seedlings randomly harvested from each block. Their needles and stems were sampled as above for
ethanol analysis at 0, 1, 2, and 4 h after treatment initiation. These samples served as the unexposed-
controls. On the following day, three styroblocks used for the 20 ethanol treatment in the main
experiment above now with dead seedlings were placed alternately between the 0 controls and
each watered with their respective ethanol solu- tions for 20 min. Two seedlings from the 0
treatment were harvested and their needles and stems sampled for ethanol analysis at 0, 1, 2, and
4 h after treatment initiation, as described previ- ously. This was the exposed-treatment. The atmo-
sphere surrounding these seedlings also was sampled for ethanol at each time. One air sample
was taken at seedling height from both ends of the three styroblocks. Air 30 cm
3
was drawn with a plastic syringe and injected into a sealed autosam-
pler vial with a needle inserted through the septum serving as an outlet for the displaced air from the
vial. The outlet and inlet needle from the syringe were immediately removed from the septum.
Ethanol in the sealed vial was analyzed as before.
All statistical analyses were made with SAS software [21] using styroblocks as the experimental
unit, with a single mean value for each parameter obtained from subsamples within blocks. Ethanol
concentrations measured 24 h after the first treat- ment, and after 1 week three treatments were
analyzed separately for needles and stems as a split-plot design with treatment as the main plot
and time as the sub-plot. Similarly, the experiment on ethanol absorption by needles was analyzed as
a split-plot design with the unexposed-control and exposed-treatment as main effects and time as the
sub-plot. Diurnal gas exchange after 1 week was analyzed as a split-plot design with treatment as
the main plot and time as a repeated measure, because these measurements were repeatedly made
on the same seedlings. Xylem pressure potentials measured at 24 h, 1 week, and 8 weeks were
analyzed separately for each time as one-way ANOVAs with treatment as the main effect. Dark
respiration after 1 week, and the final measure- ments after 8 weeks for shoot length, dry
weights, gas exchange, and tissue ethanol concen- trations were each analyzed as a one-way ANOVA
with treatment as the main effect. Where neces- sary, data were natural log transformed to meet
homogeneity of variance, and normality assump- tions. Geometric back transformed means are
presented for data that was transformed. Signifi- cant differences between means were separated
using Fisher’s Protected LSD at a = 0.05.
3. Results
Seedlings with roots exposed to 10 and 20 ethanol solutions showed severe signs of needle
burn within a week of treatment initiation. Nearly all seedlings in the 20 treatment and about half
in the 10 treatment died within this time. Brown- ing of needles started at the base and moved
toward the tip. Seedlings in the 0, 1, and 5 treatments showed no visible signs of damage
during the first week. After 2 weeks initiation, nearly 100 of the seedlings from the 20 treat-
ment and approximately 90 of the seedlings in the 10 ethanol treatment were dead.
Ethanol was rapidly taken up by seedlings and concentrations in their needles and stems corre-
sponded to the differences in ethanol treatments Fig. 1. For each tissue, ethanol concentrations
were significantly influenced by treatment solution and time since treatment initiation P B 0.001. In
both 0 and 1 treatments, needle ethanol concen- trations were significantly high at 2 h P B 0.001,
but by 4 h had dropped to their respective equi- librium levels. Needle ethanol concentrations from
the 10 ethanol treatment continued to increase up to 8 h before leveling off. In contrast, stem
ethanol concentrations increased to a maximum at 4 h for both the 1 and 10 ethanol solutions and
then leveled off. Stem ethanol concentrations in control seedlings 0 ethanol were near 0 and
remained relatively unchanged over the course of 24 h.
Ethanol readily diffused from the atmosphere into the needles Fig. 2. Ethanol concentrations
were significantly higher in needles exposed to ethanol vapors than in unexposed controls at 1, 2,
and 4 h after treatment all P values 5 0.023. Changes in ethanol concentrations within needles
followed the changes in atmospheric concentra- tions surrounding the needles. They both were
significantly high 1 h after treatment initiation and then declined significantly P 5 0.003, P 5 0.011,
respectively. Stem ethanol concentrations did not differ between control and exposed seedlings.
Fig. 2. Ethanol concentrations in needles and stems of Dou- glas-fir seedlings A and in the surrounding air B when
exposed to ethanol vapors from treatment applications. Points represent means 9 1 S.E. n = 3.
After 1 week 24 h after the third ethanol treat- ment, ethanol concentrations in needles all P
values 5 0.016 and stems all P values 5 0.013 continued to correspond to concentrations in the
treatment solutions Fig. 3. However, stem ethanol was several-fold higher than the needles
for both the 1 and 5 ethanol treatments. There was no difference between ethanol concentrations
at 07:30 and 15:30 h for needles or stems data not shown. After 1 week of treatments, the midmorn-
ing and afternoon net photosynthesis P
n
, Fig. 4A was significantly different with 0 \ 1 \ 5 ethanol
all P values 5 0.033. Stomatal conductance g
s
in the 0 treatment was significantly higher than either the 1 or 5 ethanol treatments at all times
during the day all P values 5 0.003 Fig. 4C. Although g
s
in the 1 ethanol treatment was 1.5 times higher than the 5 treatment, it was not
significantly different P = 0.139. Transpiration E differed among treatments with 0 \ 1 \ 5
throughout the day all P values 5 0.001, except at 06:40 h when E was zero for all treatments Fig.
4E. Afternoon E increased much more in the 0 treatments than in either the 1 or 5 treatments.
Dark respiration at 22:30 h was 1.42 mmol m
− 2
s
− 1
for all ethanol treatments. At the end of 8 weeks 24 h after the final
treatment, midmorning P
n
was still different Fig. 4B with 0 \ 1 \ 5 ethanol all P values 5
0.024, although the P
n
of the 1 and 5 treatments were close to zero. Stomatal conductance of con-
trols was higher than either the 1 or 5 treatments both P values = 0.002, both of which were close
to zero Fig. 4D. Transpiration by controls was higher than either the 1 or 5 treatments both
P 5 0.003, both of which were close to zero Fig. 4F.
Xylem pressure potential 24 h after the initial ethanol treatment was lowest in the 5 treatment
P 5 0.050, with no difference between the 1 treatment and controls P = 0.800 Fig. 5. After
1 week, there were small differences in XPP among treatments with 0 \ 1 \ 5 ethanol all
P 5 0.049. After 8 weeks, the XPP again showed no difference between 0 and 1 treatments P =
0.156, whereas it was significantly more negative in the 5 treatment P B 0.001 for comparisons
with 0 and 1 and most seedlings appeared dead Fig. 5. Also at 8 weeks, shoot dry weights were
higher for the controls than either the 1 or 5 treatments both P values 5 0.026; Fig. 6A, and
Fig. 3. Ethanol concentrations in needles A and stems B of Douglas-fir seedlings after 1 week of treatment with 0, 1, or
5 ethanol solutions. Vertical bars are means from measure- ments at 07:30 and 15:30 h 9 1 S.E. n = 3.
there was a weak treatment effect on shoot lengths P = 0.082; Fig. 6B with controls being the
longest. There was no difference in these growth parameters
between the
1 and
5 ethanol
treatments.
4. Discussion
