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A comparative characterization of organic matter in agrarian and forest soils from Italy
EnvironmentInternational,Vet.20, No. 3, pp. 419-424,1994 Copyright@1994ElsevierScienceLtd Printed in the USA. All rightsrestored
Pergamon
0160-4120/94 $6.00 +.00
A COMPARATIVE CHARACTERIZATION OF ORGANIC MATTER IN AGRARIAN AND FOREST SOILS FROM ITALY
A. Benedetti, F. Alianiello, S. Dell'Orco, and S. Canali Istituto $perimentale per la Nutrizione delle Piante (Ministero per le risorse Agricole, Alimenteri e Forestali), via della Navieella 2, 00184 Roma, Italy
EI 9306-154 M (Received 15 June 1993; accepted 18 December 1993)
Italian forest and agrarian soils were analyzed utilizing electrofocusing. The results showed that while the agrarian soils displayed great regularity in their profiles, the profiles from forest soils showed different characteristics. A determination of the humification parameters by the polyvinylpyrrolidone (PVP) technique was also carried out. The results agreed with those obtained from eleetrofoeusing. Soils that showed the highest amount of organic matter in peaks at the highest pH values had the highest values of the humification indices.
INTRODUCTION phoretic separation of amphoteric substances on a pH gradient. The fractionation obtained, suitably displayed by a densitometer, allows an adequate characterization of the organic matter and provides information useful for the understanding of its humification level (De Nobili 1988). In this study, electrofocusing is used to compare five forest and three agrarian soils with different pedoagronomic characteristics, with the objective of identifying similarities and differences in the EF fractionation of their organic matter. Information on soil management and on the transformations of the organic matter was sought. Results from electrofocusing were compared with those coming from the PVP technique according to the method of Sequi et al. (1986). The purposes were to evaluate the relationship between the two techniques and confirm them as sources of effective information on the humification levels of organic matter of soils.
The characterization of organic matter in soil remains open for study especially related to the separation of humic substances from inorganic matter in soil. Recently, many analytical techniques have been used to obtain a greater knowledge of the organic matter in soil. The complexity of these substances requires multiple approaches which can provide information about their origin and their humification level. Spectroscopical techniques, such as ultra violet spectroscopy (UV), Fourier transform infrared spectroscopy (FTIR), and Nuclear Magnetic Resonance (NMR) (Stevenson 1982; Senesi 1990; Malcolm 1989); separation techniques such as ultracentrifugation, viscosimetry (Chen and Schnitzer 1976), and gel chromatography (De Nobili et al. 1989); or destructive techniques such as gas chromatographic pyrolysis (Trasar-Cepada et al. 1992), and thermal differential analysis (Mitchell and Birnie 1970) have given information on humic substances. Electrofocusing (EF) is based on the electro-
419
420
A. Benedetti et al.
MA
CE
TM I
3.5
5.~
3.9 pH I
1
~
2
I
I
3
4
1
5
cm
Fig. 1. EF profiles of three agrarian soils. A = absorbance.
MATERIALS AND METHODS
Agrarian soils originated from different experimental fields of the Istituto Sperimentale per la Nutrizione delle Piante (ISNP) in the areas of Tor Mancina (TM), Roma Villa Celimontana (CE), and Mantova (MN). Forest soils were obtained from the forest areas of Monte Venere (MV), Monte Fogliano (MF), Campitelli (CA), and Tolfa (TF1 and TF2). The samples were collected from the 0-40 cm layer (except Tolfa), which is the depth of tillage in the agrarian soils and the depth of the organic layer in the forest soils. The samples from Tolfa were taken at two different depths, the TF1 layer (0-20 cm) and the TF2 layer (0-40 era). The soils were air dried, sieved to pass a 2-ram screen, and analyzed. Their
chemical-physical characteristics were determined according to the methods of the Societ~ Italiana di Scienza del Suolo (SISS 1985) (Table 1 and 2). The organic matter was extracted from the soils by a solution of NaOH (0.1 M) and Na4P20./ (0.1 M). The extracts were used for both EF fractionation and determination of degree of humification (DH) and humification rate (HR) according to the PVP method (Sequi et al. 1986). This method consists of an extraction and subsequent chromatographic separation on a PVP column of humie and fulvic acids. The quantities of humic acids (HA), fulvic acids (FA), total organic carbon (TOC), and total extractable carbon (TEC) were determined. The results were computed using the following equations:
Organic matter in Italian soils
421
RESULTS AND DISCUSSION
DH = (HA+FA)/TEC. 100 and HR = (HA+FA)/TOC. 100 A 4-g sample of soil was weighed and extracted with 100 mL of extracting solution. Subsequently, it was dialyzed and simultaneously concentrated by an ultrafiltration system (Microprodicon) on membranes of 5000 Molecular Weight Cut Off. The fractionation was performed on a polyacrylamide gel plate 0.5-ram thick in a pH range of 3.5-7.0 for 3 h under a 1500 V tension. In order to avoid contacts with the atmospheric CO 2, a plastic film was placed over the gel. At the end of the fractionation, the p l a s t i c film was removed and the pH values of the gel were measured along the plate with a surface electrode. After staining with Comassie Blue 250, the electrofocusing profiles were read by a laser gel scanner. From the same extract, DH and HR were determined. All analyses were carried out in triplicate.
