Discriminating inhibitory from enhancing effects in respirometry assays from metal polluted-sewage sludge amended soils

Applied Soil Ecology 34 (2006) 52–61 www.elsevier.com/locate/apsoil

Discriminating inhibitory from enhancing effects in respirometry assays from metal polluted-sewage sludge amended soils G. Montserrat a, E. Martı´ b,*, J. Sierra b, M.A. Garau b, R. Cruan˜as b A`rea de Microbiologia, Facultat de Cie`ncies, Universitat de Girona, Spain Laboratori d’Edafologia, Facultat de Farma`cia, Universitat de Barcelona, Av Joan XXIII s.n. 08028, Barcelona, Spain a

b

Received 8 March 2005; received in revised form 20 November 2005; accepted 19 December 2005

Abstract A respirometric experiment was used to assess the toxicity of a toxic element polluted-sewage sludge on two soil samples (SN and SO) differing, mainly, in their organic composition, texture and carbonate content. The essayed rates were 1.67, 3.34, 8.35 and 16.7% (dry weight basis), corresponding to total Zn, Cr and Ba concentrations ranging from 371, 105 and 514 to 3711, 1054 and 51.4 mg kg
1, respectively. The experiment consisted of the incubation of the amended soil samples under controlled temperature and humidity conditions, followed by the observation of both, daily and cumulative basal respiration curves, and the comparison of the curves parameters from the fitted exponential mathematical models. Water-soluble potentially toxic elements concentration was determined at the end of the incubation period to confirm the reason of the toxicity differences found in the respirometric tests. All the sludge amended samples showed an increase of soil respiration compared to the unamended control, being positively related to the waste rate. A respiration inhibition appeared later (4 and 20 days in SN and SO soils, respectively, for the lower dose) and the time of delay depended on the waste rate used, being larger as waste dose increased. The toxicity level differed in the two soils essayed, mainly due to their basal microbial activity, and to their different properties, which determine the released elements bioavailability. Water-soluble elements determination at the end of the incubation revealed the presence of soluble Zn, Cr and Ba in different concentrations in the two soils. For instance, Zn concentration was, at the highest application dose, of 131 and 23 mg kg
1, for SN and SO, respectively. So, the sludge toxicity was best observable at low rates and at incubation time periods long enough to allow the mineralisation of the waste oxidable organic matter. # 2006 Elsevier B.V. All rights reserved. Keywords: Soil respiration; Basal respiration; Potentially toxic elements; Zn; Cr; Ba; Sewage sludge; Soil toxicity

1. Introduction The monitoring of soil contamination can be performed by both microbiological and chemical methods, although these latter alone cannot provide criteria about bioavailability of the pollutants, and so, they should be less suitable for environmental purposes (Allen and Yin, 1998). The use of bioassays allows the assessment of the

* Corresponding author. Tel.: +34 93 402 4494; fax: +34 93 402 4495. E-mail address: [email protected] (E. Martı´). 0929-1393/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2005.12.005

ecotoxicological effects of pollutants in soils, and some of them have been accepted as national and international standards (Filzek et al., 2003). Two of the most important microbial processes that take place fundamentally in the soil environment are carbon and nitrogen cycles. The assessment of the effects of soil pollution at least has to consider, among others, these two main processes, which must remain as operational as possible in order to keep a good ecological equilibrium (Brohon et al., 2001; Zak et al., 1994). Carbon mineralisation can be measured by the respiratory activity; its determination consists on the evaluation of CO2 release or O2 consumption due to

