EVALUATION OF STRESS FACTORS

AFTER IRRIGATION OF WILLOW WITH

LANDFILL LEACHATE

I. DIMITRIOU, P. ARONSSON AND M. WEIH

SLU (Swedish University of Agricultural Sciences), Department of Short-Rotation

Forestry, P.O Box 7016, 75007 Uppsala, Sweden

SUMMARY: The use of nature-like systems for remediation of landfill leachate is at present of
increasing interest but the leachate’s chemical composition can cause plant damage mainly due
to high NaCl and NH4 concentrations. A pot experiment was conducted in order to assess the
applicability of leaf length and leaf fluctuating asymmetry as stress indicators after irrigation of 5
different willow clones (“78-183”, “Jorr”, “Loden”, “Olof”, “Tora”) with different leachate
mixtures (corresponding to 240, 180, and 120 mg Cl l-1). Leaf length appeared to be a useful
stress diagnostic tool showing a good correlation to growth, whereas fluctuating asymmetry
showed no such correlation. Results from a field study with willow irrigated with landfill
leachate substantiated the previous findings.

1. INTRODUCTION

Landfill operators in Sweden are increasingly interested in nature-like treatment systems that
include aeration followed by irrigation of short-rotation willow coppice (energy forest) on
restored parts of the landfills or on adjacent arable fields.
Short-rotation willow coppice (SRWC) is a commercial crop currently grown on
approximately 15000 hectares in Sweden. The average production of SRWC in Sweden is 6-10
tonnes dry matter per hectare and year, and the crop is harvested every 3-4 years and burned in
district heating plants producing heat and electricity. Around 30 natural-like systems treating
landfill leachate using irrigation of willow plantations are presently found in Sweden. Willow, if
established on a restored cap of the landfill or on adjacent fields, decreases the leachate
formation due to high evapotranspiration (Cureton et al, 1991; Ettala, 1988). By recycling
leachate water back to a willow plantation established on a landfill, the net discharge of leachate
can be close to zero due to the high evapotranspiration rate. Simultaneously, hazardous
compounds in the leachate can be taken up by willows or retained in the soil-plant system
(Aronsson & Perttu, 2001). Willow also tolerates low oxygen levels in the root zone enabling
high irrigation rates.
A series of damage due to the chemical composition of the leachate can be coused by
irrigation of plants with leachate. Leaf damage, premature leaf senescence and reduced biomass
production are some of them (Mensar et al, 1983, Wong & Leung, 1989). A number of factors

Proceedings Sardinia 2003, Ninth International Waste Management and Landfill Symposium
S. Margherita di Pula, Cagliari, Italy; 6 - 10 October 2003
 2003 by CISA, Environmental Sanitary Engineering Centre, Italy
interfere with plant vigour in a field situation , such as soil type, plant species and variety, water
quality and irrigation rates, climate, and their interactions. Consequently predictions of growth
and vigour of plants subject to leachate irrigation are difficult to make. Therefore, identification
of plants stress in the field would be of great value for determining a short-term negative reaction
that will lead to severe problems in the future. Growth and biomass production are the common
indicators of imposed stress (Larcher, 1995). For trees though, growth measurements are not
practical to measure and other factors as leaf length and leaf fluctuating asymmetry, which is a
random non-directional deviation from anticipated symmetry, have been proposed as
environmental stress indicators (Alados et al, 2001; Zvereva, 2001).
The objectives of the study were to assess different stress indicators as a means of a rapid
diagnostic tool for stress emergence to willow clones irrigated with landfill leachate. For that, a
pot experiment with five willow clones irrigated with different mixtures of landfill leachate and
field measurements in a willow plantation irrigated with landfill leachate were conducted.

