Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol83.Issue3-4.March2000:
Scientia Horticulturae 83 (2000) 339±352
Toxicity level for phytoavailable zinc in
compost±peat substrates
Annette S. Bucher*, Manfred K. Schenk
Institute of Plant Nutrition, University of Hannover, HerrenhaÈuser Straûe 2,
30449 Hannover, Germany
Accepted 17 June 1999
Abstract
Petunias were grown in compost±peat substrates with different Zn contents (basic load, 400, 800,
and 1600 mg kg±1) in order to identify toxic levels of Zn in plant dry matter and of phytoavailable
(CaCl2-extractable) Zn in compost±peat substrates. Yield reduction of petunias was only observed
at an extremely high Zn content (635 mg Zn kg±1 plant d.m.), although chlorosis was evident at
lower Zn levels (160 mg Zn kg±1 plant d.m.). Thus, the occurrence of Zn-induced chlorosis during
the growth period was chosen as the toxicity parameter. Chlorosis and plant Zn content were
reduced by additional Fe supply, although the Fe content of the plants was not affected. During a six
week growth period, petunia Zn content and chlorosis increased in the ®rst two and three weeks,
respectively, and then decreased, whereas plant Fe content decreased sharply between the second
and third week after the start of Zn treatment. Chlorosis ®rst occurred with a plant Zn content of
160 mg kg±1, corresponding to a CaCl2-extractable Zn content in the substrate of 6 mg l±1, which
was identi®ed as the toxicity level for phytoavailable Zn in compost±peat substrates. Taking a safety
factor into account, a critical level of 4.5 mg l±1 was computed, which should not be exceeded in
order to avoid Zn-induced chlorosis. # 2000 Elsevier Science B.V. All rights reserved.
Keywords: CaCl2 extraction; Fe de®ciency chlorosis; Petuniahybrida; Zn toxicity
Abbreviations: AAS, atomic absorption spectroscopy; d.m., dry matter; EDDHA, ethylenediamine
di(o-hydroxyphenylacetic acid); ICP, inductively coupled plasma (spectroscopy); PTFE, polytetrafluorethylene (Teflon); SD, standard deviation
*
Corresponding author. Tel.: 49-511-2626; fax: 49-511-3611.
E-mail address: [email protected] (A.S. Bucher).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 8 6 - 2
340
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
1. Introduction
Composts prepared from green yard waste (grass and vegetative cuttings) and
organic waste are often used as a component of substrates for pot and container
plants. However, they often contain large amounts of heavy metals (Kehres,
1991), of which Zn is the one with the highest potential toxicity, because of its
high concentration and the comparatively high phytoavailability of Zn in the
compost. Zn phytoavailability depends on the total Zn content, pH, content of
other heavy metals, and the salt content of the substrate (Herms, 1989). With
decreasing pH, intensity and selectivity of Zn sorption decrease, resulting in a
higher phytoavailability (Herms, 1982). In compost±peat substrates, phytoavailable Zn contents can be characterized well by CaCl2 extraction (Bucher and
Schenk, 1997).
Zn-induced chlorosis is known to occur with a Zn content of 200 mg kg±1 in
sugar beet leaves (Delschen and Werner, 1989) and of 300 mg kg±1 for petunias
(Alt et al., 1994) and cereals (Merkel and KoÈster, 1976), indicating different
susceptibility between plant species. The toxicity level for CaCl2-extractable Zn
in soil and substrates, based on the occurrence of chlorosis, has been shown to
vary between 6 mg l±1 for sugar beet (calculated after Delschen and Werner,
1989) and 55 mg l±1 for petunias (Alt et al., 1994).
Zn toxicity-induced chlorosis resembles Fe de®ciency chlorosis (Rufner and
Barker, 1984), which can be corrected by Fe supply (Chaney, 1993). Zn may
interfere with Fe uptake, translocation or utilization in the leaves (Chaney, 1993).
The aim of this paper was to de®ne the toxicity levels for Zn in the plant dry
matter of petunias and for CaCl2-extractable Zn in compost±peat substrates.
Further emphasis was laid on the interaction of Zn and Fe with regard to the
occurrence of Zn-induced Fe de®ciency chlorosis.
2. Material and methods
The test plant used was the heavy metal-sensitive Petunia hybrida `Express
Orchid' (Bucher and Schenk, 1997).
2.1. Growing conditions
Plants were sown and pricked out in standard peat substrate. Seedlings were
potted in all trials in plastic pots of 250 ml volume and they were grown in a
greenhouse with 14±168C day and 12±148C night heating temperature, the
ventilation temperature being 28C higher. In autumn/winter (trial II and IV)
additional arti®cial light (about 165 mE m±2 s±2 PAR for 12 h) was provided.
Treatments held at pH 4 (CaCl2) received water without carbonate hardness, in
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
341
order to maintain the low pH during the growth period. Plants were harvested
after 38 (trial I), 56 (trial II), 45 (trial III), 50 (trial IV), and 42 (trial V) days,
respectively.