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the aerobic and heterotrophic soil microbial activity. Respiration has been described as one of the key soil indicators for soil quality assessment (Eisentraeger et al., 2000; Arshad and Martin, 2002; Hollender et al., 2003). It can be performed on unamended soils (basal respiration) or on easily degradable substrate-amended soils (substrate induced respiration, SIR). According to some authors (Van Beelen and Doelman, 1997) basal respiration seems to be less sensitive to metals and organic chemicals, whilst the enhancement of respiration produced by the addition of substrate in SIR assays facilitates the measurement of the effects of the pollutants. For SIR assays, the pre-stabilisation of samples (incubation for native soil organic matter exhaustion) before substrate addition has to be done, in order to neutralize the stimulating effects of oxidable organic matter. Standardised soil microbial activity assessment methods use SIR (OECD, 2000) or a combination of basal and SIR procedures (ISO, 2002). Usually, cumulative respiration in terms of total evolved CO2/consumed O2 are considered for respiration methods, in addition, for SIR plots other parameters like growth rate, respiratory activation quotient or lag time can be used (ISO, 2002). The use of the abovementioned methods usually brings data that show the toxic effect as a decrease in the measured parameter, with some exceptions corresponding to certain type of disturbances of the soil microflora, described as the result of the energetic needs for adaptive purposes, among others (Giller et al., 1998). However, in some cases, also beneficial effects can be observed by means of an increase of the values, for instance when the waste essayed contributes with nutrients or organic matter useful to the microbial development (Sierra et al., 2001; Ortiz and Alcan˜iz, 1994). Moreover, there is the possibility of waste that share both characteristics, by the one hand some toxic components and, by the other, the presence of oxidable carbon and/or other nutrients, especially when both properties are self dependent, for instance a situation where organic matter evolution may modify the toxicants availability. When such materials are brought to a soil its response is hard to assess and, usually, the toxic effect is hidden by an enhancement of the microbial activity due to nutrient contribution. This masking effect loses intensity as mineralisation processes go on. The main aim of this study is the determination of the effect of an industrial, potentially toxic elements polluted-sewage sludge, in controlled laboratory conditions, on the global microbial status of two different soils, reflected by soil basal respiration. Particularly, the study focuses on the different ability and behaviour of

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the soils in front of the waste application due to their distinct characteristics, and also considering the dual behaviour of a waste which brings favourable properties (presence of oxidable organic matter and nutrients) along with negative conditions (potentially toxic elements content). Also, the availability of potentially toxic elements brought by the waste will be addressed and its potential relation with the effect found on microbial activity. 2. Materials and methods 2.1. Soil and waste samples Soil samples used for this study correspond to A horizons that were sieved (2 mm), hermetically closed and stored (4 8C), in order to slow down the microbial activity and keep the initial moisture content. The reference nomenclature of samples, soils classification by Soil Taxonomy System (USDA, 1999) and their main characteristics can be seen in Table 1. The waste used in this work corresponds to the sewage sludge coming from the treatment of sanitary and washing wastewater of a car factory. The waste sample was air-dried, and ground to reduce particle size (<200 mm), to get the best homogeneous mixture with soil samples. Main characteristics and minor elements content are shown in Table 1. Soils and sludge samples were characterised with methods usually used in soil analysis (Sparks et al., 1996), while total PTE concentrations were determined by X-ray fluorescence. From the data shown, some relevant properties of the sludge sample can be pointed out. First, the high organic C and N contents, ammonium and phosphorus concentrations. The waste C/N ratio (C/N 10) is suitable for biodegradation purposes and, also, to avoid nitrogen immobilisation phenomena. Also, there can be found high concentrations of toxic elements, mainly Zn, Cr and Ba, because of the origin of the sludge. 2.2. Experimental procedure Waste sample was added to the two soils at the following rates: 1.67, 3.34, 8.35 and 16.7% (waste/soil, w/w, dry weight basis). Each rate was then mixed thoroughly and distilled water was added in order to achieve the equivalent to 50% of the soil water holding capacity. The identification of the mixtures can be done by the reference of the sample, which is formed by two letters (SN or SO) that identify the soil used, then a letter, which indicates the waste dose, namely: A for 1.67%, B for 3.34%, C for 8.35% and D for 16.7%.

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Table 1 Main physical, physico-chemical and chemical properties of soil and waste samples Parameters

Parent materials Textural class (USDA) pH (1:10, w/v) Electrical conductivity 25 8C (1:10, w/v) dS m
1 CaCO3 (%) Oxidable carbon (%) (Walkley Black) Losses by ignition (%) (560 8C) Kjeldahl N g kg
1 C/N Available P mg kg
1a Ba mg kg
1b Co mg kg
1b Cr mg kg
1b Cu mg kg
1b Mn mg kg
1b Ni mg kg
1b Pb mg kg
1b Zn mg kg
1b

