1965. The nitrogen mineralised was estimated from the difference between the inorganic N content before and
after incubation. Results are expressed in mg kg
21
of oven-dried soil.
Microbial biomass carbon. Microbial biomass C was determined by the chloroform fumigation extraction
method, using 0.5 M K
2
SO
4
as extractant Vance et al., 1987. The organic C of extracts was estimated by oxidation
with potassium dichromate. The difference in C content between the fumigated and unfumigated extracts was
converted to microbial biomass C expressed in mg kg
21
of oven-dried soil by applying a factor K
c
of 0.45 Jenkin- son, 1988.
Enzymatic activities. Dehydrogenase activity was deter- mined with iodonitrotetrazolium violet INT as substrate,
incubating at pH 7.5 1 M Tris–HCl buffer and 40 8C for
1 h. The iodonitrotetrazolium formazan INTF produced was extracted with a 1:1 v:v mixture of ethanol and
dimethylformamide and measured spectrophotometrically at 490 nm Camin˜a et al., 1998. Activity was quantified
by reference to a calibration curve constructed using INTF standards incubated with soil under the same conditions
described above, and is expressed in mmol INTF g
21
h
21
. Urease activity was determined with urea as substrate,
incubating at pH 7.1 0.2 M phosphate buffer and 37 8C
for 1.5 h and measuring the NH
1 4
released with an ammo- nia-selective gas electrode METROHM Ltd., Herisau,
Switzerland. The enzymatic activity is expressed in mmol NH
3
g
21
h
21
. Acid phosphomonoesterase activity was determined with
p-nitrophenyl phosphate as substrate, incubating at pH 5.0 Modified Universal Buffer and 37
8C. After 30 min 2 M CaCl
2
was added to stop the reaction and to avoid the colouration caused by organic matter, and the p-nitrophe-
nol released was extracted with 0.2 M NaOH and measured spectrophotometrically at 400 nm Tabatabai and Bremner,
1969; Saa´ et al., 1993. b-glucosidase activity was deter- mined as described for phosphomonoesterase activity
except that the substrate was p-nitrophenyl-b-d-glucopyra- noside, the incubation time was 1 h and the p-nitrophenol
released was extracted with 0.1 M THAM–NaOH pH 12.0 Eivazi and Tabatabai, 1988. Both phosphomonoesterase
and b-glucosidase activities were quantified by reference to calibration curves constructed using p-nitrophenol stan-
dards incubated with soil under the same conditions described above and are expressed in mmol p-nitrophe-
nol g
21
h
21
. All
determinations of
enzymatic activities
were performed in triplicate, and all values reported are averages
of the three determinations expressed on an oven-dried soil basis 105
8C. 2.7. Statistical analysis
All data expressed as percentages were compared by using the test of significance of means Spiegel, 1969.
3. Results
Table 1 lists general soil properties, microbial biomass C and N mineralisation capacity, and Table 2 the measured
enzyme activities. Fig. 1 shows enzyme activities per gram of total C or total N content as percentages of the corre-
sponding quotients for the control samples. In the remainder of this section, the trends shown by these results are
described for each kind of pollution.
3.1. Tanning effluent At the site polluted by tanning effluent, the phosphomo-
noesterase activities of samples T1, T2 and T3 were 110 10
; 215 20 and 89 8
; respectively, of the activity in
C. Trasar-Cepeda et al. Soil Biology Biochemistry 32 2000 1867–1875 1869
Table 1 General characteristics of soil samples
Soil sample Total C
Total N CN
pH H
2
O pH KCl
Microbial biomass C mg kg
21
N mineralisation capacity mg kg
21
Tanning effluent T1
13.93 1.267
11 7.29
6.83 426
43.8 T2
18.91 1.803
11 4.57
4.30 517
16.1 T3
10.50 1.039
10 4.33
3.88 139
6.9 T4 control
8.91 0.756
12 6.25
5.31 999
29.9 Landfill effluent
L1 8.56
0.653 13
6.15 5.78
132 50.0
L2 7.26
0.583 13
5.93 5.49
5 26.9
L3 6.85
0.549 13
7.65 7.15
73.4 L4 control
7.59 0.475
16 5.40
4.31 700
2 2.9 Hydrocarbons
H1 6.15
0.470 13
6.75 5.75
595 2.9
H2 6.08
0.600 10
5.48 4.51
521 35.7
H3 4.56
0.410 11
6.45 5.48
376 18.6
H4 control 4.54
0.390 12
6.03 4.42
424 7.4
C. Trasar-Cepe
da et
al. Soil
Biology Biochemistry
32 2000
1867 –
1875 1870
Table 2 Activities of four enzymes in the soil samples mean S.D., and the ranges observed in native Galician soils. In parentheses percentage values relative to the control samples S.D
Soil sample Phosphomonoesterase
a
b -Glucosidase
a
Urease
b
Dehydrogenase
c
Tanning effluent T1
6.41 0.12 110 10 2.64 0.29 109 24
13.76 0.87 47 5 0.853 0.094 61 7
T2 12.49 0.42 215 20
3.32 0.20 137 27 15.44 0.64 53 5
0.616 0.031 44 3 T3
5.20 0.19 89 8 1.29 0.08 53 10
8.16 0.39 28 3 0.333 0.004 24 1
T4 control 5.85 0.50 100
2.42 0.45 100 29.28 2.39 100
1.395 0.042 100 Landfill effluent
L1 4.37 0.36 121 11
1.97 0.38 94 20 18.94 1.87 66 9
0.666 0.100 89 15 L2
1.72 0.12 49 4 0.33 0.05 16 3
11.54 0.48 40 4 0.310 0.023 42 4
L3 1.35 0.07 37 2
0.38 0.00 18 2 23.37 0.83 81 9
0.612 0.009 82 5 L4 control
3.62 0.15 100 2.10 0.19 100
28.91 2.87 100 0.745 0.047 100
Hydrocarbons H1
5.61 0.50 260 25 2.40 0.04 250 7
11.83 1.11 194 18 1.391 0.051 251 13
H2 3.70 0.15 171 9
1.57 0.14 164 15 11.51 2.34 188 38
0.858 0.041 155 9 H3
1.80 0.07 83 4 1.30 0.11 135 12
4.87 0.76 80 12 0.728 0.049 132 10
H4 control 2.16 0.08 100
0.96 0.02 100 6.11 0.00 100
0.553 0.020 100 Native soils
2.33–47.45 0.67–29.63
1.76–66.26 0.10–1.32
a
m mol p-nitrophenol g
21
h
21
.
