 1997, 1998; Burrows and Edwards, 2000). Such microcosms have usually consisted of units containing intact or mixed soil supporting indigenous multiple biotic species, and have ranged in size from a few grams of soil to cores as large as a meter in diameter. Although field observations are needed for realistic calibration and validation of the information, our integrated in vitro microcosm
Soil enzymatic activities, in particular dehydrogenase activity, have been suggested as appropriate parameters for the monitoring of soil contamination. Using agricultural soil microcosm systems, we conclude that the continuous applications of different LAS solutions at 10 and 50 mg l1 concentrations for periods of time up to 21 days significantly decrease the dehydrogenase activity, while other enzymatic activities catalysed by specific bacterial groups (alkaline phosphatase, acid phosphatase and arylsulphatase) were significantly increased. Therefore, a statistically negative correlation between dehydrogenase activity and the number of heterotrophic aerobic microorganisms was observed, suggesting an inhibition of the soil dehydrogenase activity in response to the addition of increasing concentrations of LAS in agricultural soils. LASs also induce the alteration of the phosphorous turn-over by increasing its bioavailability. Further studies are needed in order to evaluate 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 Educacio´n y Ciencia (MEC), as part of Project Reference PPQ2003-07978V02-02 (Programa Nacional de IþD). B.R. was supported by Programa Ramo´n y Cajal (MEC, Spain). C. Pozo was supported by Programa Retorno de Doctores (Junta de Andalucı´a. Spain).
References Avidano, L., Gamalero, E., Cossa, G.P., Carrazo, E., 2005. Characterization of soil health in an Italian polluted site by using microorganisms as bioindicators. Applied Soil Ecology 30, 21–33. Bandick, A.K., Dick, R.P., 1999. Field management effects on soil enzyme activities. Soil Biology & Biochemistry 31, 1471–1479. Barber, S.A., 1995. Soil Nutrient Bioavailability. A Mechanistic Approach, second ed. Wiley-Interscience, New York, 384 pp. Bremmer, J.M., 1965. Inorganic forms of nitrogen. Agronomy 9, 1179–1237. Burrows, L., Edwards, C.A., 2000. The fate and environmental effects of the fungicide carbendazim in an innovative terrestrial microcosm system. In: Proceedings of the Brighton Crop Protection Conference on Pests and Diseases, 4C-2, pp. 365–370. Carlsen, L., Metzon, M.B., Kjelsmark, J., 2002. LAS in the terrestrial environment. Science of the Total Environment 290, 225–230. CESIO, Comisio´n europea de surfactantes y sus intermediarios orga´nicos, 2006. General Assembly. Santiago de Compostela, Spain. Criquet, S., Tagger, S., Vogt, G., Le Petit, J., 2002. Endoglucanase and beta-glycosidase activities in an evergreen oak litter: annual variation and regulating factors. Soil Biology & Biochemistry 34, 1111–1120. Criquet, S., Ferre, E., Farnet, A.M., Le Petit, J., 2004. Annual dynamics of phosphatase activities in an evergreen oak litter: influence of biotic and abiotic factors. Soil Biology & Biochemistry 36, 1111–1118. Del Olmo, M., Garballo, A., Nimer, M., Lo´pez, I., De Ferrer, J.A., Vı´lchez, J.L., 2004. Development of methodologies for different degrees of resolution of linear alkylbenzene sulfonates in groundwater and wastewater by liquid chromatography using sodium dodecyl sulphate. Chromatographia 60, 157–164. Dick, R.P., 1997. Soil enzyme activities as integrative indicators of soil health. In: Pankhurst, C.E., Doube, B.M., Gupta, V.V.S.R. (Eds.), Biological Indicators of Soil Health. CAB International, Wellingford, pp. 121–156. Dodgson, K.S., White, G.F., Fitzgerald, J.W., 1982. Sulfatases of Microbial Origin. CRC Press, Boca Rato´n, Florida, USA, 199 pp. Edwards, C.A., Knacker, T.T., Pokarzhevski, A.A., Subler, S., Parmelee, R., 1997. The use of soil microcosms in assessing the effects of pesticides on soil ecosystems. In: Proceedings of the International Symposium on Use of Nuclear and Related Techniques for Studying Environmental Behaviour of Crop Protection Chemicals. Vienna, Austria, pp. 1–23. Edwards, C.A., Knacker, T., Pokarzhevskii, A.A., 1998. The prediction of the fate and effects of pesticides in the environment using tiered laboratory terrestrial model ecosystems. In: Proceedings of the Brighton Crop Protection Conference on Pests and Diseases, 4C-1, pp. 267–272.
