to denature soil enzymes. Thus, three independent treat- ments were applied to soils beneath non-acid-misted, stan-
dard-dose acid-misted, and double-dose acid-misted trees: i control; ii HgCl
2
-addition; and iii HgCl
2
-addition plus autoclaving.
Immediately after treatment, all soils were examined for resin-extractable P during a 48 h incubation by shaking
samples, 2 g, with 30-ml deionised water and one resin- bag 1-g of oven-dry-equivalent Dowex 1-X8 resins satu-
rated with Cl
2
in 50-ml centrifuge tubes in a reciprocal shaker 150 strokes min
21
. To ensure complete sterilisation of soils by the HgCl
2
and the HgCl
2
1 autoclaving treat- ments, and to mantain sterile conditions during incubation
with resins, additional HgCl
2
2500 mg kg
21
of dry soil was added along with the 30-ml of water to the correspond-
ing centrifuge tubes. After incubation, resins were extracted with 30 ml 0.5 M HCl on a reciprocal shaker for 1 h. Phos-
phate concentration in the solutions was analysed using the ascorbic acid–molybdenum blue method of John 1970.
Phosphorus extracted by resins from the control soils reveals the net balance between solubilisation of inorganic
P, mineralisation of organic P, and immobilisation of solu- tion P. Resin-extractable P from the HgCl
2
-treated samples results from the sum of solubilised P and mineralised P
HgCl
2
-sterilisation avoids microbial immobilisation of solution P. Phosphorus from the HgCl
2
-treated plus auto- claved soils comes from the solubilisation of inorganic P
only autoclaving of HgCl
2
-treated soils additionally avoids P mineralisation by phosphatase enzymes, and gives an
estimate of net P solubilisation rate. The difference in resin-extractable P between the HgCl
2
-addition and the HgCl
2
-addition plus autoclaved treatments is an estimate of gross P mineralisation rate, whereas the difference
between the HgCl
2
-addition and control treatments is an estimate of P immobilisation rate.
To check for the direct effect of HgCl
2
-addition and auto- claving on resin-extractable P, three subsamples of 2 g
moist soil from each of the control, HgCl
2
-addition, and HgCl
2
-addition plus autoclaving treatments were extracted with anion exchange resin bags as above except that extrac-
tion time was only 1 h. If there were differences in resin-P concentrations before and after HgCl
2
-addition and HgCl
2
- addition plus autoclaving, correction factors were applied in
the calculation of P transformation rates to allow for direct treatment effects Zou et al., 1992. Effects of treatments on
phosphatase activity were also tested.
2.4. Statistical analysis A 2-way ANOVA was performed to determine for the
effects of acid-misting non-acid-misting, and standard- dose acid-misting and tree height class H1–H5 on soil
and litter P fractions. A 1-way ANOVA was performed pooling data from different height classes to determine the
dose-effect of acid-mist application non-acid-misting, stan- dard acid-mist dose, and double acid-mist dose. When no
significant effect of height class was found in the previous analysis, unweighted means for all height classes combined
were calculated for the non-acid-mist and the standard acid- mist dose treatments, and compared to the double acid-mist
dose treatment. Otherwise, we used in the analysis non-acid- misting and standard acid-mist dose weighted means with
the same proportion of height classes as in the double acid- mist dose chamber 2 H3:1 H4:1 H5.
To analyse data resulting from the application of the Zou et al. 1992 method, an ANOVA was first applied to test for
significant effects of treatments on resin-extractable P comparing the amount of P extracted by resins during 1 h
in the control, HgCl
2
-treated, and HgCl
2
-treated plus auto- claved soils. When significant differences appeared, correc-
tion factors were applied to estimate net P solubilisation rate, gross P mineralisation rate, and P immobilisation rate
Zou et al., 1992.
ANOVA requirements of normality Kolmogorov–Smir- nov test and homogeneity Bartlett–Box F test were
checked at
a
:
01 :
For variables that did not meet such requirements, log x 1 1 transformation was enough to
achieve normality and homogeneity.
3. Results
3.1. Soil acidity There were no differences in loss-on-ignition between
J.A. Carreira et al. Soil Biology Biochemistry 32 2000 1857–1865 1860
Table 2 ANOVA results on effects of acid-mist treatment and tree height class A, and on acid-mist dose level applied to the tree canopy non-acid-misting, standard-
dose, and double-dose acid-misting treatments B for soil phosphatase activity and soil labile P pools of the Glencorse experiment see Section 2.4 for further details
V A
B Height class
Acid-mist treatment Interaction
Acid-mist dose level Pase activity
0.000 0.000
0.000 0.035
Pi-resin 0.110
NS
0.084
NS
0.584
NS
0.046 Pi-bicarbonate
0.009 0.533
NS
0.561
NS
0.539
NS
Po-bicarbonate 0.000
0.063
NS
0.000 0.000
P :
01; 2-way ANOVA F-tests. P
0.05.
dose-level acid-mist treatments Table 1. Soil pH 1:1 H
2
O decreased significantly
P 0
: 001 with increasing acid-
mist dose, from 5.09 in soils under non-acid-misted trees to 3.93 in soils under double-dose acid-mist treated trees.