The electrofocusing profiles of the three agrarian soils are shown in Fig. 1. There is a similarity in the agrarian soils. These profiles consist of a characteristic small band at about pH of 3.7 and a broad band, probably caused by many bands close to each other, between 4.3 and 4.8 pH. It is necessary to point out the poor amount of organic matter in most of all Italian agrarian soils, likely to have been caused by intensive cultivation, tillage, and irrigation without sufficient return of organic matter. In such a situation, labile and stabile organic matter is lost. The organic matter of these soils is probably in a condition of non-equilibrium, where the stabile fraction is poor and continuously mineralizing. The values of DH and HR are lower than those of forest soils, reflecting this situation (Table 3). Figure 2 shows the related profiles of forest soils TF2, TF1, MF, MV, and CA. The EF profiles of forest soils are more variable than for agrarian soils. The forest soils are clearly different even though some of their pedogenetic origins are the same. There is also
Table 1. Chemical-physical characteristics of the three agrarian soils. Values refer to dry matter at 105"C. LOCATION
MANTOVA
CELIMONTANA
TOR MANCINA
MN
CE
TM
Sand %
13.9
65.7
77.6
Silt %
60.1
29.0
19.7
Clay %
26.0
5.3
2.7
Texture
Loam
Silty loam
Silty loam
Sample
Field capacity % (pF = 2.5)
24.5
24.0
15.3
pH (H20 1:2.5)
8.1
8.0
7.3
Organic C % (Spdnger-Klee)
1.7
2.8
1.6
Humus % (Cxl.724)
2.9
4.8
2.8
Total N % (Kjeldhal)
0.08
0.15
0.09
C/N
21
18
17
Exchangeable K (mg/kg)
649
3833
2860
Available P (mg/kg)
182
58
79
Cationic Exchange Capacity (meq/100g)
20.3
50.6
27.8
422
A. Benedetti et al,
TF2
TF1
MF
MV
CA
3.5
'3.8
'4.1
'4.4
4.8
5.2
pH
cm Fig. 2. EF profiles of five forest soils. A = absorbanee.
a flat concentration of bands with acidic pH values, indicating poorly humified matter. The comparison between TF1 and TF2 is interesting. The sample at 0-20 cm had the same characteristics as that of 20-40 cm in the pH interval 3.5-4.2. However, going towards the neutral values (4.3-5.2) the deepest sample showed a series of bands that were lacking in the sample from the upper level. The bands in the TF2 profile in the pH range of 4.3-4.8 indicate the stabile, humified fraction of organic matter that is lacking in the TF1 soil. Present here is organic matter principally derived from recently decomposed biological residues, where the processes of humification are still at the initial stage. The MF soil is completely lacking material in the 4.3-4.8 pH range.
An evaluation of the results of PVP indicates that DH and HR of TF1 and TF2 are both high, but different. These samples represent two moments in the evolution of the organic matter of the same soil. The TF1 soil represents the younger, less-humified one, for which values of DH and HR are lower than those of the TF2, the oldest and most-humified one. The difference in these values can be related also to the differences in the EF profiles, in particular to the band in the 4.3-4.8 range of pH, that is lacking in the TF1 profile. This is confirmed by the MF soil, which lacks that band: its DH and HR values are the lowest of all the forest soils. However, comparing the values of D H and HR of agrarian soils with those of the forest soils is difficult because of the complex dynamics of mineralization of organic matter.
