Table 2 Effect of an 8 month exposure to 40 nl l
− 1
NO
2
+ SO
2
on the mean value of a dry mass g of plant parts and b the concentrations of nutrients mg g
− 1
in shoots
a
a Root mass
Treatment Total mass
Shoot mass Ratio rootshoot
0.801 Control
1.252 0.451
1.815 40 nl l
− 1
NO
2
+ SO
2
0.618 0.820
1.434 1.359
t-value, after 2-tailed t-test 3.71
0.23 1.84
2.51 0.823
0.083 0.02
0.002 Probability P
b Sulphur
Potassium Treatment
Nitrogen 0.30
3.47 6.54
Control 8.76
40 nl l
− 1
NO
2
+ SO
2
0.81 7.53
4.65 t-value, after 2-tailed t-test
7.23 7.48
0.0009 B
0.0001 B
0.0001 Probability P
a
Plants were harvested in October after 8 months treatment.
the year. For example the test temperatures in January were − 15, − 20 and − 25°C, while in
October they were − 5, − 10 and − 15°C.
2
.
3
. Assessment of frost injury After the overnight freezing cycle, the elec-
trolyte leakage from the shoots was determined from the increase in conductivity C of the
bathing solution measured using a platinum elec- trode at 20°C, after briefly shaking the vial by
hand. The first measurement C
was : 1 h after the shoots were placed in the vial while the next
was after a 5 h interval C
5
during which vials remained static at 20°C. Finally, the vials were
autoclaved at 105°C for 4 min, and after cooling, measured again. This final value, C
8
i.e. C at ‘infinity’, was an estimate of total electrolyte
content. Using the three conductivity measure- ments C
, C
5
and C
8
the electrolyte leakage coefficients k, units h
− 1
were calculated for each shoot using the following equation:
k = ln C
8
− C
C
8
− C
t
t where t = 5 h.
The electrolyte leakage coefficient k was shown previously to be a sensitive and reliable
method of assessment of frost injury in this spe- cies Caporn et al., 1994. The electrolyte leakage
results are shown in the tables as k × 1000. Statistical one and two-way analysis of variance
with frost temperature and pollution treatment as main effects in the latter was performed on loga-
rithmically-transformed values i.e. ln k × 1000. Transformation was required to normalise the
distribution of the data. All the data was analysed using the SPSS Statistical Package.
3. Results
3
.
1
. Growth and nutrition Fumigation of C. 6ulgaris with 40 nl l
− 1
NO
2
+ SO
2
for 8 months, between February and October, significantly increased P = 0.002 the
shoot dry mass by 37, produced a 15 increase in whole plant dry mass not statistically signifi-
cant P = 0.82, but had negligible effect + 2 on the size of the roots Table 2. The ratio of
rootshoot dry mass was, therefore, significantly lowered by the pollutant exposure P = 0.02. A
similar change in growth was also recorded in plants sampled 1 month later in November after 9
months exposure data not shown. In October, associated with the promotion of shoot growth in
fumigated plants, there was a large increase in the concentrations of nitrogen + 34, potassium
+ 117 and, most markedly, sulphur + 173 in the green shoots. Despite an increase in growth
during the summer there were no visible signs that
the pollution treatment extended the length of the growing season, either into the autumn or
through starting growth earlier in the spring. There were no other changes in plant appearance
caused by the gaseous treatment.
3
.
2
. Frost tolerance The rate of electrolyte leakage from the tissue
provided a rapid indication of cellular damage following exposure of excised shoot pieces to a
single overnight frost. Shoots with poor frost tolerance showed increased damage and a higher
leakage coefficient k. The first series of experi- ments studied the effect of exposure to the pollu-
tants NO
2
+ SO
2
, given to three different sets of plants, on the frost tolerance measured between
autumn and mid winter see series 1 – 3 in Table 1. Electrolyte leakage measurements revealed
that plants grown in filtered air showed a small increase in cellular damage as the test tempera-
tures were lowered from + 5 to − 25°C. In the shoots from the pollution treatments, however,
leakage was greatly increased with the severity of frosts. This was apparent during the period of
hardening October and November and in mid- winter January. At each sampling time there was
a highly significant treatment × temperature inter- action term in the analysis of variance. At all but
one of the sub-zero test temperatures − 5°C in October ion leakage was significantly increased in
the polluted plants compared with filtered con- trols Table 3. In November, after 9 months
fumigation, a significantly increased leakage was detected from shoots of polluted plants even in
the absence of frosting. Indeed, at all other sam- pling times the leakage was also increased in
polluted, non-frosted shoots, but only in Novem- ber were the changes statistically significant.
A comparison was made of the effects of apply- ing the NO
2
+ SO
2
treatments for 5 months either during the pre-winter hardening period August –
January or the post-winter de-hardening period November – April, see series 4 – 5 in Table 1.
Cellular freezing damage was significantly in- creased at all sub-zero temperatures by the
NO
2
+ SO
2
treatments over both periods Table 4.
A third comparison was made of the influence of duration of the pollution treatment on frost
hardiness measured in January. This was achieved by combining data from two of the above experi-
Table 3 Electrolyte leakage coefficient k×1000, units: h
− 1
of excised shoots of Calluna 6ulgaris following frost tests at different temperatures
a
Frost temperature °C Experiment date and treatment
− 25
− 10
− 20
− 15
+ 5
− 5
Feb–Oct 8 months 23
12.1 Filtered
– 23.9
– 18.8
16.9 24.7
50.7 Polluted
102.5 –
– –
– P
0.12 0.73
0.01 B
0.0001 Feb–Nov 9 months
11.4 16.6
20.3 –
11 Filtered
– 21.3
71.2 120.8
– –
Polluted 27.8
B 0.01
– –
0.04 B
0.0001 P
B 0.0001
Feb–Jan 11 months –
12.16 12.43
14.68 Filtered
11.23 –
45.53 –
– 21.15
Polluted 85.66
57.32 –
P B
0.0001 B
0.0001 B
0.0001 –
0.12
a
Plants were grown in filtered air containing 40 nl l
− 1
NO
2
+ SO
2
commencing February for different durations. In each case the probability of a significant difference between filtered and polluted air at each temperature is shown following a one-way analysis
of variance. The over-all effects of temperature and treatment are shown in Table 1.
Table 4 Electrolyte leakage coefficient k×1000, units; h
− 1
of excised shoots of Calluna 6ulgaris following frost tests at different temperatures
a
Experiment date and treatment Frost temperature °C
− 10
− 15
− 20
+ 5
− 25
Aug–Jan –
7.91 10.05
Filtered 12.9
6.38 –
17.07 8.26
34.80 Polluted
48.34 0.288
P –
B 0.001
B 0.001
B 0.001
No6–April 10.2
11.9 13.3
18.9 Filtered
– 13.5
Polluted 20.1
29.6 36.2
– 0.028
P B
0.001 0.673
B 0.001
–
a
Plants were grown in filtered air or 40 nl l
− 1
NO
2
+ SO
2
during either a 5 month hardening period August–January or a de-hardening period November–April. In each case the probability of a significant difference between filtered and polluted air at
each temperature is shown following a one-way analysis of variance. The over-all effect of temperature and treatment are shown in Table 1.
ments February – January; August – January with a shorter fumigation of 2 months given between
November and January series six in Table 1. Frost tolerance was measured on all the material
at the same time in January. The leakage data indicate that while fumigation of the plants for
periods of 5 or 11 months caused significant re- ductions in frost tolerance an exposure to the
pollutants for just 2 months was without signifi- cant effect Table 1.
4. Discussion