Acid-mist treatments induced little change in soil CEC, which was relatively low in all soils low buffering capa-
city. Consequently, marked changes in the proportion of individual exchangeable cations were observed. Base satura-
tion decreased from near 90 to less than 30, and the Ca 1 Mg:Al ratio decreased from 9.5 to 0.4, among the non-acid-
misting and the double-dose acid-misting treatments.
3.2. Phosphatase activity and respiration rates The effects of tree height-class, acid-mist treatment, and
interaction between them, on the potential rates of phospha- tase activity at the soil pH were all highly significant Tables
2 and 3. Mean phosphatase activity all height classes combined was lower in soils under acid-misted trees
255.1 mg pNP g
21
h
21
than in soils under non-acid-misted trees 400.6 mg pNP g
21
h
21
, although this trend was only consistent for the smallest height classes H1–H3. Soil
phosphatase activity in the double-dose acid-mist treatment was also significantly lower than in the non-acid-mist
treatment.
Soil respiration rates showed a decreasing trend although not significant with the dose of applied acid-mist from
3.69 in the non-acid-mist to 3.09 mmol CO
2
g
21
day
21
in the double-dose treatments, at 30 moisture content
J.A. Carreira et al. Soil Biology Biochemistry 32 2000 1857–1865 1861
Table 3 Phosphatase activity mg pNP h
21
g
21
dw and labile P pools g m
22
in soils under non-acid-misted, standard-dose acid-misted, and double-dose acid-misted trees of different height classes H1–H5, from the shortest to the tallest at the Glencorse field experiment. Data are means SE. When there were significant
differences between height classes, weighted means
n 16 for the ‘non-acid-misting’ and ‘standard-dose acid-mist’ treatments with the same proportion of height classes as in the ‘double-dose acid.mist’ treatment
n 4
; 2:1:1 H3:H4:H5 were calculated to assess for an acid-misting dose effect. Otherwise,
unweighted means
n 20 were used. Means with different superscript letters are significantly different at the P 0 :
05 level Tukey’s HSD following ANOVA
Acid-mist treatment Height classes
H1 H2
H3 H4
H5 All combined
unweighted means H3:H4:H5
2:1:1, weighted means Phosphatase activity
Non-acid-misting 496.8 42.8
361.6 46.4 383.0 48.1
379.9 8.3 382.0 37.6
400.6 382.0
A
17.5 Standard-dose
137.4 28.8 198.6 19.0
116.4 25.8 366.8 50.1
456.1 57.4 255.1
263.9
B
43.3 Double-dose
– –
– –
– –
279.9
AB
8.1 Resin-extractable P
Non-acid-misting 0.68 0.16
0.51 0.06 0.54 0.17
0.35 0.07 0.42 0.13
0.50
A
0.06 –
Standard-dose 0.41 0.05
0.53 0.10 0.36 0.05
0.23 0.03 0.40 0.03
0.39
AB
0.03 –
Double-dose –
– –
– –
– 0.29
B
0.04 Bicarbonate-extractable inorganic P
Non-acid-misting 1.49 0.20
1.32 0.09 1.36 0.21
1.04 0.09 0.99 0.09
– 1.19
A
0.09 Standard-dose
1.29 0.10 1.34 0.10
1.22 0.06 0.94 0.09
1.17 0.04 –
1.14
A
0.04 Double-dose
– –
– –
– –
1.30
A
0.03 Bicarbonate-extractable organic P
Non-acid-misting 2.59 0.06
2.58 0.06 2.43 0.13
2.09 0.03 1.89 0.12
– 2.21
A
0.08 Standard-dose
3.12 0.21 2.28 0.07
2.14 0.13 2.23 0.07
2.47 0.08 –
2.25
A
0.06 Double-dose
– –
– –
– –
2.94
B
0.13
Fig. 2. CO
2
release mmol CO
2
g
21
day
21
at 30 moisture content a, and increment in CO
2
release expressed as of initial after increasing moist- ure content from 15 to 30 b, in soils under non-acid-misted, standard-
dose acid-misted, and double-dose acid-misted trees of the Glencorse field experiment.