Organic matter in Italian soils
423
Table 2. Chemical-physical characteristics of the five forest soils. Values refer to dry matter at 105'C. LOCATION Sample Vegetative covering
I
CAMPITELLI
TOLFA 1
TOLFA 2
VENERE
MF
CA
TFI
TF2
MV
Beech
Beech
Mixed turkey
Mixed turkey oak
Beech
oak
FOGLIANO
Sand %
76
54
46
32
67
Silt %
22
30
29
30
31
Clay %
2
16
25
38
2
Texture
Silty loam
Silty loam
Loam
Clayey loam
Silty loam
pH (H20 1:2.5)
5.5
5.1
6.9
6.5
5.7
Organic C (Springer-Klee)
12.1
6.0
9.5
4.1
6.4
Humus % (Cxl.724)
20.9
10.3
16.4
7.1
11
Total N % (Kjeldhal)
0.81
0.41
0.58
0.31
0.62
C/N
14.9
14.6
16.4
13.6
10.3
Exchangeable K (mg/kg)
1102
221
1686
1039
624
Available P (mg/kg)
13.5
16.3
25
13.6
9.4
Cationic Exchange Capacity (meq/10Og)
65
34.3
50
52.5
56.9
E l e c t r o f o c u s i n g profiles o f soil organic matter can provide information on soil management. Agrarian soils show great similarity, while forest soils are variable. It is possible to single out a range (4.3-4.8) of pH in the EF profile where highly humified matter
is present. This range corresponds to highest pH values with large quantities of organic matter. A comparison of EF with the PVP method confirms the suitability of both methods to give information on the humification level of organic matter of soils.
Table 3. Total organic carbon (TOC), total extractable carbon (TEC), degree of humification (DH), and humification rate (HR) of the soils,
CA
MF
MV
TFI
TF2
MN
CE
TM
TOC %
6.0
12.1
6.4
9.5
4.1
1.7
2.8
1.6
TEC %
3.0
3.8
5.6
5.9
2.6
1.2
1.9
1.2
C % (HA+FA)
2.0
1.9
3.9
4.4
2.2
0.4
1.0
0.6
DH %
66.7
49.3
69.5
62.1
84.6
33.3
52.6
50.0
HR %
33.3
16.0
61,0
46.3
53.7
23.5
35.7
37.5
Sample
424
REFERENCES Chen, Y.; Schnitzer, M. Viscosity measurements on soil humic substances. Soil Sci. Soc. Am. J. 40: 866-872; 1976. De Nobili, M. J. Electrophoretic evidence of the integrity of humic substances separated by means of electrofocusing. Soil Sci. 39: 437-445; 1988. De Nobili, M.; Gjessing, E.; Sequi, P. Size and shapes of humic substances by gel chromatography. In: Humic Substances II. New York: Wiley Interscience; 1989. Malcolm, R.L. Application of solid-state 13C NMR spectroscopy to geochemical studies of humic substances. In: Humic Substances II. New York: Wiley Interscience; 1989. Mitchell, B.D.; Birnie, A.C. Section C: Biological materials. In: McKenzie, R.C., ed. Differential thermal analysis 1.London/New York: Academic Press; 1970.
