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Suitability of a winery-sludge as soil amendment
Bioresource Technology 49 (1994) 173-178 © 1994 Elsevier Science Limited Printed in Great Britain. All rights reserved 0960-8524/94/$7.0(l 0960-g524(94)00023-9 ELSEVIER
SUITABILITY OF A WINERY-SLUDGE AS SOIL A M E N D M E N T A. Saviozzi,* R. Levi-Minzi, R. Riffaldi & R. Cardelli lnst#ute of Agricultural Chemistry, University of Pisa, via S. Michele degli Scalzi, 2, .56124Pisa, Italy (Received 22 March 1994; revised version received 25 May 1994; accepted 31 May 1994)
Abstract
A laboratory study was carried out to assess the suitability as soil amendment of a sludge obtained through aerobic depuration of wastewaters from a winery ('winery-sludge'). During a 180-day incubation period, changes in chemical and biological properties of a soil amended with 0"5 and 2"5% of sludge were evaluated. A t the end of the experiment, winery-sludge increased the amount of available N, P, K and S, organic and potentially mineralizable carbon, and total microbial activity, while pH, biomass C, dehydrogenase activity, decomposition rate, water-soluble sugars, phenolic compounds and chemical oxygen demand (COD) were not affected by the sludge application. A germination test indicated that the winery-sludge did not show toxicity even immediately after its addition to soil. The great build-up of salinity in soil amended with the highest dose could give rise to risks to crops and environment, so there is a need for field tests. Key words: Winery-sludge, organic matter, soil amendment.
legislation (.law no. 99, dated 27 January 1992), adapted to conform to EEC directives concerning sludge application to agricultural land. Experimental results giving information on the suitability of winerysludge as soil amendment, and for its use without risks to crops or the environment, are needed. Research concerning the impact of some types of winery wastes on soil characteristics has been carried out (Cabrera et al., 1986; Cruz et al., 1991; Morisot, 1986; Strivastava & Sahai, 1987), but reliable data on the effect of sludges obtained through depuration of wastewaters from wineries (winery-sludge) on the chemical and biological properties of soil are lacking. This report is concerned with a laboratory investigation on the effects of the disposal into soil of winerysludge as a possible means of recycling and fertilization. METHODS
The soil used was collected from the top 0-15 cm of a representative soil of a wine-producing region located near Piacenza. For analysis soil samples were air-dried and passed through a 1 mm sieve. The sludge was supplied by a wastewater deputation system belonging to the wine-producing factory 'Asti Barbera', near Asti (Piedmont). The aerobic sludge was collected at the end of a sedimentation phase of about 4 months, lyophilized and screened through a 1-mm sieve. In this dry sludge, organic C was determined after removing carbonate C (Nelson & Sommers, 1982) by dry combustion (inducaion furnace 900 CS, Eltra). Total N and pH were determined by standard methods. Total P and K were brought into solution by acid digestion and determined colorimetrically by the ascorbic acid method (John, 1970) and by atomic absorption spectroscopy (AAS), respectively; SO~-, CI , HPO~- were detected by ion chromatography of an aqueous extract (sludge/extractant 1:25). On the same extract were evaluated: electrical conductivity using a cell calibrated with NaCI standards; phenolic compounds, expressed as coumaric acid, by a modification of the method of Folin, according to Kuwatsuka and Shindo (1973); sugars, expressed as glucose, by the phenol method (Dubois et al., 1956). Sludge was mixed with soil at rates of 0 (control), 0"5 and 2.5% of dry matter on a dry soil basis. The
INTRODUCTION The extension of industrial processing of agricultural products has given rise to the accumulation of large amounts of wastes which are hard to dispose of. Treatment and disposal of wastewaters from the wine-producing industry are becoming a serious problem in wine-producing countries, where the volume of effluent produced is great. Because of their potential polluting power, direct discharge of the wastewaters, without preliminary treatment, to land, surface waters, streams, lakes and the sea, is prohibited by Italian legislation (law no. 319, dated 10 May 1976). Most treatment plants for wastewaters coming from winery operations are faced with the problem of disposal of sludges; their land application appears an attractive solution, because of the need for low disposal costs and for the recycling of both organic matter and nutritive elements in the soil-crop system. Digested sewagesludges, from wastewater depuration of municipal and industrial plants, can be applied to the land if the material meets criteria established by current Italian *To whom correspondence should be addressed. 173
174
A. Saviozzi, R. Levi-MinzL R. Riffaldi, R. Cardelli
soil-sludge mixtures, as well as the control, were placed in 300-ml beakers, into which water was added at appropriate intervals to reach and maintain a moisture content of 50% of the maximum water-holding capacity (MWHC)(determined by making a saturation paste). The mixtures were incubated in triplicate at 25 _+2°C and samples were taken for analysis after, 1, 5, 19, 47, 89 and 180 days. On the aqueous extract obtained by shaking the soil samples with water for 1 h (soil/extractant 1:5) the following were monitored by the procedures previously described: pH, electrical conductivity (EC), SO] , phenolic compounds, sugars. On the same extracts, NO3-N was determined by ion chromatography, and chemical oxygen demand (COD) by the method reported by CNR-IRSA (1978). Available P was measured according to the Olsen method. Exchangeable K was evaluated by AAS on samples which had been extracted for 2 h with 1 N CH3COONH 4 at pH 7.0 (soil/extractant 1:50). Total organic C was determined by the method previously described; dehydrogenase activity according to the method described by Casida et al. (1964); biomass C by the chloroform fumigation technique (Jenkinson & Powlson, 1976); and hydrolytic activity as absorbance at 490 nm of a filtrate from a soil suspension incubated with fluorescein diacetate at 24°C (Schnurer & Rosswall, 1982). Finally, a germination index, with water as reference, was tested on the aqueous extract (soil/water 1:1) according to the method of Zucconi et aL ( 1981 ). An analysis of variance was performed by the ANOVA test (Zar, 1984) on the mean value of each variable for all levels of added sludge and, where main effects or interactions were significant, a multiple comparison least significant difference of F (LSD'F) test was performed. The incubation period for evaluating the rate of decomposition in the soil of sludge consisted of mixing, at 25°C and at 50% of MWHC, 100 g of soil with 0, 0"5 and 2"5% of dry sludge in 300ml glass containers, in which were placed vials holding 20 ml of 0"5 M NaOH to trap the evolved CO2. The excess alkali was back-titrated with standard 0"5 M HCI after precipitating the carbonate with a 1"5 M BaCL solution. The decomposition of the material in soil was monitored daily for 10 days and at longer time intervals thereafter. All treatments were carried out with three replicates; the coefficient of variation was always lower than 3%. A non-linear, least squares regression analysis, provided by CoSTAT (COHORT Software, 1990), was used to calculate parameters from cumulative data of C mineralization. The coefficient of determination (R -') was used for evaluating the quality of fit. RESULTS AND DISCUSSION In Table 1 are given the properties of soil and sludge, while Table 2 presents some changes in soil-sludge
