adsorption capacity and the rate of organic phosphorus mineralisation. In addition, associated N saturation creates
enhanced demand for P by the trees relative to the supply from the soil. The combination of these two factors will
induce P deficiency and reduce tree growth Fig. 1.
To test this hypothesis, the effects of acid-mist applied to tree canopies, on tree P nutrition, and soil P cycling and
availability, were assessed in a field manipulation experi- ment in Scotland. Acid-mist treatments had been applied for
four consecutive years to a Sitka spruce plantation and had caused a significant reduction in stem diameter growth
compared to non-acid-misted trees Sheppard et al., 1995. Foliar analysis were carried out in an attempt to relate such
reduction in stem area to the induction of nutrient deficien- cies including P, but results were inconclusive Sheppard
et al., 1995. However, Carreira et al. 1997 applied a more sensitive root bioassay technique based on the metabolic
uptake of
32
P by excised roots and demonstrated that acid- mist treated trees were under P nutritional stress relative to
non-acid-misted trees. Additionally, acid-mist treatments were found to affect soil acidity lower pH and marked
changes in the cation exchange complex composition and processes involved in the inorganic P subcycle higher soil P
adsorption capacity Carreira et al., 1997. Here, we report on changes in processes involved in the soil organic P
cycling phosphatase activity and organic P mineralisation.
2. Materials and methods
2.1. Study site and experimental design The study site was a 12 × 12 block 2.5 m spacing, Sitka
spruce Picea sitchensis Bong. Carr, single clone, 19- year-old plantation, growing on a brown earth soil at
Glencorse Mains, south of Edinburgh Scotland. The site had been previously used to evaluate the whole tree
response of mature Sitka to acid-mist Sheppard et al., 1994, 1995. Trees were grouped into five height classes,
from the shortest H1, mean 3.8 m to the tallest H5, mean 5.5 m, along a 68 diagonal slope across the plot. Two blocks
of four trees per height class were enclosed in open-top chambers equipped with side roller blinds that were pulled
down only during period of treatment application. Twice a week from July to December 1990, and from April to Octo-
ber 1991–92–93, chambers received the equivalent of 2 mm precipitation as acid-mist consisting of an equimolar
mixture of H
2
SO
4
and NH
4
NO
3
at pH 2.5 3.2 mM H
1
, and 1.6 mM each of NH
1 4
; NO
2 3
and SO
22 4
;
simulating stan- dard, acidic, cloud water composition Crossley et al.,
1991. An extra chamber, enclosing a mix of tree height classes, was used to apply an unreplicated, double dose,
acid-mist treatment 8 mm of mistweek. Eight individual trees for each height class, not enclosed within chambers,
were selected as control non-acid-misted trees. Control trees received non-acid pH 5.0 mist at 2 mm per spraying
in 1993. Detailed information on the experimental layout, treatment applications, and previous results of the Glencorse
study can be found elsewhere Crossley et al., 1991; Shep- pard et al., 1995; Carreira et al., 1997.
2.2. Soil sampling and general analyses One year after the end of treatment applications, four soil
samples litter plus 0–5 cm depth were collected using a 11.5 cm diameter core sampler from each treatment block 5
tree height classes × 2 acid-mist doses — no acid-mist and standard acid-mist dose —, and 1 double acid-mist dose
with mixed height classes. Within each acid-misting cham- ber, soil samples were taken at random but, to avoid edge
J.A. Carreira et al. Soil Biology Biochemistry 32 2000 1857–1865 1858
Fig. 1. Hypothesised pathways by which atmospheric acidifying inputs to a forest ecosystem may induce P nutritional stress due to i changes in soil acidity and inorganic and organic P cycling, and ii increased P demand by the plants associated to N saturation.
effects, collection was restricted to the inner “quadrat” between the stems of the four trees enclosed in each cham-
ber. Similarly, soil samples were collected from within a 1.0 m radius in a direction selected at random beneath
each of four, individual, non-acid-misted trees in each height class.
Litter and underlying mineral soil from each core sample were separated. Each bulk soil sample was weighed, sieved
2 mm, and the volume of the .2 mm fraction recorded to calculate bulk density. Soil subsamples were oven-dried
1058C, 24 h to calculate moisture content, and then ignited in a muffle 5508C, 2 h to calculate loss-on-ignition. Water
holding capacity WHC was estimated from bulk density BD using the equation: WHC 61.89
p
BD
21.099
r
2
:
96 ;
n 100
; P
, 0 :
001 ;
developed for forest soils Harrison and Bocock, 1981; Harrison, unpublished data.