A. Benedetti et al,
Senesi, N. Molecular and quantitative aspects of the chemistry of fulvic acid and its interaction with metal ions and organic chemicals--I: the electron spin resonance approach. Anal. Chim. Acta 232: 51-75; 1990. Sequi P., De Nobili, M.; Leita, L.; Cercignani, G. A new index of humification. Agrochimica 30: 175-179; 1986. SISS (Societ~tItaliana di Scienza del Suolo). Metodi normalizzati di analisi del suolo. Bologna: Edagricole; 1985. Stevenson, F.J. Humus chemistry: Genesis, composition, and reactions. New York: Wiley Interscience; 1982. Trasar-Cepada, M.C.; Ceccanti, B.; Leir~s, M.C.; Calcinai M.; Gil-Sotres F. Pyrolysis/gas chromatography characterization of organic matter in lignite mine soils at different evolution ages. In: Senesi, N.; Miano, T.M., eds. Proc. 6th IHSS meeting on humic substances in the global environment and implications for human health; Monopoli, Italy; 1992. Amsterdam: Elsevier; 1994. (In press)
Pergamon
0160-4120/94 $6.00 +.00
A COMPARATIVE CHARACTERIZATION OF ORGANIC MATTER IN AGRARIAN AND FOREST SOILS FROM ITALY
A. Benedetti, F. Alianiello, S. Dell'Orco, and S. Canali Istituto $perimentale per la Nutrizione delle Piante (Ministero per le risorse Agricole, Alimenteri e Forestali), via della Navieella 2, 00184 Roma, Italy
EI 9306-154 M (Received 15 June 1993; accepted 18 December 1993)
Italian forest and agrarian soils were analyzed utilizing electrofocusing. The results showed that while the agrarian soils displayed great regularity in their profiles, the profiles from forest soils showed different characteristics. A determination of the humification parameters by the polyvinylpyrrolidone (PVP) technique was also carried out. The results agreed with those obtained from eleetrofoeusing. Soils that showed the highest amount of organic matter in peaks at the highest pH values had the highest values of the humification indices.
INTRODUCTION phoretic separation of amphoteric substances on a pH gradient. The fractionation obtained, suitably displayed by a densitometer, allows an adequate characterization of the organic matter and provides information useful for the understanding of its humification level (De Nobili 1988). In this study, electrofocusing is used to compare five forest and three agrarian soils with different pedoagronomic characteristics, with the objective of identifying similarities and differences in the EF fractionation of their organic matter. Information on soil management and on the transformations of the organic matter was sought. Results from electrofocusing were compared with those coming from the PVP technique according to the method of Sequi et al. (1986). The purposes were to evaluate the relationship between the two techniques and confirm them as sources of effective information on the humification levels of organic matter of soils.
The characterization of organic matter in soil remains open for study especially related to the separation of humic substances from inorganic matter in soil. Recently, many analytical techniques have been used to obtain a greater knowledge of the organic matter in soil. The complexity of these substances requires multiple approaches which can provide information about their origin and their humification level. Spectroscopical techniques, such as ultra violet spectroscopy (UV), Fourier transform infrared spectroscopy (FTIR), and Nuclear Magnetic Resonance (NMR) (Stevenson 1982; Senesi 1990; Malcolm 1989); separation techniques such as ultracentrifugation, viscosimetry (Chen and Schnitzer 1976), and gel chromatography (De Nobili et al. 1989); or destructive techniques such as gas chromatographic pyrolysis (Trasar-Cepada et al. 1992), and thermal differential analysis (Mitchell and Birnie 1970) have given information on humic substances. Electrofocusing (EF) is based on the electro-
419
420
A. Benedetti et al.
MA
CE
TM I
3.5
5.~
3.9 pH I
1
~
2
I
I
3
4
1
5
cm
Fig. 1. EF profiles of three agrarian soils. A = absorbance.
MATERIALS AND METHODS
Agrarian soils originated from different experimental fields of the Istituto Sperimentale per la Nutrizione delle Piante (ISNP) in the areas of Tor Mancina (TM), Roma Villa Celimontana (CE), and Mantova (MN). Forest soils were obtained from the forest areas of Monte Venere (MV), Monte Fogliano (MF), Campitelli (CA), and Tolfa (TF1 and TF2). The samples were collected from the 0-40 cm layer (except Tolfa), which is the depth of tillage in the agrarian soils and the depth of the organic layer in the forest soils. The samples from Tolfa were taken at two different depths, the TF1 layer (0-20 cm) and the TF2 layer (0-40 era). The soils were air dried, sieved to pass a 2-ram screen, and analyzed. Their
chemical-physical characteristics were determined according to the methods of the Societ~ Italiana di Scienza del Suolo (SISS 1985) (Table 1 and 2). The organic matter was extracted from the soils by a solution of NaOH (0.1 M) and Na4P20./ (0.1 M). The extracts were used for both EF fractionation and determination of degree of humification (DH) and humification rate (HR) according to the PVP method (Sequi et al. 1986). This method consists of an extraction and subsequent chromatographic separation on a PVP column of humie and fulvic acids. The quantities of humic acids (HA), fulvic acids (FA), total organic carbon (TOC), and total extractable carbon (TEC) were determined. The results were computed using the following equations:
Organic matter in Italian soils
421
RESULTS AND DISCUSSION
DH = (HA+FA)/TEC. 100 and HR = (HA+FA)/TOC. 100 A 4-g sample of soil was weighed and extracted with 100 mL of extracting solution. Subsequently, it was dialyzed and simultaneously concentrated by an ultrafiltration system (Microprodicon) on membranes of 5000 Molecular Weight Cut Off. The fractionation was performed on a polyacrylamide gel plate 0.5-ram thick in a pH range of 3.5-7.0 for 3 h under a 1500 V tension. In order to avoid contacts with the atmospheric CO 2, a plastic film was placed over the gel. At the end of the fractionation, the p l a s t i c film was removed and the pH values of the gel were measured along the plate with a surface electrode. After staining with Comassie Blue 250, the electrofocusing profiles were read by a laser gel scanner. From the same extract, DH and HR were determined. All analyses were carried out in triplicate.