2.MATERIALS AND METHODS

2.1 Experimental design

An experiment was conducted outdoors at Uppsala (59°49´N, 17°39´E), central Sweden. On 21

May 2002, 5 cm-long willow cuttings were planted in 1-dm3 pots filled with sand, having a
water holding capacity of 0.2 l. In the bottom of each pot a 20-micron nytrel mesh was placed to
prevent roots from growing outside the pots. In total, 225 pots were used in a fully randomized
factorial block design with five blocks, five willow clones, four different irrigations (i.e. landfill
leachate in three concentrations, and a liquid fertiliser as control), and two pots per clone and
treatment in each block. The clones used were “78-183” (Salix viminalis, commonly used as a
reference clone in the Swedish willow breeding programme), and the four bred clones “Jorr” (S.
viminalis), “Loden” (S. dasyclados), “Olof” (S. viminalis x S. schwerinii), and “Tora” (S.
schwerinii x S. viminalis). From 5 May until 16 June, the pots were watered daily with tap water.
On 16 June, initial harvest of five pots per clone was conducted. On 17 June, the main
experiment was started with application of landfill leachate, which lasted until 20 August. The
landfill leachate came from a commercial landfill at Högbytorp, central Sweden. The chemical
composition of the leachate is shown in Table 1. The three leachate concentrations resulted by
mixing 1 unit by volume of leachate with 2, 3, and 5 units of tapwater, respectively (resulting in
240, 180, and 120 mg Cl l-1). For the control treatment a liquid fertiliser (Blomstra, Cederroth
International AB) was used diluted to a concentration of 51 mg N-tot l-1 (40% NH4-N, 60% NO3-
N), and with the other plant nutrients in balanced proportions to N (see Table 1). During
weekdays, 200 ml of leachate mixtures or liquid fertiliser were applied manually. Tap water
provided to the plants during weekends with sprinklers.
Table 1. Chemical composition of the pure leachate and the nutrient supply with liquid fertiliser
Pure leachate Supply Supply Supply Supply control
Element conc. (mg l-1) 1:2 (g) 1:3 (g) 1:5 (g) (g)
Tot.-N 278 0.74 0.56 0.37 0.41
Of which NH4-N 267 0.71 0.54 0.36 0.16
NO3-N 2.3 0.006 0.005 0.003 0.25
TOC 330 0.88 0.66 0.44 -
Tot.-P 4.26 0.01 0.008 0.006 0.08
Of which PO4-P 2.1 0.005 0.004 0.003 0.08
K 359 0.956 0.717 0.478 0.344
Cl 723 1.93 1.45 0.96 -

2.2 Sampling and measurements

At both harvests, initial and final, each plant was separated into leaves, shoots, cutting and roots
and dry weights were measured after drying at 70 ºC for 4 days. At the final harvest, 10 leaves
per plant were systematically sampled (i.e. from the whole shoot axis) for measurements of mean
leaf length (LL) and leaf fluctuating asymmetry (FA), where LL was defined as the mean length
of the left and right side of the leaf blade, and FA was calculated according to:

WL − WR
FA = 2 ∗
WL + WR

where WL and WR denote the width of the left and right sides of the leaf blade at the mid point
from the base to the top of the leaf blade (Palmer & Strobeck 1986). At the initial harvest, the
mean values of the five plants per clone were calculated and used in the further calculations. At
the final harvest, the mean values of the two plants per clone, treatment and block were used as
the experimental units (i.e. n=5). In order to compensate for the very marked differences in
initial plant development between clones, the concept of relative growth rate (RGR) was adopted
for shoot dry weight, and the RGR from initial to final harvest was calculated according to Hunt
(1982):

(ln A f − ln Ai )
RGR =
(t f − ti )

where Af denotes the measured trait at final (f) harvest (mean value of the two plants of same
clone, treatment and block), Ai denotes the initial (i) harvest (the mean of the five plants per
clone), and t the time in weeks. In the following text, the relative growth rate of shoot is referred
to as RGRS.