SN

SO

HumicLithic dystroxerept

Typic xerorthent

Metamorphic schists Silt loam 6.0 0.100 n.d. 5.0 16.85 5.08 9.85 25.4 252 7.3 15 17 653 b. d. l. 26 86

Recarbonated ochre silt Silty clay loam 8.0 0.150 19.6 3.3 11.7 4.10 8.05 16.8 113 1.1 43 92 684 22 19 124

Waste

– – 7.3 3.29 22.3 14.2 45.7 13.32 10.66 346.9 3078 26 6311 279 999 99 398 22222

b. d. l., below detection limit. a SN, Osmond–Bray; SO, Olsen–Watanabe. b Elemental analysis by X-ray fluorescence.

The soil–sludge mixtures were prepared by duplicate and were kept at 30 8C in the dark. For basal respirometric experiments, CO2 release was determined according to Anderson (1982). Carbon dioxide evolved from samples was determined daily at the initial phase of incubation and then progressively spaced, reaching 2 weeks periods at the end of incubations, which lasted 100 days. Results are given as mg CO2 per 100 g of dry soil. The data obtained will be shown in this work by means of plots representing cumulative respiration (CR) or equivalent daily respiration (EDR). The latter parameter corresponds to the mean CO2 evolved in each incubation interval (between two successive measurements), and is assigned to the midpoint between the two data collection times (Martı´, 1998). Respiratory activity of the soil–sludge mixtures was compared to that of not amended soil (control soil). Potentially toxic elements availability was determined at the beginning and at the end of the incubation by water extraction 1:20 (w/v) of the mixtures corresponding to the highest and the lowest sludge rates. The concentrations of potentially toxic elements (Cr, Ba and Zn) were quantified in the extracts by ICP-MS.

effects on respiration of waste amended soil. This was done by the modelisation of the respiration process, by simple lineal regression and ulterior analysis of variance, which allowed the comparison of the response of the amended versus control soil. The software used for this purpose was S-plus Version 2000 (Venables and Ripley, 1996). In a previous study, done with the two soils and four different industrial wastes, using controls and four different amendment doses, by duplicate, two models were found which describe the behaviour of daily and cumulative respiration plots (Montserrat, 2000). For equivalent daily respiration the following exponential adjustment was done: EDR ðmg CO2 100 g soil
1 day
1 Þ ¼ a expð
btÞ In this expression the parameter a represents the initial respiration and
b the decrease of the daily respiration rate with time (t). In Fig. 1 the adjustment between experimental and fitted CO2 values can be observed. As it can be seen, the adjustment is much better in low respiration values, which in this curve type correspond to the last period of incubation. For the cumulative curves the following expression was found:

2.3. Statistical analysis and curves adjustment

CR ðmg CO2 100 g soil
1 Þ ¼ cð1
exp
dt Þ

The aim of the statistical analysis in this work was to assess the nature and significance of the observed

It agrees with a kinetic exponential model, usually used to describe mineralisation processes in soils

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ANOVA analysis, in which a comparison was done among all the curves corresponding to mixtures and control soil, for the 95% confidence level. So, for each curve, calculated p-values below 0.05 establish the existence of significant differences among mixtures and control, being the curve described by the values obtained for a and b or c and d (Table 2). 3. Results 3.1. Respirometry

Fig. 1. Experimental (EV) vs. exponential fitted (FV) values of the EDR curves.

Fig. 2. Experimental (EV) vs. exponential fitted (FV) values of the CR curves.

´ lvarez and A ´ lvarez, 2000). The parameter c (A represents the maximum amount of CO2 releasable in infinite time period and d the rate of cumulative respiration increase with time, tending to c. The plots of experimental versus fitted values are shown in Fig. 2. Using these two models to describe the soil respiration behaviour, the assessment of the waste effect was done by