2.2. Treatments
1. Compost trials (trials I±IV). Mixtures of 40 vol% compost and 60 vol% peat
were used as substrates. Compost was prepared from green yard waste (grass and
vegetative cuttings). Zn sulfate was added before the waste was composted in
order to achieve similar Zn forms as in compost made from polluted raw material.
Zn addition was varied to match the contents occurring in commercial composts.
The target Zn values of the compost±peat substrates (400, 800, 1600 mg kg±1)
were not met exactly, as shown in Table 1.
Composting until maturity of the compost lasted for 14.5±19 weeks. In trials I,
II, and III, pH was adjusted to 4, 5, 6, and 7 according to buffer curves by using
H2SO4 and CaO. Substrates in trial IV were adjusted to pH 4. The target pH
values were obtained on average with an accuracy of 0.22 and ±0.37 pH units.
During the trials, pH increased on average by about 0.42 units at the time of
harvest. Basic fertilization consisted of 200 mg N l±1 substrate given in the form
of NH4NO3. No further nutrients were necessary as basic fertilization, since they
were present in the compost. During the growth period, plants were fertigated
with a nutrient solution containing (in mg l±1) 75 N, 15.26 P, 91 K, 0.6 Fe, 0.15 B,
0.01 Cu, 0.25 Mn, 0.025 Mo, and 0.05 Zn. In addition to fertilization, 1 mg Fe per
plant as solution of Fe-EDDHA was applied once after one week of cultivation as
is usual in horticultural practice (Fe treatments). Some further treatments were
included which did not receive the additional Fe supply (±Fe treatments). In trial
III these ±Fe treatments were only conducted at pH 4 and 6.
2. Peat trial (trial V). The Zn contents of petunias during the growth period
were investigated by growing plants in peat with varied Zn supply, with adjusted
substrate pH to 5.2 by the addition of 2.5 g CaCO3 l±1 peat. Zinc sulfate was
added before potting the plants in order to achieve the target total Zn contents
(800, 1600 mg kg±1). In addition, the peat substrate was fertilized with (in mg lÿ1):
225 N, 150 P, 225 K, 30 Mg, 1.125 Fe, and other micronutrients. Fertigation
was the same as for the compost±peat substrates, but no additional Fe was
supplied.
2.3. Experimental design and statistics
Trials were conducted using a completely randomized block design with three
replicates of each treatment. Ten plants were used for each treatment. Analysis of
variance, Tukey test, and linear regressions (Pearson) were computed, using the
statistical package SAS for Windows (1995).
342
Target Zn contents
(mg kg±1 substrate d.m.)
Basic load
400
800
1600
a
b
Aqua regia soluble Zn contents (mg kg±1 substrate d.m.)a
Trial I
(compost±peat
substrate)
Trial II
(compost±peat
substrate)
Trial III
(compost±peat
substrate)
Trial IV
(compost±peat
substrate)
Trial V
(peat)
157
552
975
±b
±
390
695
±
76
420
700
±
92
±
675
1306
26
±
886
1911
Average of all treatments with different pH.
Treatment in this trial not available.
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
Table 1
Target and aqua-regia-soluble Zn contents in the compost±peat substrates and in peat substrate
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
343
2.4. Chemical analysis and observation of plants
The pH in 0.01 M CaCl2 solution was determined in all substrates at the start of
the culture and at harvesting. Substrate samples were taken before potting the
plants and after drying and grinding, 2.5 g substrate was digested in aqua regia
(10.5 ml HCl 3.5 ml HNO3). Zn in the digest was detected by atomic
absorption spectroscopy (Hoffmann, 1991). Extractable (phytoavailable) Zn
contents in substrates were determined by extracting 1 g dried, sieved (2 mm
mesh, stainless steel) substrate with 10 ml 0.1 M CaCl2, and shaking for 1 h
(KoÈster and Merkel, 1982).
Plant samples were oven-dried at 60±708C to determine plant dry matter. Dried,
ground plant matter was digested under pressure in PTFE vessels with nitric acid.
Zn and Fe were analyzed using AAS and ICP. Plants were harvested weekly for
chemical analysis for trial V and at the end of the culture for the other trials.
Chlorosis of the plants was monitored weekly in all trials and is reported as a
percentage of chlorotic plants, without consideration of the severity of chlorosis.
3. Results
No signi®cant effect of Zn supply on plant dry matter yield was found in most
trials (see Table 2), except trial IV. In this case, plant dry matter decreased when
treated with the highest Zn supply. This occurred with a plant Zn content of
635 mg Zn kg±1 d.m. and a CaCl2-extractable Zn content of 69 mg Zn l±1
compost±peat substrate. The highest Zn contents without yield reduction were
471 mg Zn kg±1 plant d.m. for the plants grown in the compost±peat substrates
and 550 mg Zn kg±1 plant d.m. for the plants grown in the peat-only substrate at
the end of the experiment.