2.3 Complementary field study

For comparison, field measurements of whole-season shoot growth and mid-season leaf length
(LL) and leaf fluctuating asymmetry (FA) were conducted in a large-scale willow plantation
irrigated with the same landfill leachate from the landfill at Högbytorp as was used in the pot
trial. The stand was established in 2001 by planting of approximately 15000 cuttings of the clone
“Tora” (which was also tested in the pot trial) in a double row system. In early spring 2002, the
plants were cut back at a height of approximately 0.4 m in order to promote sprouting and speed
up canopy closure. In spring 2002, the plants resprouted, and on 26 June sprinkler irrigation with
landfill leachate started and commenced until 1 October. The average daily dose of leachate was
2.6 mm resulting in an accumulated dose of 263 mm containing some 530 kg N-tot ha-1. The
chemical composition of the leachate was similar to the concentrated leachate used in the pot
experiment (see Table 1). The willow plantation covers an area of 6.5 hectares, of which a 0.1-ha
plot was not irrigated and considered as a reference area (see also Figure 3).
Measurements of LL and FA were conducted on 2 and 22 July 2002. On each sampling
occasion, four sampling sites were selected in the irrigated part of the field. The centre of each
site corresponded with the location of an irrigation gun. At each site, measurements were made
in two transects, i.e. along and across the plant rows, on plants at 1, 2, 3, 5, and 7 (across) or 8
(along) m distance from the centre of the sampling site. From each sampled plant (n=40) three
fully developed leaves near the top from the northern, eastern, southern, and western side of the
plant were collected and pooled to a general sample. For each plant the average LL and FA was

Table 2. Significance levels for the experimental treatments. The non-significant (p>0.05)
sources of variation are not presented (see text)
Source of Response
Variation LL FA
Clone *** ***
Treatment *** ns
Clone x Treatment ** ns
* 0.01 < p < 0.05, ** 0.001 < p < 0.01, *** p < 0.001, ns p > 0.05

measured and calculated according to the same method as described above. Also in the reference
area the average LL and FA of randomly selected plants (n=12) were determined. In November
2002, an estimation of the whole-season shoot growth was conducted. For this, the diameter at
55 cm height of all shoots within 52 3-m plant row sections was measured with a calliper. A set
of 20 shoots of all diameter classes was then destructively harvested. The diameter was measured
and the shoot dry weight was determined after drying to constant weight at 70 °C. A two-
parameter power function of the form:

2.4071
SDW = 0.1446 * d 55

was the established using non-linear regression and used to calculate the dry weight of the shoots
measured in the field. The field coordinates and the position of the sampled row sections were
determined using GPS.

2.4 Statistical analyses

Significant differences between treatments as regards all measured responses were tested by
ANOVA GLM. The probability values of the sources of variation which where non-significant
(p>0.05), i.e. the “block” variable and its interactions, were not included in the table below (see
Table 2) but were included in the analyses.
3. RESULTS

3.1 Pot trial

The results show very clear treatment effects compared with the control for leaf length (LL),
whereas the differences between the three leachate concentrations were small and typically non-
significant (see Tables 2 and 3). Clone differences were evident for both LL and fluctuating
asymmetry (FA) (see Table 2). For FA however, treatment differences were not evident.
Furthermore, for FA the treatment response did not varied significantly between the clones (see
Figure 1 and Table 2, clone x treatment interaction).

Table 3 - Mean values ± SEM for all clones of LL and FA for the different treatments (n=25)
LL FA
Treatment (mm) (-)
1:2 60.76 ± 2.18 0.116 ± 0.009
1:3 61.93 ± 1.85 0.110 ± 0.007
1:5 56.02 ± 1.82 0.116 ± 0.009
Control 89.35 ± 4.40 0.102 ± 0.008

The average LL of control plants was also significantly higher than that of leachate irrigated
plants (89.3 and 59.6 mm, respectively, see Table 3). Between the respective values for FA no
such differences were mentioned. LL varied considerably between clones, and was also well
correlated with RGRS for all clones. Linear regressions of RGRS as a function of LL were highly
significant (p<0.001) for all clones. The predictive power (i.e. the adjusted R2) for “78-183”,
“Jorr”, “Loden”, “Olof” and “Tora” was 0.53, 0.77, 0.57, 0.87 and 0.59 (n=20) (see Figure 2),
but was however rather poor for “78-183”, “Loden” and “Tora”. The biggest reduction in LL
between the two “extreme” treatments (1:2 and control) was reported for “Jorr” and “Olof” and
the smallest reduction was mentioned for “Tora” and “Loden” (see Figure 1).