Data from respiratory activity determinations are shown in Figs. 3 and 4. The values correspond to the mean of two duplicates, with mean standard deviation of 2.43 and 3.03, for EDR and CR plots, respectively. Regarding the waste effect assessment, several parameters can be chosen in order to describe the inhibitory effect. First, the final cumulative amount of carbon dioxide released by the mixtures compared with the corresponding control soil (from CR plots). Second, in both, EDR and CR plots, the time needed for each mixture to change its behaviour from enhancing to inhibiting the respiration comparing with the corresponding control soil, so, the point in which the curve for each mixture crosses downwards with the control one. This intersection should mean, for EDR curves, that the respiration rate of the mixture reaches levels below control, and this should correspond, in the CR plots, to a decrease in the slope compared to the control one, by which, in the course of time, the two curves tend to their intersection. Regarding the two different soils, the first observation to be done is their different behaviour. Soil SN has higher respiration rates than soil SO. The final medium cumulative amounts of carbon dioxide released for the control soils are 2572 and 1724 mg CO2 100 g soil
1, respectively. 3.1.1. SN mixtures The respiratory activities of the SN soil-waste mixtures are, at the initial periods, higher than that of

Table 2 Parameters a and b from the adjusted curves of EDR plots for SN mixtures

Control SN SN-A SN-B SN-C SN-D *

a

a
acontrol

pa

b

b
bcontrol

pb

78.62 111.51 154.86 196.84 367.32

– +32.89 +76.24 +118.22 +288.70

– 0.0907 0.0006* <0.0001* <0.0001*

0.0312 0.0806 0.1091 0.0939 0.1215

– +0.0494 +0.0779 +0.0627 +0.0903

– 0.0300* 0.0018* 0.0004* <0.0001*

p-Values <0.05 indicate significant differences for the parameter among mixtures and control.

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Fig. 3. Experimental plots of the EDR and CR respirometric curves for soil SN.

Fig. 4. Experimental plots of the EDR and CR respirometric curves for soil SO.

The values of the fitted curves parameters, a and b, corresponding to EDR plots of SN mixtures are shown in Table 3. Significant differences (indicated by pvalues) among mixtures and control can be found in all the application doses for both parameters (a and b), except for the parameter a in the lower dose mixture. The differences consist of increases in all cases (a
acontrol > 0 and b
bcontrol > 0). Regarding cumulative respiration plots for the same soil mixtures (Fig. 3), the inhibition should be

the control soil, for all the application rates. This enhancement of respiration is directly proportional to the sludge dose, as can be seen in SN EDR plots (Fig. 3). As time passes, the respiratory activity decreases, this decrease is larger for the mixtures than for the control soil. The lower dose of sludge (SN-A) causes a fall of the soil respiration that situates it below that of the control since the 4th day of incubation, as higher doses produce proportional delays in this inhibition (with a maximum of 20 days in the upper dose). Table 3 Parameters c and d from adjusted curves of CR plots for SN mixtures

Control SN SN-A SN-B SN-C SN-D *

c

c
ccontrol

pc

d

d
dcontrol

pd

3072 2154 2447 2933 3293

–
918
625
139 +221

– 0.0005* 0.0151* 0.5594 0.3271

0.020 0.028 0.029 0.037 0.070

– +0.008 +0.009 +0.017 +0.050

– 0.0661 0.0295* <0.0001* <0.0001*

p-Values <0.05 indicate significant differences for the parameter among mixtures and control.

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Table 4 Parameters a and b from adjusted curves of EDR plots for SO mixtures

Control SO SO-A SO-B SO-C SO-D *

a

a
acontrol

pa

b

b
bcontrol

pb

64.57 91.15 97.83 179.4 267.37

– +26.58 +33.26 +114.83 +211.80

– 0.1459 0.0587 <0.0001* <0.0001*

0.0452 0.0695 0.0578 0.0958 0.0816

– +0.0243 +0.0126 +0.0466 +0.0364

– 0.3343 0.5574 0.0317* 0.0441*

p-Values <0.05 indicate significant differences for the parameter among mixtures and control.

in this case, this decrease does not lead to respiration values below the control in the time of the experiment, so, the curves tend to approach that of the control without crossing it. The fitted EDR curve parameters can be observed in Table 4. Significant differences in both a and b values can only be observed for the two upper doses. In this soil, these differences are due only to enhancements of the respiration (a
acontrol > 0). The fitted parameters of the CR curve (Table 5) show no significant differences except for an increase of the respiration in the highest dose.

observable as an intersection between the curve of the mixture and the control one, and consequently as negative values of the rates of final cumulative carbon dioxide released referred to control. As it has been mentioned above, this crossing is obviously preceded by a change in the slopes that lead to lower values in the mixtures compared with the control. The intersection among curves occurs about the 11th and 24th day, respectively, for mixtures SN-A and SN-B. For this soil sample, the values of c and d parameters of the cumulative curve adjustment are shown in Table 3. The maximum carbon dioxide releasable from samples is indicated by the c parameter. The comparisons done among mixtures and control for this parameter indicate significant differences only in the two lowest doses, for which a decrease in the maximum releasable CO2 can be seen (c
ccontrol < 0). On the contrary, for the d parameter, doses B to D show significant differences, all of them being above the control value (difference >0).