Table I. Selected chemical and physical properties of the soil and winery-sludge
Property
Soil
Sludge
pH Electrical conductivity (dS m - ~) Organic C (%) Total N (%) Sand (%) Silt (%) Clay (%) Total P (%) Total K (%) SO4 (ppm) C1- (ppm) HPO4- (ppm) Phenolic compounds (%) Sugars (%)
7"8
8"9 1.2 32.1 5'0
0.9 0.1 56"0 32.0 12.0
0"7 1"0 785"0 353"0 489"0 1"0 2"6
during the incubation. Since the material added to soil had a pH of 8-9, soil pH would be expected to increase as a result of sludge amendment; nevertheless, in the early stages of incubation the pH was not affected by the addition of the material, perhaps because of the buffer power of this heavy-textured soil. Later, pH decreased, especially for the highest dose applied, without ever reaching levels shown one day after sludge addition. The drop in pH observed could result from both the continuous increase in nitrate formation and the activity of microorganisms which break down complex carbonaceous materials to organic acids. Table 2 also shows the increase of EC values over time for both doses applied. Cabrera et al. (1986) found a similar result for a soil treated with anaerobically digested marcs. Significant differences from untreated soil were detectable almost immediately after the application of the 2"5% dose, but only after about 3 months for the lowest addition. According to Davidescu and Davidescu (1982), 387 and 579 dS m -1 values found about 3 months after the amendment with the highest dose of sludge could be considered a hazardous level of salt concentration in the soil solution. However, the EC increase also observed in the control is probably more pronounced in laboratory experiments than in the field, where leaching can translocate salts downwards in the soil profile. The low agronomic value and the phytotoxicity of organic materials are often related to a strong immobilization of nitrate in the soil (Perez & Gallardo-Lara, 1987; Riffaldi et al., 1993; Saviozzi et al., 1991 ). In incubation experiments performed to study the effects of the application to soil of sludges from olive-oil processing, Riffaldi et al. (1993) found a decrease in nitrate for about 6 weeks, after which time nitrate concentration in soil began to increase. In the present study, a continuous increase of NO3-N content as the incubation period increased was found both in the control and in treated soil. It must be emphasized that the pH values of the soil, always over 7-5, and the low C/N ratio of the material (6-1 ) were the
Winergy-sludge disposed on soil
175
Table 2. Changesin pH, EC and some fertility parameters during incubation of soil amended with various amounts of sludge"
Sludge added
Analytical properties
0 0.5 2.5
pH
0 0-5 2.5
EC (dS m-1)
0 0"5 2.5
NO3-N (#g g- ~)
0 0'5 2.5
Available P (/~gg- J)
0 0'5 2.5
Exchangeable K (me 100 g- t)
0 0"5 2-5
SO4 (/~gg- t)
Days after additions 1
5
19
47
89
7"8a 7"8a 7.8a
7"8a 7"8a 7,6c
7.8a 7.7b 7"6c
7'8a 7.7b 7.6c
7.8a 7.7b 7"6c
105.0h 110.0h 155.0efgh
107,0h 125,0gh 202,0ef
128.0h 179.0efg 345.0b
138.0gh 206.0de 387.0b
164.0efgh 264.0cd 579.9a
4.0h 3.0h 1.5h
7"3h 8.5h 17.0gh
11-7gh 17.5gh 45.5de
28"5fg 47.7de 121.2c
34.9ef 63.3d 142.7b
59.0d 113.6c 258'1a
12.0g 28-6e 92-5a
12.4g 23-4ef 89-0a
12.2g 22-8ef 82-3b
12.4g 24.8ef 67-6c
0.20ef 0.23c 0.31 a 6"lj 21.0hij 49'6ghi
114.0h 145.0fgh 274.0c
0.20ef 0.23c 0-30ab 6.7j 31.0hij 173"6e
0.19f 0.22cd 0-30ab 8'3j 52"3gh 247-7d
0.19f 0.23c 0"29b 10"9ij 78"6fg 354-2c
11.8g 19.4f 70-3cd 0-19f 0-22cd 0-30ab 13'5hij 100-0f 417"4b
180 7.8a 7.7b 7"7b
12. i g 20.2f 62.8d 0.19f 0.2 lde 0"29b 27"0hij 114-8f 522-0a
"Values with different letters indicate the differences at a 5% probability level according to LSD'F multiple range test, considering separately the interaction between sludge amount and time for each homogeneous section of the table.
optima for the nitrification process. The percentages of the added N, which mineralized as nitrate from the two treatments at the end of the incubation period, were similar (about 11%) and almost double that of the control. This would indicate that the organic substrate, when added to the soil, had not yet reached a sufficiently high degree of maturity, and therefore a large part of the easily decomposable fraction of the residue was still present. Soil-extractable P increased significantly with sludge additions (Table 2); in the later stages, a progressive decrease was found, probably due to adsorption by soil colloids. However, after about 6 months, available P in the amended soil was still significantly different from the control. It is usually accepted that potassium in agricultural wastes behaves as an effective fertilizer, given that the element in wastes is totally soluble and available. A comparative analysis of the treatments shows that the addition of the winery-sludge to soil produced a significant increase in exchangeable K but, contrary to available P, the extent of variation was not substantially affected by the incubation time. The addition of the sludge to soil led to an increase in sulphate content which was almost proportional to the doses applied. For both dosed and control soils, there was a linear tendency of increase in SO ] content with time, perhaps due to substrate mineralization. This was consistent with the trend of nitrate already discussed. Therefore, from a practical point of view, the application of winery-sludge increases the availability of N,
P, K and S in the soil, thus showing a high fertilizing potentiality. Table 3 shows that in spite of a generally constant decrease in organic C, its content in amended soil was still significantly different from the control, and more than 85% of the organic C added remained at the end of the experiment. These results suggest that the winery-sludge can contribute to raising the organic matter in the soil and lessening its decline in intensively cultivated areas. The decline of organic C in untreated soil should be noted: both temperature and moisture level were probably in the optimum range for microbial activities, so producing an accelerated rate of substrate mineralization. Moreover, although our soil was only coarsely sieved, some organic materials may have been made susceptible to rapid mineralization. The level of water-soluble sugars (Table 3), including degradation products of many complex sugars, was higher in soil amended with the 2.5% dose than in the 0-5% treatment and control, but only up to day 19 of the incubation. In every treatment, only small amounts of sugars were found during the later stages of incubation, showing that these substances represented an easily decomposed fraction. Table 3 also lists the behaviour of COD and phenolic compounds, usually considered responsible for some unfavourable effects on plant growth and yield (Kuiters & Sarink, 1987). The initial amounts of these were linearly related to the quantity of sludge applied. Differences in COD with the control disappeared on day 5 for the lowest dose and on day 19 for the highest amount of sludge applied to the soil.