3.2 Field measurements

The shoot growth during first year of irrigation varied markedly within the field (see Figure 3),
but was on average 2.4 tones dry matter ha-1 in the irrigated part of the field. In the non-irrigated
reference area the corresponding growth was considerably higher, i.e. 5.7 tones dry matter ha-1.
The measurement of LL and FA in the irrigated and non-irrigated reference plot of the field
showed no significant differences in LL at the first sampling occasion on 2 July (mean LL 121.0
mm and 123.3 mm for irrigated and reference plots, respectively). However, FA was highest in
the reference plot (0.112) as compared with the irrigated part of the field (0.083). At the second
sampling occasion roughly three weeks later, LL was significantly higher (p<0.021) in the
reference plot (181.1 mm) as compared with that in the irrigated part (163.0 mm). However,
there were no significant differences in FA at the second sampling occasion (0.062 and 0.068 for
reference plot and irrigated part, respectively).
 78-183
120 JORR 0.20
LODEN
100 OLOF
TORA 0.15
80
LL (mm)

FA
60 0.10

40
0.05
20

0 0.00
1:2 1:3 1:5 Control 1:2 1:3 1:5 Control

Figure 1. Leaf length (LL, in mm) and fluctuating asymmetry (FA) at final harvest, in relation to
treatment for each clone (n=5). Error bars indicate SEM.

1.0
Relative Shoot Dry Weight (wk )
-1

0.8

0.6

78-183
0.4 JORR
LODEN
OLOF
0.2 TORA

20 40 60 80 100 120 140

Leaf Length (mm)

Figure 2. Relative growth rate (RGR, in wk-1) of shoot dry weight (S) in relation to mean leaf
length (LL) of each clone at final harvest. R2 values are 0.53, 0.77, 0.57, 0.87 and 0.59
(n=20) for “78-183”, “Jorr”, “Loden”, “Olof” and “Tora” respectively
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Figure 3. Graphical display of the shoot dry weight in the willow plantation at Högbytorp based
on smoothened data from 52 sampled row-sections (indicated by dots). The rhomboid
area corresponds with the non-irrigated reference area, and the stars around it indicate
the position of the sprinklers closest to the reference area.