The electrofocusing profiles of the three agrarian soils are shown in Fig. 1. There is a similarity in the agrarian soils. These profiles consist of a characteristic small band at about pH of 3.7 and a broad band, probably caused by many bands close to each other, between 4.3 and 4.8 pH. It is necessary to point out the poor amount of organic matter in most of all Italian agrarian soils, likely to have been caused by intensive cultivation, tillage, and irrigation without sufficient return of organic matter. In such a situation, labile and stabile organic matter is lost. The organic matter of these soils is probably in a condition of non-equilibrium, where the stabile fraction is poor and continuously mineralizing. The values of DH and HR are lower than those of forest soils, reflecting this situation (Table 3). Figure 2 shows the related profiles of forest soils TF2, TF1, MF, MV, and CA. The EF profiles of forest soils are more variable than for agrarian soils. The forest soils are clearly different even though some of their pedogenetic origins are the same. There is also
Table 1. Chemical-physical characteristics of the three agrarian soils. Values refer to dry matter at 105"C. LOCATION
MANTOVA
CELIMONTANA
TOR MANCINA
MN
CE
TM
Sand %
13.9
65.7
77.6
Silt %
60.1
29.0
19.7
Clay %
26.0
5.3
2.7
Texture
Loam
Silty loam
Silty loam
Sample
Field capacity % (pF = 2.5)
24.5
24.0
15.3
pH (H20 1:2.5)
8.1
8.0
7.3
Organic C % (Spdnger-Klee)
1.7
2.8
1.6
Humus % (Cxl.724)
2.9
4.8
2.8
Total N % (Kjeldhal)
0.08
0.15
0.09
C/N
21
18
17
Exchangeable K (mg/kg)
649
3833
2860
Available P (mg/kg)
182
58
79
Cationic Exchange Capacity (meq/100g)
20.3
50.6
27.8
422
A. Benedetti et al,
TF2
TF1
MF
MV
CA
3.5
'3.8
'4.1
'4.4
4.8
5.2
pH
cm Fig. 2. EF profiles of five forest soils. A = absorbanee.
a flat concentration of bands with acidic pH values, indicating poorly humified matter. The comparison between TF1 and TF2 is interesting. The sample at 0-20 cm had the same characteristics as that of 20-40 cm in the pH interval 3.5-4.2. However, going towards the neutral values (4.3-5.2) the deepest sample showed a series of bands that were lacking in the sample from the upper level. The bands in the TF2 profile in the pH range of 4.3-4.8 indicate the stabile, humified fraction of organic matter that is lacking in the TF1 soil. Present here is organic matter principally derived from recently decomposed biological residues, where the processes of humification are still at the initial stage. The MF soil is completely lacking material in the 4.3-4.8 pH range.
An evaluation of the results of PVP indicates that DH and HR of TF1 and TF2 are both high, but different. These samples represent two moments in the evolution of the organic matter of the same soil. The TF1 soil represents the younger, less-humified one, for which values of DH and HR are lower than those of the TF2, the oldest and most-humified one. The difference in these values can be related also to the differences in the EF profiles, in particular to the band in the 4.3-4.8 range of pH, that is lacking in the TF1 profile. This is confirmed by the MF soil, which lacks that band: its DH and HR values are the lowest of all the forest soils. However, comparing the values of D H and HR of agrarian soils with those of the forest soils is difficult because of the complex dynamics of mineralization of organic matter.