A. SaviozzL R. Levi-Minzi, R. Riffaldi, R. Cardelli
176
T h e losses of phenolic compounds parallel the losses of sugars; phenolic substances were undetectable on day 19 in all treatments. Results Obtained in this experiment suggest that incorporation in soil of winerysludge about 20 days before drilling would eliminate possible negative effects on the plants. T h e lack of toxicity of the material was confirmed by the findings of the germination test on Lepidium sativum (data not shown); the germination responses of different doses of sludge applied did not show significant differences from the control. Biochemical activities (Table 4) are a good general measure of organic matter turnover in soil. In comparison to the control assay, only the amendment with the highest dose of sludge resulted in a marked increase (1 day after addition) in the activity of dehydrogenase, the enzyme responsible for biological oxidation of all organic compounds. On the other hand, at the beginning of incubation there was enough easily degradable material coming from the added sludge to support a higher biochemical activity than
that of the control. Subsequently, apart from an initial rapid decrease of dehydrogenase activity, c o m m o n to both doses applied and to the control but differing in amount, the rate of decrease of the activity remained reasonably constant over the entire period, until the same value was shown for all treatments 6 months after sludge addition. The fluorescein diacetate (FDA) test was checked, to a limited extent, 5 and 180 days from sludge disposal. T h e ability to hydrolyse the FDA, which determines active fungi (Soderstrom, 1977), bacteria (Brunins, 1980; Lundgren, 1981) and, more recently, total microbial activity (Schnurer et al., 1985), was found generally to increase with soil organic matter content (Schnurer et al., 1985). No evident influence of the 0"5% treatment on F D A hydrolysis was noted in the experiment, while the 2"5% treatment gave significantly higher values than the 0-5% treatment and control, and this difference remained at the end of incubation. Data for biomass C on the day 1 of incubation were greatly influenced by the amendment, resulting in too
Table 3. Changesins•me•rganicc•mp•nentsduringincubati•n•fs•ilamendedwithvari•usam•unts•fsludge
Sludge added (%)
Analytical properties
a
Days after additions
0 0.5 2.5
Organic C (%)
0 0-5 2-5
Sugars (as glucose) (/zg g- ~)
0 0.5 2.5 0 0'5 2'5
1
5
19
47
89
180
0.93ghi 1.1 le 1.79a
0.89hij 1.07el 1.70ab
0.88hij 1.08e 1.67be
0-86ij 1.03efg 1-60bed
0-83ij 1.04ef 1.58ed
0.79j 0.98fgh 1.54d
39-5b 43.9b 52-9a
0.2d 5-2d 20-5c
0.2d 0-ld 2-8d
n.d. n.d. 0-1d
n.d. n.d. n.d.
n.d. n.d. n.d.
COD (mg l- J)
4"0d 5.2c 9.6a
3"9d 3-9d 7.5b
3-7d 3-9d 4.0d
3'7d 3.8d 3.1de
3-6d 3.0de 2.9de
2.0e 2.5e 2-0e
Phenolic compounds (as coumaric acid)(/~g g- ~)
0"9c 2"8b 9"2a
0.2c 0'3c 1'4c
0' 1c 0' l c 0"3c
n.d. n.d. 0"lc
n.d. n.d. n.d.
n.d. n.d. n.d.
"Values with different letters indicate the differences at a 5% probability level according to LSD'F multiple range test, considering separately the interaction between sludge amount and time. n.d., Not detectable.
Table4. Changes in biochemical activities during incubation of soil amended with various amounts of sludge"
Sludge added
Analytical properties
Days after additions
(%)
1
5
0 0-5 2"5
Dehydrogenase activity (pg g soil- l h- l of TPF )
0"8c 0.9c 2'8a
0"6cd 0-8c l'4b
0 0'5 2-5
FDA hydrolysis (OD units h- ~g- ~dry wt)
----
0"082a 0.084a 0.123b
0 0.5 2-5
Biomass C (/~g g ~)
----
426.0e 436-0e 980.0d
19
47
89
180
0"4de 0.4de l'4b
0.5de 0"5de 1"3b
0.4de 0-4de 0'9c
0.5de 0"5de 0"6cd
----
----
----
477.0e 448.0e 1003-0d
1147.0c 1309.0b 1395.0b
1159.0c 1281.0bc 1542.0a
0"080a 0.099a 0.170c 986.0d 960.0d 1002.0d
~Values with different letters indicate the differences at 5% probability level according to LSD'F multiple range test, considering separately the interaction between sludge amount and time for each homogeneous section of the table. --, Not tested.
Winergy-sludge disposed on soil many wide variations among the replicates, and are thus not reported in Table 4. Only the application of sludge at the 2-5% rate markedly increased the size of biomass C on the day 5, as a consequence of the increased amount of available organic substrate. A general increase of the biomass, especially between the days 47 and 89, was observed for all treatments. Towards the end of the incubation period, however, a stabilization phase was established and no differences were observed among the treatments. It is probable that after 180 days the microorganisms were in equilibrium with their resources and no drastic biomass changes could thus be expected. The usefulness of an organic material as fertilizer is affected by the rate at which it decomposes when added to soil. The evolution of CO2 is generally considered an index of the microbial activity that primarily regulates the decomposition rate of any kind of organic material in soil. Our incubation experiment demonstrated that the CO2 evolution from the soil-sludge mixtures was maximum in the first few days following the incorporation of sludge into the soil, and then decreased (Fig. 1). High initial levels of microbial activity in both treatments were probably supported by readily available C from easily decomposed C components, such as water-soluble sugars, amino acids, etc. Thereafter, the lower decomposition rates reflect the greater resistance of recalcitrant materials. The total loss of CO2-C (Fig. 1) over the incubation period amounted to about 3% of the added organic carbon, and this figure was similar to that found by Levi-Minzi et al. (1990) when soil was incubated with municipalrefuse compost and cattle farmyard-manure in a similar laboratory investigation. Remarkably higher percentages were found in soil amended with aerobic sewage sludge, pig-slurry, rye straw, poultry manure (Levi-Minzi et al., 1990) and sludge coming from oliveoil processing (Saviozzi et al., 1993a); these latter results may be the response to the more readily-available C in the easily decomposing compounds in these organic materials. On the basis of these findings, winery-sludges seem to be promising materials for improving the organic matter level of a soil and maintaining its biological activity. A mathematical description of the mineralization curves was used to analyze the cumulative CO2-C data through the first-order, non-linear regression model:
177
Parameters calculated according to the model are also reported in Table 5. As expected, the amount of carbon that can be mineralized (Co) and the rapidly mineralizing sub-fraction (C1) increased with increasing addition of sludge. Positive C~ values of the treated soil indicate that no lag-phase in the initial period of incubation occurred (Ellert & Bettany, 1988), so confirming the lack of toxic effects due to the application of the material. Finally, Table 5 reports data of the decomposition rate (k) of the control and treated soil. As can be seen, sludge applied at the two doses had no appreciable influence upon the rates of CO2-C evolution. Although the sludge produced some beneficial changes in the soil, in these laboratory conditions the incorporation of the highest dose of sludge increased soil salinity to a hazardous level and, for this reason, in order to assess the suitability of the winery-sludge for agricultural purposes, it would be advisable to verify the extent of salinization under field conditions. ACKNOWLEDGEMENTS This research was supported by the MURST (Grant 40%).