There were no significant differences between acid-mist dose-level treatments for soil moisture at the field
23
: 2
1 :
2 gravimetric water content, for soil bulk density 0.76 g cm
23
, and for soil WHC 83.7 gravimetric water content. Effective cation exchange capacity CEC
e
was measured on air-dried soil samples as the sum of bases plus exchangeable acidity displaced by 0.1 M BaCl
2
Hendershot and Duquette, 1986. pH and P fractionation analyses were carried out on fresh soil samples. Soil pH was
measured in 1:1 H
2
O McLean, 1982. Labile P fractions were obtained by sequentially extracting soil samples with
anion-exchange resins Dowex 1-X8 and 0.5 M NaHCO
3
Hedley et al., 1982. Results are expressed as g P m
22
of 0– 5 cm depth soil using bulk density values of individual soil
samples. 2.3. Phosphatase activity and net P transformation rates
Air-dried soil samples were wetted 25 gravimetric water content; 30 WHC and pre-incubated at 208C for
21 days prior to the assessment of phosphatase activity. Phosphomonoesterase activity was determined following
the method of Tabatabai 1994. Since the activity of phos- phatase enzymes is strongly affected by pH, standard assays
are usually carried out at pH 6.5 or pH 11.0 the average optimal pH for acid and alkaline phosphatase activity in a
wide range of soils to allow for inter-studies comparisons in terms of potential phosphatase activity. However, we were
interested on effects induced by soil acidification under acid-mist application. On this basis, rather than assaying
all soils at a fixed pH in a buffered solution, we performed non-buffered assays in order to make pH conditions resem-
ble the observed pattern of soil pH variation between acid- mist treatments Table 1. The possibility that further pH
changes occurred during the 1 h assay incubation cannot be fully discounted, although we expect that relative pH differ-
ences between acid-mist treatments were maintained. These considerations should be taken into account when compar-
ing the results reported here with those from other studies on potential activity at optimal pH. In any case, the results from
brief, laboratory enzyme assays must not be interpreted beyond their scope as qualitative indicators of the changes
induced by chronic, acid-mist inputs.
The effects of acid-misting tree canopies on potential soil P transformation rates was evaluated using a modification of
the radiation–autoclaving–incubation procedure of Zou et al. 1992. Three subsets of two replicated, air-dried, height
class-composite soil samples corresponding to the non-acid- mist, standard acid-mist dose and double acid-mist dose
treatments were pre-incubated aerobically at 208C and 15 water content approximately 20 WHC for a period
of three weeks to equilibrate microbial activity to each sample potential under these temperature and water poten-
tial conditions. This pre-incubation period aimed to over- come transient effects of environmental conditions at the
sampling date and sample pre-processing, and to ensure that differences in microbial activity were due to the
‘permanent’ effects of chronic, acid-mist treatments through the acidification of soils. Respiration rates were measured at
the end of the pre-incubation period using an aqueous alkali trapping method Zibilske, 1994. Then, one subset of
samples, used as control, was brought to 30 gravimetric moisture content approximately 35 of WHC with deio-
nised water. The other two of the three subsets of soils were sterilised. To sterilise soils, we used HgCl
2
-addition at a rate of 1500 mg kg
21
of dry soil Wolf and Skipper, 1994, instead of G-radiation Zou et al., 1992. To apply the
HgCl
2
-treatment, the corresponding samples were evenly sprinkled with the appropriate volume of a concentrated
HgCl
2
solution to bring soils to 35 of WHC. All samples were then incubated for an additional 7 days, and respiration
rates were measured again to check for the degree of micro- bial sterilisation achieved by the HgCl
2
treatment. Then, one of the two HgCl
2
-treated subsets of samples was autoclaved
J.A. Carreira et al. Soil Biology Biochemistry 32 2000 1857–1865 1859
Table 1 General properties of soils under non-acid-misted, standard-dose acid-misted, and double-dose acid-misted trees of the Glencorse field experiment. Data are
means obtained pooling data from different tree-height classes see Section 2.4 for further details. Means with different superscript letters within each column are significantly different at the P 0
: 05 upper case letters or at the P 0
: 01 lower case letters level Tukey’s HSD following ANOVA. The significance
of effects of tree-height class is indicated beside the name of the corresponding soil property : P 0
: 01; NS: non-significant effect, P . 0
: 05
Acid-mist treatment Loss-on-ignition
Soil pH
NS
1:1soil:water CECe
NS
cmol
c
kg
21
Base saturation
NS
Ca 1 Mg:Al
NS
Non-acid-misted 9.26
Aa
5.09
Aa
7.02
Aa
89.8
Aa
9.47
Aa
Standard-dose 8.68
Aa
4.76
Bb
7.15
Aa
76.3
Bb
3.53
Bb
Double-dose 8.86
Aa
3.93
Cc
6.09
Ba
28.0
Cc
0.41
Cb
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