3.2. Zn, Cr and Ba availability In order to assess the possible influence of potentially toxic elements (PTE) availability on respiratory activity the concentration of water-soluble Zn, Cr and Ba before and after samples incubation were determined. Table 6 shows these concentrations before and after the incubations of the amended soils. As it is shown, Zn concentration is more than 30-fold higher after than before the incubation, for the mixture SN-D (130.8 mg kg
1) and more than 5-fold higher for SOD (22.64 mg kg
1). Little concentrations of Cr and Ba can also be found after the incubation, increasing their concentrations compared to the initial ones (most being below detection limit). Other elements were discarded in these considerations due to the low concentrations found in the water extracts after incubation, which remained below the detection limits.

3.1.2. SO mixtures For soil SO, the EDR experimental curves (Fig. 4) of doses SO-A and SO-B are very close to control, and only minor differences can be seen, as higher respiration values than the control in the first 3 weeks, with a decrease that situates them slightly below the control in later time periods. For this soil the mixtures with sludge at the two highest concentrations induce an increase in respiration in the first time of the incubation that subsequently tends to a decrease, but, Table 5 Parameters c and d from adjusted curves of CR plots for SO mixtures

Control SO SO-A SO-B SO-C SO-D *

c

c
ccontrol

pc

d

d
dcontrol

pd

2193 1551 1861 1863 2510

–
642
332
330 +317

– 0.4654 0.7101 0.7006 0.7041

0.017 0.8634 0.822 1.276 2.469

– 0.8464 0.8050 1.2590 2.4520

– 0.3993 0.4227 0.2109 0.0159*

p-Values <0.05 indicate significant differences for the parameter among mixtures and control.

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Table 6 Concentrations (mg kg
1) of water-soluble Zn, Cr and Ba in the minimum and maximum concentration mixtures, before and after the incubation process

Zn Cr Ba

Initial SN-A

Initial SO-A

Incubated SN-A

Incubated SO-A

Initial SN-D

Initial SO-D

Incubated SN-D

Incubated SO-D

0.40 N.D. N.D.

0.36 N.D. N.D.

3.61 0.12 1.46

0.45 0.07 1.67

3.80 N.D. 0.07

3.1 N.D. N.D.

130.80 1.24 2.01

22.64 0.41 2.25

N.D., not detected.

4. Discussion 4.1. Respirometry Firstly, an assumption has been done, namely that the waste, because of its organic nature, almost in all cases enhances the respiration of the soil related to the control at least in the first time periods. Generally, the addition of sewage sludge increases the respiration rates proportionally, as its easily degradable organic matter induces an increase of the short term mineralisation process in the soil (Ortiz and Alcan˜iz, 1994). Other reasons for this enhancement might be considered in this case, given the high levels of potentially toxic elements in the waste and the high application rates. By the one hand, those, pointed out by other authors (Ba˚a˚th, 1989; Giller et al., 1998), which explain the increase in respiration rates by partial sterilisation, energy needs for toxicity compensations or adaptations, etc. in the event of toxicity to the microflora. By the other hand, it might be that the sewage sludge should contribute with its own microbial activity to the carbon dioxide evolved. The first explanation can be discarded by the consideration of the respirometric response intensity of the mixtures compared to the control and its clear dependence on the dose. The second question was cleared in previous works (Martı´, 1998), in which an experiment performed with sterilised and not sterilised waste did not show significant differences in the respiration rates. As it can be observed, when the easily oxidable organic matter is exhausting, the respiration of the soil decreases at different velocities, depending on the soil and on the waste rate, reaching with time values below those of the control. Regarding the two soils essayed, the different respiration levels of the non-amended soils are clearly related to the differences in their organic matter content and nature, and in their microbial activity. As the results show, for SN mixtures the initial increases in the respiration rate tend to decrease and, in further incubation time periods, some inhibition effects can be observed. The reason for this delayed toxic effect suffered by the soil microflora could be the concentra-