Table 5. Parameter estimates and coefficients of determination according to the first-order model for C mineralization of the sludge-soil systems Sludge applied
Co o
C~
k
Re
0 0"5 2"5
32"4 38"4 59"7
1"6 1"7 2"9
- 0"104 - 0"095 - 0"092
0'995 0'996 0'995
(%)
~For meaning of symbols see text.
60-
-4
40-
. ° " . o j . ~" ,
C, = C,,(1 - e
k,)+ Cl (Jones, 1984)
where C,= cumulative amount of C mineralized after time t (mg 100 g- ~of amended soil); t = time from start of incubation when t = 0 (days); Co=potentially mineralizable C (mg 100 g-i of amended soil); C~ =easily mineralizable C (mg 100 g-i of amended soil); k = rate constant. Such a model, already found to be the most suitable among several kinetic models in describing the decomposition processes of many types of organic material (Saviozzi et al., 1993b), also shows, in this investigation, R ~-values that are always higher than 0"99 (Table 5).
0"~"
0
o 0
.
~
-
c
g
ca20-~ E
o 1 o~
10-
[
0
~ 0
Fig. 1.
2
+
s o i l -0- soil + 0 . 5 % , , , , , 4
6
8
10
12
sludge , , 14
16
days
-~- soil + 2 . 5 % sludge ~ , , , ~ 18
20
22
24
26
0 28
f loss during the decomposition of the sludge-soil
system. Continuous lines indicate the CO2-C evolved on the soil basis (left scale); dotted lines indicate the C02-C evolved as percent of the total organic carbon (right scale).
178
A. Saviozzi, R. Levi-Minzi, R. Riffaldi, R. Cardelli
REFERENCES Brunins, G. (1980). Technical aspects of the use of 3',6' diacetyl fluorescein for vital fluorescent staining of bacteria. Curr. MicrobioL, 4, 321-3. Cabrera, E, Fernandez, M., Diaz, E. & Osta, F. (1986). Use as fertilizers of wastes originating in the anaerobic digestion of vegetation waters and vinasses. In Int. Symp. on Olive By-Products Valorization. FAO, Sevilla, Spain, pp. 353-4. Casida, L. E., Jr, Klein, D. A. & Santoro, T. (1964). Soil dehydrogenase activity. Soil Sci., 98, 371-6. CNR-IRSA (1978). Metodi analitici per le acque. Quarderno, 11 (2), 137-46. COHORT Software (1990). CoSTAT Stastical Software, Berkeley, CA. Cruz, R., Righetto, A. M. & Nogueira, M. A. (1991). Experimental investigation of soil and groundwater impacts caused by vinasse disposal. Water Sci. and Technol., 24, 77-85. Davidescu, D. & Davidescu, V. (1982). Evaluation of Fertility by Plant and Soil Analysis. Abacus Press, Tunbridge Wells, Kent, UK. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, E A. & Smith, E (1956). Colorimetric method for determination of sugars and related substances. Anal. Chem., 28, 350-6. Ellert, B. H. & Bettany, J. R. (1988). Comparison of kinetic models for describing net sulfur and nitrogen mineralization. Soil Sci. Soc. Am. J., 52, 1692-702. Jenkinson, D. S. & Powlson, D. S. (1976). The effects of biocidal treatments on metabolism in soil. V. A method for measuring soil biomass. Soil Biol. Biochem., 8, 209-13. John, M. K. (1970). Colorimetric determination of phosphorus in soil and plant materials with ascorbic acid. Soil Sci., 109, 214-18. Jones, C. A. (1984). Estimation of an active fraction on soil nitrogen. Comm. Soil. Sci. Plant Anal., 15, 23-32. Kuiters, A. T. & Sarink, H. M. (1987). Effects of acids on growth, mineral composition and chlorophyll content in some herbaceous woodland species. Zeitsch. Pflanzen. and Bodenk., 15, 84-8. Kuwatsuka, S. & Shindo, H. (1973). Behavior of phenolic substances in the decaying process of plants. Identification and quantitative determination of phenolic acids in rice straw and its decayed products by gas chromatography. Soil Sci. and Plant Nutr., 19, 219-27.
Levi-Minzi, R., Rifffaldi, R. & Saviozzi, A. (1990). Carbon mineralization in soil amended with different organic materials. Agric. Ecosys. Environ., 31,325-35. Lundgren, B. (1981). Fluorescein diacetate as a strain of metabolically active bacteria in soil. Oikos, 36, 17-22. Morisot, A. (1986). Utilisation agricole de quelques d6chets de distilleries de vins rouges. Agronomie, 6,203-12. Nelson, D. W. & Sommers, L. E. (1982). Total carbon, organic carbon and organic matter. In Methods of Soil Analysis, Part 2, ed. A. L. Page et al. Am. Soc. Agron., Madison, WI, pp. 539-94. Perez, J. D. & Gallardo-Lara, F. (1987). Effects of the application of wastewater from olive processing on soil nitrogen transformation. Comm. Soil Sci. Plant Anal., 18, 1031-5. Riffaldi, R., Levi-Minzi, R., Saviozzi, A., Vanni, G. & Scagnozzi, A. (1993). Effect of the disposal of sludge from olive processing on some soil characteristics: laboratory experiments. Water, Air and Soil Pollut., 69, 257-64. Saviozzi, A., Levi-Minzi, R., Riffaldi, R. & Lupetti, A. (1991 ). Effetti dello spandimento di acque di vegetazione sul terreno agrario. Agrochimica, 35, 135-48. Saviozzi, A., Riffaldi, R., Levi-Minzi, R., Scagnozzi, A. & Vanni, G. (1993a). Decomposition of vegetation-water sludge in soil. Bioresource Technol., 44,223-8. Saviozzi, A., Levi-Minzi, R. & Riffaldi, R. (1993b). Mineralization parameters from organic materials added to soil as a function of their chemical composition. Bioresource Technol., 45, 131-5. Schnurer, J. & Rosswall, T. (1982). Fluorescein diacetate hydrolysis as a measure of total microbial activity in soil and litter. App. Environ. Microb., 43, 1256-61. Schnurer, J., Clarholm, M. & Rosswall, T. (1985) Microbial biomass and activity in an agricultural soil with different organic matter contents. Soil Biol. Biochem., 17, 611-18. Soderstrom, B. E. (1977). Vital staining of fungi in pure cultures and in soil with fluorescein diacetate. Soil Biol. Biochem., 9, 59-63. Strivastava, N. & Sahai, R. (1987). Effects of distillery waste on the performance of Cicer arietinum L. Environ. Pollut., 43, 91-102., Zar, J. H. (1984). Biostastical Analysis. Prentice Hall, Englewood Cliffs, NJ. Zucconi, E, Pera, A., Forte, M. & De Bertoldi, M. (1981). Evaluating toxicity of immature compost. BioCycle, 22, 54-7.