b
m mol NH
3
g
21
h
21
.
c
m mol INTF g
21
h
21
the control non-polluted sample. b-Glucosidase activity rose to 109 24 in T1 and to 137 27 in T2, and
declined to 53 10 in T3. Urease and dehydrogenase activities fell in all the samples polluted with tanning
effluent: to 47 5 T1, 53 5 T2 and 28 3 T3 of the control for urease and to 61 7 T1,
44 3 T2 and 24 1 T3 of the control for dehydrogenase. All those variations were statistically
significant, except in the case of phosphomonoesterase activity in T1 and T3 samples and of b-glucosidase
activity in T1 sample.
When the enzyme activities are expressed relative to total C or N content Fig.1, the polluted soil samples show
significantly lower values than the control in all cases but phosphomonoesterase activity in T2 sample. In particular,
none of the phosphomonoesterase or b-glucosidase values exceeds the control value, and the b-glucosidase values
exhibit the same trend T1 . T2 . T3 as the urease and dehydrogenase values. Both phosphomonoesterase and b-
glucosidase values, relative to the control, although not significantly different are lower when expressed with
respect to N than when expressed with respect to C Phos- phomonoesterase: T1N 65 6
; T1C 70 6; T2N
90 8 ;
T2C 100 9; T3N 65 6 ;
T3C 76 7
: b
-Glucosidase: T1N 65 17 ;
T1C 70 18; T2 N 58 11
; T2C 67 13; T3N 39 8
; T3C 44
9 :
However, there is no such difference for urease and dehydrogenase, which for a given sample are also similar
one to another when expressed with respect to C or N T1, between 28 3 and 38 4 for both urease and dehydro-
genase; T2 and T3, between 20 2 and 25 2 for urease, and between 17 0 and 19 1 for dehydrogenase.
C. Trasar-Cepeda et al. Soil Biology Biochemistry 32 2000 1867–1875 1871
Fig. 1. Enzyme activities per g of total C or total N content as percentages of the corresponding quotients for the control samples. MonoC, GluC, UreC and DesC: activities of phosphomonoesterase, b-glucosidase, urease and dehydrogenase relative to total soil C, respectively; MonoN, GluN, UreN and DesN:
activities of phosphomonoesterase, b-glucosidase, urease and dehydrogenase relative to total soil N, respectively.
3.2. Landfill effluent At the site polluted by landfill effluent Table 2, the
phosphomonoesterase activities of samples L1, L2 and L3 were, respectively, 121 11
; 49 4 and 37 2 of the
activity in the control sample L4. All the other activities were lower in polluted samples than in the control, and in all
these cases the fall was greatest for sample L2. However, there were marked quantitative differences among the trends
displayed: for b-glucosidase, the fall to 94 20 of control in L1 was not significant, but there was a drastic fall to 16
3 and 18 2 of control in L2 and L3, respectively; for urease, the fall was moderate in L1
66 9 of control,
marked in L2
40 4 and only slight in L3
81 9; while for dehydrogenase, the marked fall in L2
42 4 of
control was accompanied by only slight falls in L1
89 15 and L3
82 5
: When the activities are expressed relative to C and N
contents Fig. 1 they exhibit patterns similar to those described above. However, in contrast to the absolute
values, for L1 the phosphomonoesteraseN value is lower than in the control
88 8 and the phosphomonoester-
aseC value is only slightly higher than in the control
106 10
: The b-glucosidaseC
82 17 and b-glucosidase
N
68 15 values for L1 are lower than those for L4. 3.3. Hydrocarbons
With the sole exceptions of the phosphomonoesterase and urease activities in sample H3, all the enzyme activities
were significantly higher than control values in all the hydrocarbon polluted samples Table 2. The greatest
increases were shown by sample H1, which had values ranging from 194 18 of control for urease to 260
25 of control for phosphomonoesterase. Urease and phos- phomonoesterase activities were somewhat lower, though
still very high, in sample H2
188 38 and 171 9 of control, respectively, but were both lower than the control
values in H3
80 12 and 83 4 ;
respectively. b- Glucosidase and dehydrogenase behaved quite similarly in
all three samples, with values relative to the control of, respectively, 250 7 and 251 13 in H1, 164 15 and
155 9 in H2, and 135 12 and 132 10 in H3; like urease and phosphomonoesterase activities, they decreased
in the order H1
. H2 . H3. When the activities are expressed relative to C and N
contents Fig. 1, the phosphomonoesterase and urease activities of H3 were again the only cases of values lower
than the corresponding control. In contrast, the lowest non- control values for b-glucosidase and dehydrogenase were
recorded for H2.
4. Discussion