76
´ nchez-Peinado et al. / Soil Biology & Biochemistry 41 (2009) 69–76 M. Sa
Elsgaard, L., Petersen, S.O., Debosz, K., Kristiansen, I.B., 2001a. Effects of linear alkylbenzene sulfonates (LAS) on soil microbiology. Tenside Surfactants Detergents 38, 94–97. Elsgaard, L., Petersen, S., Debozs, K., 2001b. Effects and risk assessment of linear alkylbenzene sulfonates in agricultural soil. 1. Short-term effects on soil microbiology. Environmental Toxicology and Chemistry 20, 1656–1663. Garcı´a, C., Gil, F., Herna´ndez, T., Trasar, C., 2003. Te´cnicas de ana´lisis de para´metros bioquı´micos en suelos: medida de actividades enzima´ticas y biomasa microbiana. Ediciones Mundi-Prensa, Madrid, 371 pp. Gonza´lez, S., Navarro, M., Lo´pez, J.I., Rad, C., 2003. Determinacio´n de la actividad arilsulfatasa del suelo. In: Garcı´a, C., Gil, F., Herna´ndez, T., Trasar, C. (Eds.), Te´cnicas de ana´lisis de para´metros bioquı´micos en suelos: medida de actividades enzima´ticas y biomasa microbiana. Ediciones Mundi-Prensa, Madrid, pp. 79–99. Hartemink, A.E., 2005. Nutrient stocks, nutrient cycling and soil changes in cocoa ecosystems – a review. Advances in Agronomy 86, 227–253. Jacobsen, A.M., Mortensen, G.K., Hansen, H.C.B., 2004. Degradation and mobility of linear alkylbenzene sulfonate and nonylphenol in sludge-amended soil. Journal of Environmental Quality 33, 232–240. Jensen, J., Løkke, H., Holmstrup, M., Krogh, P.H., Elsgaard, L., 2001. Effects and risk assessment of linear alkylbenzene sulfonates in agricultural soil. 5. Probabilistic risk assessment of linear alkylbenzene sulfonates in sludge-amended soils. Environmental Toxicology and Chemistry 20, 1690–1697. Jensen, J., Smith, S.R., Krogh, P.H., Versteeg, D.J., Temara, A., 2007. European risk assessment of LAS in agricultural soils revisited: species sensitivity distribution and risk estimates. Chemosphere 69, 880–892. Jimenez, L., Breen, A., Thomas, N., Federle, T.W., Sayler, G.S., 1991. Mineralization of linear alkylbenzene sulfonate by a 4-member aerobic bacterial consortium. Applied and Environmental Microbiology 57, 1566–1569. Kertesz, M.A., Cook, A.M., Leisinger, T., 1994. Microbial metabolism of sulphur- and phosphorous-containing xenobiotics. FEMS Microbiology Reviews 15, 195–215. Lobo, M.C., Sastre, I., Vicente, M.A., 2000. Las enzimas como medida del impacto ambiental en los suelos. In: Garcı´a, C., Herna´ndez, M.T. (Eds.), Investigacio´n y ˜ a. CEBAS-CSIC, Murcia, pp. perspectivas de la enzimologı´a de suelos en Espan 297–352. Malkomes, H.P., Wo¨hler, B., 1983. Testing and evaluating some methods to investigate side effects of environmental chemicals on soil microorganisms. Ecotoxicology and Environmental Safety 7, 284–294. Margesin, R., Schiner, F., 1998. Biodegradation of the anionic surfactant sodium dodecyl sulphate at low temperatures. International Biodeterioration and Biodegradation 41, 139–143. Mortensen, G.K., Elsgaard, H., Ambus, P., Jensen, E.S., Groen, C., 2001. Influence of plant growth on degradation of LAS in sludge-amended soil. Journal of Environmental Quality 30, 1266–1270. Nannipieri, P., Pedrazzini, F., Arcaa, P.G., Piovanelli, C., 1979. Changes in amino acids, enzyme activities, and biomass during soil microbial growth. Soil Science 127, 26–34. Natural Resources Conservation Service, 1999. Soil taxonomy, a basic system of soil classification for making and interpreting soil surveys. Agriculture Handbook, vol. 436, second ed. Washington, DC, USA, 869 pp.