SUITABILITY OF A WINERY-SLUDGE AS SOIL A M E N D M E N T A. Saviozzi,* R. Levi-Minzi, R. Riffaldi & R. Cardelli lnst#ute of Agricultural Chemistry, University of Pisa, via S. Michele degli Scalzi, 2, .56124Pisa, Italy (Received 22 March 1994; revised version received 25 May 1994; accepted 31 May 1994)
Abstract
A laboratory study was carried out to assess the suitability as soil amendment of a sludge obtained through aerobic depuration of wastewaters from a winery ('winery-sludge'). During a 180-day incubation period, changes in chemical and biological properties of a soil amended with 0"5 and 2"5% of sludge were evaluated. A t the end of the experiment, winery-sludge increased the amount of available N, P, K and S, organic and potentially mineralizable carbon, and total microbial activity, while pH, biomass C, dehydrogenase activity, decomposition rate, water-soluble sugars, phenolic compounds and chemical oxygen demand (COD) were not affected by the sludge application. A germination test indicated that the winery-sludge did not show toxicity even immediately after its addition to soil. The great build-up of salinity in soil amended with the highest dose could give rise to risks to crops and environment, so there is a need for field tests. Key words: Winery-sludge, organic matter, soil amendment.
legislation (.law no. 99, dated 27 January 1992), adapted to conform to EEC directives concerning sludge application to agricultural land. Experimental results giving information on the suitability of winerysludge as soil amendment, and for its use without risks to crops or the environment, are needed. Research concerning the impact of some types of winery wastes on soil characteristics has been carried out (Cabrera et al., 1986; Cruz et al., 1991; Morisot, 1986; Strivastava & Sahai, 1987), but reliable data on the effect of sludges obtained through depuration of wastewaters from wineries (winery-sludge) on the chemical and biological properties of soil are lacking. This report is concerned with a laboratory investigation on the effects of the disposal into soil of winerysludge as a possible means of recycling and fertilization. METHODS
The soil used was collected from the top 0-15 cm of a representative soil of a wine-producing region located near Piacenza. For analysis soil samples were air-dried and passed through a 1 mm sieve. The sludge was supplied by a wastewater deputation system belonging to the wine-producing factory 'Asti Barbera', near Asti (Piedmont). The aerobic sludge was collected at the end of a sedimentation phase of about 4 months, lyophilized and screened through a 1-mm sieve. In this dry sludge, organic C was determined after removing carbonate C (Nelson & Sommers, 1982) by dry combustion (inducaion furnace 900 CS, Eltra). Total N and pH were determined by standard methods. Total P and K were brought into solution by acid digestion and determined colorimetrically by the ascorbic acid method (John, 1970) and by atomic absorption spectroscopy (AAS), respectively; SO~-, CI , HPO~- were detected by ion chromatography of an aqueous extract (sludge/extractant 1:25). On the same extract were evaluated: electrical conductivity using a cell calibrated with NaCI standards; phenolic compounds, expressed as coumaric acid, by a modification of the method of Folin, according to Kuwatsuka and Shindo (1973); sugars, expressed as glucose, by the phenol method (Dubois et al., 1956). Sludge was mixed with soil at rates of 0 (control), 0"5 and 2.5% of dry matter on a dry soil basis. The
INTRODUCTION The extension of industrial processing of agricultural products has given rise to the accumulation of large amounts of wastes which are hard to dispose of. Treatment and disposal of wastewaters from the wine-producing industry are becoming a serious problem in wine-producing countries, where the volume of effluent produced is great. Because of their potential polluting power, direct discharge of the wastewaters, without preliminary treatment, to land, surface waters, streams, lakes and the sea, is prohibited by Italian legislation (law no. 319, dated 10 May 1976). Most treatment plants for wastewaters coming from winery operations are faced with the problem of disposal of sludges; their land application appears an attractive solution, because of the need for low disposal costs and for the recycling of both organic matter and nutritive elements in the soil-crop system. Digested sewagesludges, from wastewater depuration of municipal and industrial plants, can be applied to the land if the material meets criteria established by current Italian *To whom correspondence should be addressed. 173
174
A. Saviozzi, R. Levi-MinzL R. Riffaldi, R. Cardelli
soil-sludge mixtures, as well as the control, were placed in 300-ml beakers, into which water was added at appropriate intervals to reach and maintain a moisture content of 50% of the maximum water-holding capacity (MWHC)(determined by making a saturation paste). The mixtures were incubated in triplicate at 25 _+2°C and samples were taken for analysis after, 1, 5, 19, 47, 89 and 180 days. On the aqueous extract obtained by shaking the soil samples with water for 1 h (soil/extractant 1:5) the following were monitored by the procedures previously described: pH, electrical conductivity (EC), SO] , phenolic compounds, sugars. On the same extracts, NO3-N was determined by ion chromatography, and chemical oxygen demand (COD) by the method reported by CNR-IRSA (1978). Available P was measured according to the Olsen method. Exchangeable K was evaluated by AAS on samples which had been extracted for 2 h with 1 N CH3COONH 4 at pH 7.0 (soil/extractant 