4. DISCUSSION

Growth has been considered as the most reliable indicator of stress emergence. Larcher (1995)
denotes that growth processes are especially sensitive to the effects of salt, therefore growth rates
provide reliable criteria for assessing salt stress. However, if growth is used as stress indicator, a
certain delay in identifying and assessing stress will take place. Additionally, for measuring
growth differences between a control and a treatment with landfill leachate, destructive methods
may have been required, which in most cases is impractical.
By finding a reliable and practical tool to assess plant stress in a willow plantation treated
with landfill leachate during the growing (and irrigation) season, adjustments of irrigation
regimes for avoiding extensive damage would be possible. The results indicate a positive
correlation between leaf length (LL) and relative shoot dry weight (RGRS), despite the high
variability between clones and the poor predictive power for some of them. RGRS is a good
measure of overall plant growth and vitality, and the use of LL in field conditions as a rapid
stress estimator seems appropriate -after appropriate calibration and taking into account clone
specificity as well. Morabito et al. (1994) and Sun & Dickinson (1995) found LL to be a good
determinant of saline stress and that it plays an important role in affecting the long-term
performance of plants, due to its direct effect on photosynthetic available leaf area. Since the LL
was lower in the 1:5 treatment than in the 1:2 treatment, nutrient deficiency and not only salt
stress may have contributed to LL reduction. Therefore, if LL was to be used for assessing stress
level and not only as a stress indicator, the plant nutritional status should be considered for a
better explanation of a possible reduction in leaf length. In addition, clone differences in terms of
response to reduction in LL were evident. Certain clones (“Jorr” and “Olof”) were affected more
comparing the 1:2 and control treatments than the other clones. Therefore, if using LL as a stress
indicator, one should compare plants of the same clone for assuming stress emergence taking
into account the different responses of the different clones.
In contrast to LL, leaf fluctuating asymmetry (FA) does not seem to be an appropriate
assessment tool of stress, at least in short-term experiments. Despite findings that suggest FA as
a tool to compare the strength of different stress agents for willow (Zvereva et al., 1997),
differences between the four treatments were not found and no general pattern was observed.
However, FA differed significantly between clones, which come in accordance with the findings
by Hochwender & Fritz (1999), who claimed that FA is more likely to be genetically controlled
rather than environmentally. Our results on FA are similar with those of Anne et al. (1998), who
found that FA failed to identify saline stress whereas less canalised traits, e.g. leaf shape, did
detect changes attributed to stress.
According to our results, the degree of dilution resulting in different leachate concentrations is
of minor importance for the plants, considering the small and non-significant differences in the
LL between all the leachate treatments. However, in our experiment we may have engaged in a
situation with two factors counteracting each other, since no additional source of plant nutrients
-except the leachate- was added. That is, application of a leachate as strong as in the 1:2-
treatment might have affected the plants negatively if considering the high ionic strength of the
leachate, and diluting the leachate would then result in a lower stress on the plants. The poor
growth in the 1:3 and 1:5 treatments in our study could in consequence be attributed to another
type of stress, nutrient insufficiency. From Table 1, the differences between the treatments as
regards addition of many macro nutrients are stated. Especially P seems to be crucial in this
respect. If growth and chemical composition of the control plants are considered as “optimum”
conditions, then the plants subject to the 1:3 and especially 1:5 treatments might have suffered
from P-deficiency. However, in a field situation, the plants have access to a fairly large pool of
plant nutrients in the soil and the nutrient insufficiency would not possibly be the main reason of
stress. Yet, this makes obvious that the long-term nutritional demand of plants when used for
phytoremediation must be taken into account. It also highlights that since there is a range of
other factors interacting with each other, it is difficult to assess at which concentrations Cl or Na
become harmful to plants,.
In the leachate-irrigated field a large differences in growth between the irrigated and the non-
irrigated parts of the field were evident. The growth in the reference part (5.7 tonnes DM ha-1)
was comparably good for first year after planting, whereas the growth in the irrigated part (2.4
tonnes DM ha-1) was lower than expected. Since there was only one reference area, we cannot
exclude the possibility that the differences in growth were due to factors other than irrigation
with landfill leachate. As examples, some parts of the field appeared to be browsed, and in other
parts weed growth was probably reducing growth. However, at the first sampling occasion on 2
July one week after start of irrigation, there were no differences in LL between the irrigated and
the non-irrigated parts of the field, whereas three weeks later at the second sampling occasion
there were clear differences in LL. This could indicate that irrigation was an important reason to
the growth differences during season and strengthen the presumption that LL is an appropriate
rapid stress indicator.
5. CONCLUSIONS

Irrigation of willows with a high-ionic landfill leachate results in plant stress. For the five clones
tested, there is a good correlation between leaf length and relative shoot dry weight, an indication
that leaf length could be further developed as a tool to assess plant stress and thus to adjust
irrigation regimes in phytoremediation systems. Leaf fluctuating asymmetry, on the other hand,
is not correlated with plant growth.
The results from experimental-scale pots in terms of growth response and leaf shape seem to
be valid also for a field situation. However, the results do not show at which leachate
concentration plant stress becomes apparent.

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