Ne´ble, S., Calvert, V., Le Petit, J., Criquet, S., 2007. Dynamics of phosphatase activities in a cork oak litter (Quercus suber L.) following sewage sludge application. Soil Biology & Biochemistry 39, 2735–2742. Nimer, M., Ballesteros, O., Navalo´n, G., Crovetto, C., Verge, C., Lo´pez, I., Berna, J.L., 2007. New simple treatment for determination of linear alkylbenzene sulfonate (LAS) in agricultural soils by liquid chromatography with fluorescence detection. Analytical and Bioanalytical Chemistry 387, 2175–2184. Olsen, S.R., Dean, L.A., 1965. Phosphorous. In: Page, A.L., Miller, R.H., Keeney, D.R. (Eds.), Methods of Soil Analysis: Part 2. Chemical and Microbiological Properties. American Society of Agronomy, Soil Science Society of America, Madison, WI, USA, pp. 1035–1049. Overbeck, J., 1991. Early studies on ecto- and extracellular enzymes in aquatic environments. In: Chro´st, R.J. (Ed.), Microbial Enzymes in Aquatic Environments. Springer, New York, pp. 1–5. Penteado, J.C.P., Seoud, O.A.E., Carvalho, L.R.F., 2006. Linear alkylbenzene sulfonates: chemistry, environmental impact and analysis. Quimica Nova 29, 1038–1046. Pratt, P.F., 1954. Potassium release from exchangeable and non-exchangeable forms in soils. Ohio Agricultural Experiment Station Resource Bulletin 47, 747. Prats, D., Lopez, C., Vallejo, D., Varo, P., Leon, V.M., 2006. Effect of temperature on the biodegradation of linear alkylbenzene sulfonate and alcohol ethoxylate. Journal of Surfactants Detergents 9, 69–75. Rodier, J., 1989. Ana´lisis de Aguas. Ed. Omega, Barcelona, Spain, pp. 1059–1060. Sa´nchez-Peinado, M., Gonza´lez-Lo´pez, J., Rodelas, B., Galera, V., Pozo, C., Martı´nezToledo, M.V., 2008. Effect of linear alkylbenzene sulfonates on the growth of aerobic heterotrophic cultivable bacteria isolated from an agricultural soil. Ecotoxicology 17, 549–557. Schwuger, M.J., Bartnik, F.G., 1999. Interaction of anionic surfactants with proteins, enzymes and membranes. In: Gloxhuber, C. (Ed.), Anionic Surfactants – Biochemistry, Toxicology, Dermatology. Marcel Dekker Inc., New York, USA, pp. 1–49. Sinsabaugh, R.L., 1994. Enzymic analysis of microbial pattern and process. Biology and Fertility of Soils 17, 69–74. Solbe`, J., Berna, J.L., Cavalli, L., Feitjtel, T.C.J., Fox, K.K., Heinze, J., Marshall, S.J., de Wolf, W., 2000. Terrestrial risk assessment of LAS in sludge-amended soils. In: 5th World Cesio Congress, vol. 2, pp. 1433–1438. Speir, T.W., Ross, D.J., 1978. Soil phosphatase and sulphatase. In: Burns, R.G. (Ed.), Soil Enzymes. Academic Press, London, pp. 197–250. Stryler, L., 1995. Biochemistry, fourth ed. Freeman and Company, New York, 632 pp. Swisher, R.D., 1987. Surfactants Biodegradation, second ed. Marcel-Dekker Inc., New York, USA, 1085 pp. Tabatabai, M.A., 1982. Soil enzymes. In: Page, A.L., Miller, R.H., Keeney, D.R. (Eds.), Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties, second ed. Soil Science Society of America, Inc., Madison, WI, USA, pp. 902–947. Van Straalen, N.M., Van Gestel, C.A.M., 1993. Soil invertebrates and micro-organisms. In: Calow, P. (Ed.), Handbook of Ecotoxicology, vol. 1. Blackwell Scientific, Oxford, U.K., pp. 251–277. Vong, P.-C., Lasserre-Joulin, F., Guckert, A., 2002. Mobilization of labelled organic sulphur in rhizosphere of rape and barley and in non-rhizosphere soil. Journal of Plant Nutrition 25 (10), 2191–2204.