1:50). Total organic C was determined by the method previously described; dehydrogenase activity according to the method described by Casida et al. (1964); biomass C by the chloroform fumigation technique (Jenkinson & Powlson, 1976); and hydrolytic activity as absorbance at 490 nm of a filtrate from a soil suspension incubated with fluorescein diacetate at 24°C (Schnurer & Rosswall, 1982). Finally, a germination index, with water as reference, was tested on the aqueous extract (soil/water 1:1) according to the method of Zucconi et aL ( 1981 ). An analysis of variance was performed by the ANOVA test (Zar, 1984) on the mean value of each variable for all levels of added sludge and, where main effects or interactions were significant, a multiple comparison least significant difference of F (LSD'F) test was performed. The incubation period for evaluating the rate of decomposition in the soil of sludge consisted of mixing, at 25°C and at 50% of MWHC, 100 g of soil with 0, 0"5 and 2"5% of dry sludge in 300ml glass containers, in which were placed vials holding 20 ml of 0"5 M NaOH to trap the evolved CO2. The excess alkali was back-titrated with standard 0"5 M HCI after precipitating the carbonate with a 1"5 M BaCL solution. The decomposition of the material in soil was monitored daily for 10 days and at longer time intervals thereafter. All treatments were carried out with three replicates; the coefficient of variation was always lower than 3%. A non-linear, least squares regression analysis, provided by CoSTAT (COHORT Software, 1990), was used to calculate parameters from cumulative data of C mineralization. The coefficient of determination (R -') was used for evaluating the quality of fit. RESULTS AND DISCUSSION In Table 1 are given the properties of soil and sludge, while Table 2 presents some changes in soil-sludge
Table I. Selected chemical and physical properties of the soil and winery-sludge
Property
Soil
Sludge
pH Electrical conductivity (dS m - ~) Organic C (%) Total N (%) Sand (%) Silt (%) Clay (%) Total P (%) Total K (%) SO4 (ppm) C1- (ppm) HPO4- (ppm) Phenolic compounds (%) Sugars (%)
7"8
8"9 1.2 32.1 5'0
0.9 0.1 56"0 32.0 12.0
0"7 1"0 785"0 353"0 489"0 1"0 2"6
during the incubation. Since the material added to soil had a pH of 8-9, soil pH would be expected to increase as a result of sludge amendment; nevertheless, in the early stages of incubation the pH was not affected by the addition of the material, perhaps because of the buffer power of this heavy-textured soil. Later, pH decreased, especially for the highest dose applied, without ever reaching levels shown one day after sludge addition. The drop in pH observed could result from both the continuous increase in nitrate formation and the activity of microorganisms which break down complex carbonaceous materials to organic acids. Table 2 also shows the increase of EC values over time for both doses applied. Cabrera et al. (1986) found a similar result for a soil treated with anaerobically digested marcs. Significant differences from untreated soil were detectable almost immediately after the application of the 2"5% dose, but only after about 3 months for the lowest addition. According to Davidescu and Davidescu (1982), 387 and 579 dS m -1 values found about 3 months after the amendment with the highest dose of sludge could be considered a hazardous level of salt concentration in the soil solution. However, the EC increase also observed in the control is probably more pronounced in laboratory experiments than in the field, where leaching can translocate salts downwards in the soil profile. The low agronomic value and the phytotoxicity of organic materials are often related to a strong immobilization of nitrate in the soil (Perez & Gallardo-Lara, 1987; Riffaldi et al., 1993; Saviozzi et al., 1991 ). In incubation experiments performed to study the effects of the application to soil of sludges from olive-oil processing, Riffaldi et al. (1993) found a decrease in nitrate for about 6 weeks, after which time nitrate concentration in soil began to increase. In the present study, a continuous increase of NO3-N content as the incubation period increased was found both in the control and in treated soil. It must be emphasized that the pH values of the soil, always over 7-5, and the low C/N ratio of the material (6-1 ) were the
Winergy-sludge disposed on soil
175
Table 2. Changesin pH, EC and some fertility parameters during incubation of soil amended with various amounts of sludge"
Sludge added
Analytical properties
0 0.5 2.5
pH
0 0-5 2.5
EC (dS m-1)
0 0"5 2.5
NO3-N (#g g- ~)
0 0'5 2.5
Available P (/~gg- J)
0 0'5 2.5
Exchangeable K (me 100 g- t)
0 0"5 2-5
SO4 (/~gg- t)
Days after additions 1
5
19
47
89
7"8a 7"8a 7.8a
7"8a 7"8a 7,6c
7.8a 7.7b 7"6c
7'8a 7.7b 7.6c
7.8a 7.7b 7"6c
105.0h 110.0h 155.0efgh
107,0h 125,0gh 202,0ef
128.0h 179.0efg 345.0b
138.0gh 206.0de 387.0b
164.0efgh 264.0cd 579.9a
4.0h 3.0h 1.5h
7"3h 8.5h 17.0gh
11-7gh 17.5gh 45.5de
28"5fg 47.7de 121.2c
34.9ef 63.3d 142.7b
59.0d 113.6c 258'1a
12.0g 28-6e 92-5a
12.4g 23-4ef 89-0a
12.2g 22-8ef 82-3b
12.4g 24.8ef 67-6c
0.20ef 0.23c 0.31 a 6"lj 21.0hij 49'6ghi
114.0h 145.0fgh 274.0c
0.20ef 0.23c 0-30ab 6.7j 31.0hij 173"6e
0.19f 0.22cd 0-30ab 8'3j 52"3gh 247-7d
0.19f 0.23c 0"29b 10"9ij 78"6fg 354-2c
11.8g 19.4f 70-3cd 0-19f 0-22cd 0-30ab 13'5hij 100-0f 417"4b
180 7.8a 7.7b 7"7b
12. i g 20.2f 62.8d 0.19f 0.2 lde 0"29b 27"0hij 114-8f 522-0a
"Values with different letters indicate the differences at a 5% probability level according to LSD'F multiple range test, considering separately the interaction between sludge amount and time for each homogeneous section of the table.