tions of potentially toxic elements, mainly Zn, Cr and Ba, found in the waste composition. These elements make their effect as the waste organic matter mineralisation goes on, and so when the toxic elements bound to the organic matter are released. Obviously, higher doses of waste produce a greater delay in the toxicity appearance, as there is a larger amount of organic matter to be degraded. So, the organic matter mineralisation obscures, for some time, the toxic effect on respiration caused by the potentially toxic elements brought with the waste. Nutrient limitations may be discarded as alternative reasons of the respiratory inhibition, because the waste N and P supply seems to be enough to keep the C mineralisation ratio shown at the beginning. Regarding the parameters of the fitted curve, the comparison between mixtures and control show the enhance of soil respiration due to the contribution with organic matter by the waste, mainly during the first days of the incubation, and it is represented by the a parameter. As time passes, there is a decrease in the respiration rate, indicated by b. It should be logical that samples that show increased a values also show increased b values in EDR curves, thus describing the trend towards the re-establishment of the control regular respiration values at the end of the experiment. This is the behaviour observed in SN mixtures except for the lowest dose one. In this case there is a significant decrease of the respiration rate (b > 0) but the little differences noted in the a parameter are not significant. So, the organic matter brought by the waste does not cause a significant effect in the initial soil respiration (a), which remains similar to control. However, towards the end of the experiment, the respiration rate in this mixture decreases faster compared with the control soil (shown by a significant positive difference in b), thus, it is indicating the appearance of certain degree of toxicity, and it is only observable in the lowest application dose. For the cumulative curves of SN soil, seemingly, only two of the mixtures behave as inhibitors of respiration: SN 50 and SN 100, in the course of the experiment. Surely, for higher rates this event should take place in larger time periods. This trend may be

G. Montserrat et al. / Applied Soil Ecology 34 (2006) 52–61

predicted on the basis of the decrease in the slope of the curves that occurs towards the end of the incubation period. Also in this case, a toxic effect can be seen in the lower dose, by means of a clear decrease in the final releasable carbon dioxide, not affected significantly by an initial induction due to organic matter (d). The dose of 100 provokes initial enhancement of the respiration, with a subsequent deceleration that conduces to a lower than control estimated maximum carbon dioxide produced, both of them significant. This means that there is a significant inhibitory effect in the two lower doses, despite the initial no-effect or significant stimulation observed in these samples. The two highest application rates only allow seeing the first stimulatory effect (high d values), while an inhibitory effect on final releasable carbon dioxide cannot be observed in the experimental incubation time. In SO mixtures there is neither a significant initial enhancement of the respiration in the two lower doses nor a significant ulterior decrease in respiration related to control that could indicate the existence of some kind of toxicity. The only effect that can be seen is the enhancement of respiration caused by the organic matter contribution, mainly in the upper rates. This is better observable in EDR plots and estimated parameters, as the effects shown by those corresponding to CR are most of them not significant. The response to labile organic matter is lower than in SO. In spite of that, some inhibition can be guessed from c values (Table 5), which are below those of the control, although the differences are not statistically significant ( pc parameters), probably due to the variability among replicates, which enlarges the significance range of the values. This variability is less relevant when EDR plots are considered and also in SN soil. Generally, the time intervals needed to reveal toxicity in CR curves are larger than those in the EDR curves are. This means that EDR plots can indicate toxicity with shorter analysis time. The inhibition seen in the respiratory activity is stronger in SN soil than in SO soil, because of the higher basal biological activity and organic matter content of the first soil sample, along with other physical–chemical properties. This sample has no limitation of organic matter, and so, the organic content brought by the sludge makes relatively little influence on its biological activity, whereas the toxicity given by the potentially toxic elements is the most important variation on its equilibrium. The other soil sample, SO, besides its lower biological activity and organic matter content, shows a notable increase of respiration following the