Organic matter in Italian soils
423
Table 2. Chemical-physical characteristics of the five forest soils. Values refer to dry matter at 105'C. LOCATION Sample Vegetative covering
I
CAMPITELLI
TOLFA 1
TOLFA 2
VENERE
MF
CA
TFI
TF2
MV
Beech
Beech
Mixed turkey
Mixed turkey oak
Beech
oak
FOGLIANO
Sand %
76
54
46
32
67
Silt %
22
30
29
30
31
Clay %
2
16
25
38
2
Texture
Silty loam
Silty loam
Loam
Clayey loam
Silty loam
pH (H20 1:2.5)
5.5
5.1
6.9
6.5
5.7
Organic C (Springer-Klee)
12.1
6.0
9.5
4.1
6.4
Humus % (Cxl.724)
20.9
10.3
16.4
7.1
11
Total N % (Kjeldhal)
0.81
0.41
0.58
0.31
0.62
C/N
14.9
14.6
16.4
13.6
10.3
Exchangeable K (mg/kg)
1102
221
1686
1039
624
Available P (mg/kg)
13.5
16.3
25
13.6
9.4
Cationic Exchange Capacity (meq/10Og)
65
34.3
50
52.5
56.9
E l e c t r o f o c u s i n g profiles o f soil organic matter can provide information on soil management. Agrarian soils show great similarity, while forest soils are variable. It is possible to single out a range (4.3-4.8) of pH in the EF profile where highly humified matter
is present. This range corresponds to highest pH values with large quantities of organic matter. A comparison of EF with the PVP method confirms the suitability of both methods to give information on the humification level of organic matter of soils.
Table 3. Total organic carbon (TOC), total extractable carbon (TEC), degree of humification (DH), and humification rate (HR) of the soils,
CA
MF
MV
TFI
TF2
MN
CE
TM
TOC %
6.0
12.1
6.4
9.5
4.1
1.7
2.8
1.6
TEC %
3.0
3.8
5.6
5.9
2.6
1.2
1.9
1.2
C % (HA+FA)
2.0
1.9
3.9
4.4
2.2
0.4
1.0
0.6
DH %
66.7
49.3
69.5
62.1
84.6
33.3
52.6
50.0
HR %
33.3
16.0
61,0
46.3
53.7
23.5
35.7
37.5
Sample
424
REFERENCES Chen, Y.; Schnitzer, M. Viscosity measurements on soil humic substances. Soil Sci. Soc. Am. J. 40: 866-872; 1976. De Nobili, M. J. Electrophoretic evidence of the integrity of humic substances separated by means of electrofocusing. Soil Sci. 39: 437-445; 1988. De Nobili, M.; Gjessing, E.; Sequi, P. Size and shapes of humic substances by gel chromatography. In: Humic Substances II. New York: Wiley Interscience; 1989. Malcolm, R.L. Application of solid-state 13C NMR spectroscopy to geochemical studies of humic substances. In: Humic Substances II. New York: Wiley Interscience; 1989. Mitchell, B.D.; Birnie, A.C. Section C: Biological materials. In: McKenzie, R.C., ed. Differential thermal analysis 1.London/New York: Academic Press; 1970.
A. Benedetti et al,
Senesi, N. Molecular and quantitative aspects of the chemistry of fulvic acid and its interaction with metal ions and organic chemicals--I: the electron spin resonance approach. Anal. Chim. Acta 232: 51-75; 1990. Sequi P., De Nobili, M.; Leita, L.; Cercignani, G. A new index of humification. Agrochimica 30: 175-179; 1986. SISS (Societ~tItaliana di Scienza del Suolo). Metodi normalizzati di analisi del suolo. Bologna: Edagricole; 1985. Stevenson, F.J. Humus chemistry: Genesis, composition, and reactions. New York: Wiley Interscience; 1982. Trasar-Cepada, M.C.; Ceccanti, B.; Leir~s, M.C.; Calcinai M.; Gil-Sotres F. Pyrolysis/gas chromatography characterization of organic matter in lignite mine soils at different evolution ages. In: Senesi, N.; Miano, T.M., eds. Proc. 6th IHSS meeting on humic substances in the global environment and implications for human health; Monopoli, Italy; 1992. Amsterdam: Elsevier; 1994. (In press)