optima for the nitrification process. The percentages of the added N, which mineralized as nitrate from the two treatments at the end of the incubation period, were similar (about 11%) and almost double that of the control. This would indicate that the organic substrate, when added to the soil, had not yet reached a sufficiently high degree of maturity, and therefore a large part of the easily decomposable fraction of the residue was still present. Soil-extractable P increased significantly with sludge additions (Table 2); in the later stages, a progressive decrease was found, probably due to adsorption by soil colloids. However, after about 6 months, available P in the amended soil was still significantly different from the control. It is usually accepted that potassium in agricultural wastes behaves as an effective fertilizer, given that the element in wastes is totally soluble and available. A comparative analysis of the treatments shows that the addition of the winery-sludge to soil produced a significant increase in exchangeable K but, contrary to available P, the extent of variation was not substantially affected by the incubation time. The addition of the sludge to soil led to an increase in sulphate content which was almost proportional to the doses applied. For both dosed and control soils, there was a linear tendency of increase in SO ] content with time, perhaps due to substrate mineralization. This was consistent with the trend of nitrate already discussed. Therefore, from a practical point of view, the application of winery-sludge increases the availability of N,
P, K and S in the soil, thus showing a high fertilizing potentiality. Table 3 shows that in spite of a generally constant decrease in organic C, its content in amended soil was still significantly different from the control, and more than 85% of the organic C added remained at the end of the experiment. These results suggest that the winery-sludge can contribute to raising the organic matter in the soil and lessening its decline in intensively cultivated areas. The decline of organic C in untreated soil should be noted: both temperature and moisture level were probably in the optimum range for microbial activities, so producing an accelerated rate of substrate mineralization. Moreover, although our soil was only coarsely sieved, some organic materials may have been made susceptible to rapid mineralization. The level of water-soluble sugars (Table 3), including degradation products of many complex sugars, was higher in soil amended with the 2.5% dose than in the 0-5% treatment and control, but only up to day 19 of the incubation. In every treatment, only small amounts of sugars were found during the later stages of incubation, showing that these substances represented an easily decomposed fraction. Table 3 also lists the behaviour of COD and phenolic compounds, usually considered responsible for some unfavourable effects on plant growth and yield (Kuiters & Sarink, 1987). The initial amounts of these were linearly related to the quantity of sludge applied. Differences in COD with the control disappeared on day 5 for the lowest dose and on day 19 for the highest amount of sludge applied to the soil.
A. SaviozzL R. Levi-Minzi, R. Riffaldi, R. Cardelli
176
T h e losses of phenolic compounds parallel the losses of sugars; phenolic substances were undetectable on day 19 in all treatments. Results Obtained in this experiment suggest that incorporation in soil of winerysludge about 20 days before drilling would eliminate possible negative effects on the plants. T h e lack of toxicity of the material was confirmed by the findings of the germination test on Lepidium sativum (data not shown); the germination responses of different doses of sludge applied did not show significant differences from the control. Biochemical activities (Table 4) are a good general measure of organic matter turnover in soil. In comparison to the control assay, only the amendment with the highest dose of sludge resulted in a marked increase (1 day after addition) in the activity of dehydrogenase, the enzyme responsible for biological oxidation of all organic compounds. On the other hand, at the beginning of incubation there was enough easily degradable material coming from the added sludge to support a higher biochemical activity than
that of the control. Subsequently, apart from an initial rapid decrease of dehydrogenase activity, c o m m o n to both doses applied and to the control but differing in amount, the rate of decrease of the activity remained reasonably constant over the entire period, until the same value was shown for all treatments 6 months after sludge addition. The fluorescein diacetate (FDA) test was checked, to a limited extent, 5 and 180 days from sludge disposal. T h e ability to hydrolyse the FDA, which determines active fungi (Soderstrom, 1977), bacteria (Brunins, 1980; Lundgren, 1981) and, more recently, total microbial activity (Schnurer et al., 1985), was found generally to increase with soil organic matter content (Schnurer et al., 1985). No evident influence of the 0"5% treatment on F D A hydrolysis was noted in the experiment, while the 2"5% treatment gave significantly higher values than the 0-5% treatment and control, and this difference remained at the end of incubation. Data for biomass C on the day 1 of incubation were greatly influenced by the amendment, resulting in too
Table 3. Changesins•me•rganicc•mp•nentsduringincubati•n•fs•ilamendedwithvari•usam•unts•fsludge
Sludge added (%)
Analytical properties
a
Days after additions
0 0.5 2.5
Organic C (%)
0 0-5 2-5
Sugars (as glucose) (/zg g- ~)
0 0.5 2.5 0 0'5 2'5
1
5
19
47
89
180
0.93ghi 1.1 le 1.79a
0.89hij 1.07el 1.70ab
0.88hij 1.08e 1.67be
0-86ij 1.03efg 1-60bed
0-83ij 1.04ef 1.58ed
0.79j 0.98fgh 1.54d
39-5b 43.9b 52-9a
0.2d 5-2d 20-5c
0.2d 0-ld 2-8d
n.d. n.d. 0-1d
n.d. n.d. n.d.
n.d. n.d. n.d.
COD (mg l- J)
4"0d 5.2c 9.6a
3"9d 3-9d 7.5b
3-7d 3-9d 4.0d
3'7d 3.8d 3.1de
3-6d 3.0de 2.9de
2.0e 2.5e 2-0e
Phenolic compounds (as coumaric acid)(/~g g- ~)
0"9c 2"8b 9"2a
0.2c 0'3c 1'4c
0' 1c 0' l c 0"3c
n.d. n.d. 0"lc
n.d. n.d. n.d.
n.d. n.d. n.d.
"Values with different letters indicate the differences at a 5% probability level according to LSD'F multiple range test, considering separately the interaction between sludge amount and time. n.d., Not detectable.
Table4. Changes in biochemical activities during incubation of soil amended with various amounts of sludge"
Sludge added
Analytical properties
Days after additions
(%)
1
5
0 0-5 2"5
Dehydrogenase activity (pg g soil- l h- l of TPF )
0"8c 0.9c 2'8a
0"6cd 0-8c l'4b
0 0'5 2-5
FDA hydrolysis (OD units h- ~g- ~dry wt)
----
0"082a 0.084a 0.123b
0 0.5 2-5
Biomass C (/~g g ~)
----
426.0e 436-0e 980.0d
19
47
89
180
0"4de 0.4de l'4b
0.5de 0"5de 1"3b
0.4de 0-4de 0'9c
0.5de 0"5de 0"6cd
----
----
----
477.0e 448.0e 1003-0d
1147.0c 1309.0b 1395.0b
1159.0c 1281.0bc 1542.0a
0"080a 0.099a 0.170c 986.0d 960.0d 1002.0d
~Values with different letters indicate the differences at 5% probability level according to LSD'F multiple range test, considering separately the interaction between sludge amount and time for each homogeneous section of the table. --, Not tested.