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amendment with sludge. Also, there is a release of potentially toxic elements, but this soil sample has some characteristics that offer it a better buffering capacity, as high clay and carbonate contents and pH value. As it has been reported above, the soil SO does not show the toxicity of the waste, as the only effect that can be observed is an enhancement of the respiratory activity due to the organic contribution. The properties of this soil allow it to neutralize a possible toxicity effect of the waste applied, at least in the time elapsed in the incubation experiment. As it can be seen from the results, the possible toxicity produced by wastes is better demonstrated by the lower doses, as the enhance of the respiration caused by the organic matter masks the possible inhibition of soil microbial activity due to the presence of higher sludge rates. The release of toxic elements and the benefit brought by the organic matter are better explained by the lower doses of waste, in which the respiration is inhibited in a higher degree than other doses. Regarding the information supplied in the present work by the different models for respiratory activity data presentation (EDR and CR), some indications must be done. First, the fact that EDR is able to reflect the inhibition of the current soil microflora respiration in a clearer way than cumulative plots and in shorter periods of time. This is because the CR graphic accumulates the effect of the first large organic matter mineralisation rates, and this initial stimulatory effect has a certain degree of inertia that affects ulterior respiration curve behaviour. By the other hand, CR presentation is able to show the global affection and trends of the C mineralisation process. Soil respiration is usually represented in the literature by means of cumulative plots, although the differential form (EDR) is recommended in some cases (Hess and Schmidt, 1995), ´ lvarez because of statistical reasons. Other authors (A ´ and Alvarez, 2000) do not find differences among the use of integrated (CR) or differential (EDR) forms. In this work the better performance of EDR than CR plots is based, as explained above, on their ability to reflect, in a minimum incubation time, the toxicity of the waste. For instance, an inhibition seen by means of EDR, followed by an adaptation period and an accelerated enhancement of respiration might result in similar cumulative CO2 released at the end of the period, compared to the control. In this case, the inhibition should exist, though the carbon cycle should not be seemingly affected, as there is a recovery of the function (final CO2 evolved, and parameter c in CR plots). The affection of the carbon mineralisation ability of the soil

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can also be assessed by means of substrate induced respiration measurements. In the event of performing this test, it should be applied, in the type of wastes considered in this work, once the stimulatory effect of the organic matter has finished (OECD, 2000), so, when the respiration rate becomes constant after the initial enhancement, and this will depend on the waste dose. Similar considerations should be taken into account if ISO method 17155 (ISO, 2002) has to be performed. The different respiration rate levels reached by the control and the mixtures in the last period of the respirometric incubations (or the different slopes of the cumulative curve in the same period) are closely related to the respiration rate values obtained from substrate induced respiration experiments. This direct relation between SIR results and the basal respiration rate in the last incubation period can be deduced from the results shown in some works (Dai et al., 2004). As mentioned above, the SIR results can reflect the ability of a soil sample to perform carbon mineralisation, and this ability, in the samples used in this work, should depend of this new ‘‘basal’’ state achieved after the organic matter consumption, not before. So, an impairment of the carbon mineralisation function caused by the application of this waste may be seen by the negative differences between the respiration rate of the mixtures, and by the lower slope values of the cumulative curves in their latest sections, compared with those of the control. The use of other respirometric parameters based on earlier time periods of the incubation should be avoided for toxicity assessment purposes in this type of waste. Regarding the minimum time needed for the incubation to see the effects, when the waste mixed with soil has positive properties (enhancing respiration), as nutrients, and also toxic characteristics (inhibiting respiration), as metals, the duration time of the experiment is a variable which has to be perfectly established, as the initial enhancement of microbial activity could mask the toxicity, and usual incubation times like 10 or 30 days would not be enough to reveal toxicity. This observation does not agree with some recommendations about respirometric methodologies, which suggest short incubation periods (Van Beelen and Doelman, 1997; ISO, 2002). Because of the same reason, the rates established and the incubation time are intimately related in this type of wastes. As waste dose increases, the time needed to see real toxic effects will increase, to avoid toxicity masking by the organic matter effect on respiration. This observation agrees with that from Van Beelen and Doelman (1997), who indicate that high substrate concentration may reduce