Fig. 2a. The increment in soil microbial activity measured as respiration rate in response to changing the sample
moisture from 20 to 35 of water holding capacity was significantly higher in soils under non-acid-misted-trees
185 increment from initial respiration rate than in soils under acid-misted soils about 90 increment Fig. 2b.
3.3. Labile P fractions The concentration of the most easily available inorganic
P fractions resin-extractable P, expressed as mg P g
21
dw soil; data not shown decreased significantly from soils
under the smallest to soils under the tallest trees
P :
001 ;
and was a 30 lower in soils under standard-dose acid-misted trees than in soils under non-acid-misted trees
P
: 04
: No significant effects of tree height-class were
observed if data are expressed on an area basis Tables 2 and 3. However, the size of the resin-extractable P pool in 0–
5 cm depth soils significantly
P 0 :
046 and linearly decreased with increasing acid-mist dose levels from
0.50 g m
22
in soils under non-acid-misted trees to 0.29 g m
22
in soils under double-dose acid-misted trees Fig. 3. In contrast, the amount of labile organic P
extracted with bicarbonate significantly increased in soils under acid-mist treated trees from 2.21 g m
22
in soils under non-acid-misted trees to 2.94 g m
22
in soils under double-dose acid-misted trees, representing a 33
increment in the pool size; Fig. 3. The amount of bicarbo- nate-extractable inorganic P was significantly higher in soils
under the smallest than under the tallest trees. Acid-misting did not affect the amount of bicarbonate-extractable inor-
ganic P.
3.4. P transformation rates 3.4.1. Direct effects of HgCl
2
addition and autoclaving treatments
The effects of HgCl
2
addition and autoclaving on resin- extractable P in soils under non-acid-misted, standard-dose
and double-dose acid-misted trees were not significant 1.9 and 1.3 mg of increment in resin-extractable P g
21
in the HgCl
2
-addition and HgCl
2
-addition plus autoclaving treat- ments compared to the control treatment, respectively.
Autoclaving was effective in denaturing soil phosphatase
J.A. Carreira et al. Soil Biology Biochemistry 32 2000 1857–1865 1862
Fig. 3. Pool sizes of soil labile P fractions as a function of tree-height class a and acid-mist dose level b. Data are expressed as the difference from the non-acid-misting treatment.
Fig. 4. Net solubilisation, gross mineralisation, and immobilisation rates of P in soils under non-acid-misted, standard-dose acid-misted, and double-dose acid- misted trees of the Glencorse field experiment. Circles are P pools and arrows indicate processes. Numbers in circles are the net changes in P pool sizes
mg P g
21
soil. Numbers by arrows are P transformation rates in mg P g
21
soil days
21
. Different letters for the same process indicate significant differences among acid-misting treatments Tukey’s HSD following ANOVA.
enzymes as indicated by a reduction of soil phosphatase activity to zero in autoclaved samples. The addition of
HgCl
2
did not affect soil phosphatase activity mean phos- phatase activities of non-treated and HgCl
2
-treated soils were 420.8 and 431.5 mg pNP g
21
h
21
, respectively. Addi- tion of HgCl
2
followed by a one-week incubation at 35 WHC markedly inhibited respiration rates in all soils from
60 to 90 inhibition respect to the corresponding non- HgCl
2
-treated soils, although did not reduced it to zero. Thus, in order to ensure that microbial immobilisation of
solution P was precluded in the HgCl
2
-treated soils during the
48 h incubation
with resins,
additional HgCl
2
2500 mg kg
21
of dry soil was added to the corresponding distilled water–resin bag–soil slurries see Section 2.
3.4.2. Net solubilisation, gross mineralisation and immobilisation rates
Net P solubilisation rates were higher than gross P miner- alisation and net P immobilisation rates Fig. 4. Net P
solubilisation rates were significantly lower
a
:
05 in soils
under double-dose
acid-misted trees
7.3 mg P g
21
day
21
than in soils under non-acid-misted trees 11.9 mg P g
21
day
21
. Gross P mineralisation rates were more than four times greater in the non-acid-misting treat-
ment than in the double-dose acid-misting treatment
P :
08 :
Net P immobilisation rates were very low in all cases. Net changes in soil solution P were all positive and
decreased significantly with increasing dose of acid-mist application. Net changes in organic P pools were all nega-
tive. Soils under non-acid-misted trees showed the highest net decrease in organic P, whereas soils under double-dose
acid-misted trees showed almost no variation in organic P pools. However, the effect of acid-mist dose did not
achieved significance at the
a
:
05 level.
4. Discussion