Winergy-sludge disposed on soil many wide variations among the replicates, and are thus not reported in Table 4. Only the application of sludge at the 2-5% rate markedly increased the size of biomass C on the day 5, as a consequence of the increased amount of available organic substrate. A general increase of the biomass, especially between the days 47 and 89, was observed for all treatments. Towards the end of the incubation period, however, a stabilization phase was established and no differences were observed among the treatments. It is probable that after 180 days the microorganisms were in equilibrium with their resources and no drastic biomass changes could thus be expected. The usefulness of an organic material as fertilizer is affected by the rate at which it decomposes when added to soil. The evolution of CO2 is generally considered an index of the microbial activity that primarily regulates the decomposition rate of any kind of organic material in soil. Our incubation experiment demonstrated that the CO2 evolution from the soil-sludge mixtures was maximum in the first few days following the incorporation of sludge into the soil, and then decreased (Fig. 1). High initial levels of microbial activity in both treatments were probably supported by readily available C from easily decomposed C components, such as water-soluble sugars, amino acids, etc. Thereafter, the lower decomposition rates reflect the greater resistance of recalcitrant materials. The total loss of CO2-C (Fig. 1) over the incubation period amounted to about 3% of the added organic carbon, and this figure was similar to that found by Levi-Minzi et al. (1990) when soil was incubated with municipalrefuse compost and cattle farmyard-manure in a similar laboratory investigation. Remarkably higher percentages were found in soil amended with aerobic sewage sludge, pig-slurry, rye straw, poultry manure (Levi-Minzi et al., 1990) and sludge coming from oliveoil processing (Saviozzi et al., 1993a); these latter results may be the response to the more readily-available C in the easily decomposing compounds in these organic materials. On the basis of these findings, winery-sludges seem to be promising materials for improving the organic matter level of a soil and maintaining its biological activity. A mathematical description of the mineralization curves was used to analyze the cumulative CO2-C data through the first-order, non-linear regression model:
177
Parameters calculated according to the model are also reported in Table 5. As expected, the amount of carbon that can be mineralized (Co) and the rapidly mineralizing sub-fraction (C1) increased with increasing addition of sludge. Positive C~ values of the treated soil indicate that no lag-phase in the initial period of incubation occurred (Ellert & Bettany, 1988), so confirming the lack of toxic effects due to the application of the material. Finally, Table 5 reports data of the decomposition rate (k) of the control and treated soil. As can be seen, sludge applied at the two doses had no appreciable influence upon the rates of CO2-C evolution. Although the sludge produced some beneficial changes in the soil, in these laboratory conditions the incorporation of the highest dose of sludge increased soil salinity to a hazardous level and, for this reason, in order to assess the suitability of the winery-sludge for agricultural purposes, it would be advisable to verify the extent of salinization under field conditions. ACKNOWLEDGEMENTS This research was supported by the MURST (Grant 40%).
Table 5. Parameter estimates and coefficients of determination according to the first-order model for C mineralization of the sludge-soil systems Sludge applied
Co o
C~
k
Re
0 0"5 2"5
32"4 38"4 59"7
1"6 1"7 2"9
- 0"104 - 0"095 - 0"092
0'995 0'996 0'995
(%)
~For meaning of symbols see text.
60-
-4
40-
. ° " . o j . ~" ,
C, = C,,(1 - e
k,)+ Cl (Jones, 1984)
where C,= cumulative amount of C mineralized after time t (mg 100 g- ~of amended soil); t = time from start of incubation when t = 0 (days); Co=potentially mineralizable C (mg 100 g-i of amended soil); C~ =easily mineralizable C (mg 100 g-i of amended soil); k = rate constant. Such a model, already found to be the most suitable among several kinetic models in describing the decomposition processes of many types of organic material (Saviozzi et al., 1993b), also shows, in this investigation, R ~-values that are always higher than 0"99 (Table 5).
0"~"
0
o 0
.
~
-
c
g
ca20-~ E
o 1 o~
10-
[
0
~ 0
Fig. 1.
2
+
s o i l -0- soil + 0 . 5 % , , , , , 4
6
8
10
12
sludge , , 14
16
days
-~- soil + 2 . 5 % sludge ~ , , , ~ 18
20
22
24
26
0 28
f loss during the decomposition of the sludge-soil
system. Continuous lines indicate the CO2-C evolved on the soil basis (left scale); dotted lines indicate the C02-C evolved as percent of the total organic carbon (right scale).
178
A. Saviozzi, R. Levi-Minzi, R. Riffaldi, R. Cardelli
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Levi-Minzi, R., Rifffaldi, R. & Saviozzi, A. (1990). Carbon mineralization in soil amended with different organic materials. Agric. Ecosys. Environ., 31,325-35. Lundgren, B. (1981). Fluorescein diacetate as a strain of metabolically active bacteria in soil. Oikos, 36, 17-22. Morisot, A. (1986). Utilisation agricole de quelques d6chets de distilleries de vins rouges. Agronomie, 6,203-12. Nelson, D. W. & Sommers, L. E. (1982). Total carbon, organic carbon and organic matter. In Methods of Soil Analysis, Part 2, ed. A. L. Page et al. Am. Soc. Agron., Madison, WI, pp. 539-94. Perez, J. D. & Gallardo-Lara, F. (1987). Effects of the application of wastewater from olive processing on soil nitrogen transformation. Comm. Soil Sci. Plant Anal., 18, 1031-5. Riffaldi, R., Levi-Minzi, R., Saviozzi, A., Vanni, G. & Scagnozzi, A. (1993). Effect of the disposal of sludge from olive processing on some soil characteristics: laboratory experiments. Water, Air and Soil Pollut., 69, 257-64. Saviozzi, A., Levi-Minzi, R., Riffaldi, R. & Lupetti, A. (1991 ). Effetti dello spandimento di acque di vegetazione sul terreno agrario. Agrochimica, 35, 135-48. Saviozzi, A., Riffaldi, R., Levi-Minzi, R., Scagnozzi, A. & Vanni, G. (1993a). Decomposition of vegetation-water sludge in soil. Bioresource Technol., 44,223-8. Saviozzi, A., Levi-Minzi, R. & Riffaldi, R. (1993b). Mineralization parameters from organic materials added to soil as a function of their chemical composition. Bioresource Technol., 45, 131-5. Schnurer, J. & Rosswall, T. (1982). Fluorescein diacetate hydrolysis as a measure of total microbial activity in soil and litter. App. Environ. Microb., 43, 1256-61. Schnurer, J., Clarholm, M. & Rosswall, T. (1985) Microbial biomass and activity in an agricultural soil with different organic matter contents. Soil Biol. Biochem., 17, 611-18. Soderstrom, B. E. (1977). Vital staining of fungi in pure cultures and in soil with fluorescein diacetate. Soil Biol. Biochem., 9, 59-63. Strivastava, N. & Sahai, R. (1987). Effects of distillery waste on the performance of Cicer arietinum L. Environ. Pollut., 43, 91-102., Zar, J. H. (1984). Biostastical Analysis. Prentice Hall, Englewood Cliffs, NJ. Zucconi, E, Pera, A., Forte, M. & De Bertoldi, M. (1981). Evaluating toxicity of immature compost. BioCycle, 22, 54-7.