mineralisation tests sensitiveness, in substrate added tests. 4.2. Zn, Cr and Ba availability As expected, the results show that soluble potentially toxic elements are released during incubation, and it allows us to suppose that a parallel increase in the exchangeable PTE fraction has also occurred. The effect gets stronger for the soil with higher level of basal biological activity (SN). This could be explained by the release of Zn from organic matter-bound positions while mineralisation takes place. Moreover, the toxicity of the released metal depends finally on the receiving soil properties, which affect metal availability (Khan and Scullion, 2002). The soil SO performs better buffering tasks, due to its higher clay and carbonate contents, and higher pH values, which can promote binding or precipitation of soluble Zn, and Cr. Soluble Ba concentration is slightly higher in SO, as its high Ca concentration saturates the exchange matrix and competes with Ba for the carbonates precipitation. In this case this agrees with the results of soil respiration. The soil SN has higher biological activity than SO, so the mineralisation processes lead to the release of PTE, which act as toxic to the soil microflora. The lower biological activity of soil SO, and its carbonate content and pH value make this soil less sensible to the toxic effect of the waste. 5. Conclusions The results of this work show that the equations used to adjust the respiration curves and the curves parameters are useful to assess the behaviour of waste application on soils, especially considering that it is hard to describe the ambivalent behaviour of the waste essayed by means of the parameters currently used in respirometry. The time required to appreciate the inhibitory effects of some type of wastes depends on the basal biological activity of the reference soil, as well as its physical– chemical properties. Also, it depends on the waste properties, for instance, the concentration of organic matter, which is able to mask respiration inhibitions due to the enhancement caused by its degradation. Consequently, the time of the incubation should be large enough, or the waste rate little enough, to achieve the waste organic matter degradation and the release of potentially toxic elements, which should began to exert their toxic influence on the soil microflora. As excessively long incubation times in small-scale

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experiments may lead to the modification of soil properties, the adjustment of the experimental doses should help in some concrete cases. In any case, the use of daily respiration curves allows seeing inhibitory effects, in the waste type used, in shorter incubation periods than cumulative ones. So, the assessment of toxicity of ambivalent wastes on soil by means of respirometry is better done using low application rates and daily respiration curves. The curve parameters of the exponential adjustment of daily plots help in this assessment, better than cumulative ones. References Allen, H.E., Yin, Y., 1998. Combining chemistry and biology to derive soil quality criteria for pollutants. In: Proceedings 16th World Congress on Soil Science, Montpellier, France. ´ lvarez, R., A ´ lvarez, C.R., 2000. Soil organic matter pools and their A associations with carbon mineralisation kinetics. Soil Sci. Soc. Am. J. 64, 184–189. Anderson, J.P.E., 1982. Soil respiration. In: Page, et al. (Eds.), Methods of Soil Analysis, Part II. Chemical and Microbiological Properties. second ed. ASA, SSSA, Madison, Wisconsin. Arshad, M.A., Martin, S., 2002. Identifying critical limits for soil quality indicators in agro-ecosystems. Agric. Ecosyst. Environ. 88, 153–160. Ba˚a˚th, E., 1989. Effects of heavy metals in soil on microbial processes and populations (a review). Water Air Soil Pollut. 47, 335–379. Brohon, B., Delolme, C., Gourdon, R., 2001. Complementarity of bioassays and microbial activity measurements for the evaluation of hydrocarbon-contaminated soils quality. Soil Biol. Biochem. 33, 883–891. Dai, J., Becquer, T., Rouiller, J.H., Reversat, G., Reversat, F.B., Lavelle, P., 2004. Influence of heavy metals on C and N mineralisation and microbial biomass in Zn-, Pb-, Cu- and Cd-contaminated soils. Appl. Soil. Ecol. 25, 99–109. Eisentraeger, A., Maxam, G., Rila, J.P., Dott, W., 2000. A stepwise procedure for assessment of the microbial respiratory activity of soil samples contaminated with organic compounds. Ecotoxicol. Environ. Saf. 47, 65–73. Filzek, P., Hund-Rinke, K., Simon, M., Huettner, S., Grabert, E., Jander, J.P., Eisentraeger, A., 2003. Optimisation and utilisation of ecotoxicological bioassays for quality assessment of soils and soil materials by on-site detection. In: Proceedings of the 8th Inter-

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