In all trials, Zn toxicity was visible as interveinal chlorosis of the leaves, often
as a transient symptom. Fig. 1(a) and (b) show a typical course of chlorosis during
the growth period, using trial III as an example. During the ®rst three weeks after
the start of Zn treatment, the percentage of chlorotic plants increased drastically.
In the second half of the study, chlorosis decreased, resulting in mainly nonchlorotic plants at the end of the culture. Chlorosis was much more severe at pH 4
(Fig. 1(a)) compared to pH 6 (Fig. 1(b)), since the phytoavailable (CaCl2extractable) Zn content of the compost±peat substrate was almost ®ve times
higher at pH 4. Supply of 1 mg Fe as Fe±EDDHA per plant, as is usual in
horticultural practice, reduced chlorosis appreciably at both pH 4 and 6.
Although chlorosis was reduced by additional Fe supply, no signi®cant effect of
Fe supply and pH on plant Fe content was observed (Fig. 2(a)). On the other
hand, the Zn content in shoots of petunias was signi®cantly (p 0.01) reduced by
additional Fe supply at all pH levels (Fig. 2(b)). Plant Zn content decreased with
344
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
Table 2
Dry matter yield of petunias as in¯uenced by Zn supply (trials I±V)
Zn supply (mg kg±1
substrate d.m.)
Dry matter yield (g plant±1)
pH 4
pH 5
pH 6
pH 7
Trial I
Basic load
400
800
2.59aa
2.54a
2.68a
2.61a
2.62a
2.62a
2.46a
2.40a
2.32a
2.47a
2.74a
2.58a
Trial II
400
800
2.18a
2.11a
2.31a
2.16a
1.80a
2.19a
1.39a
1.41a
Trial III
Basic load
400
800
3.11a
3.09a
3.35a
3.30a
3.23a
3.31a
3.07a
3.04a
2.97a
2.99a
2.87a
3.29a
Trial IV
Basic load
800
1600
2.27a
2.31a
1.78b
Trial V
Basic load
800
1600
a
3.02a
2.84a
2.81a
Within one pH degree means with the same letter are not signi®cantly different.
increasing pH, since the CaCl2-extractable Zn content of the compost±peat
substrates decreased. No correlation between plant Zn and plant Fe content was
observed in the investigated plants.
The occurrence of chlorosis at any time during the growth period was chosen as
the parameter for the de®nition of toxicity levels for Zn contents in petunia dry
matter and for CaCl2-extractable Zn in compost±peat substrates, since it was the
most sensitive parameter. Summarizing the results of all four compost±peat
substrate trials (trials I±IV), chlorosis ®rst occurred at a plant Zn content of
160 mg Zn kg±1 d.m., although non-chlorotic plants were observed at higher plant
Zn contents (Fig. 3).
For all trials, the plant Zn content of petunias was closely correlated (r2 0.94)
to the CaCl2-extractable Zn content of compost±peat substrates (Fig. 4(a)),
whereas the correlation was not as good (r2 0.67) in the range below 13 mg
CaCl2-extractable Zn l±1 (Fig. 4(b)). Chlorosis ®rst occurred at approximately
6 mg CaCl2-extractable Zn l±1 compost±peat substrate.
In trial V, plant Zn and Fe contents were analyzed and chlorosis estimated at
weekly intervals during the growth period. The increase in petunia dry matter
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
345
Fig. 1. Occurrence of chlorosis in petunias during the culture period as in¯uenced by the Zn
content (76 and 700 mg kg±1, aqua regia soluble) of compost±peat substrates and Fe supply at pH 4
(a), and pH 6 (b) (trial III).
grown in peat with Zn supply was almost linear during the observed period and
was unaffected by the Zn level (Fig. 5(a)). Chlorosis did not occur in the basic
load treatment but increased with Zn supply to a level where 90% of the plants
were chlorotic (Fig. 5(c)). Chlorosis increased during the ®rst three weeks of the
growth period and then decreased until the end of the experiment.
The Zn contents of petunias followed a similar course during the growth period
(Fig. 5(b)), but the highest plant Zn contents were determined one week before
346
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
Fig. 2. In¯uence of Fe supply on Fe (a), and Zn (b) content of petunias after six weeks treatment
with a Zn supply of 695 mg kg±1 substrate d.m. (aqua regia soluble) at varied pH (trial II).
maximum chlorosis of plants. Plant Zn content then decreased until the end of the
experiment, followed one week later by the decrease in chlorosis. Although plant
Zn contents were high during the growth period, no dry matter yield reduction
was observed in any of the treatments.
Plant Fe content decreased between the second and third week after the start of
Zn treatment to one third of the initial content. The Fe content was at a similar
level in all Zn treatments (Fig. 5(d)).
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
347
Fig. 3. Occurrence of chlorosis of petunias depending on plant Zn content and toxicity level for Zn
in petunias (trials I±IV).
4. Discussion and conclusions
4.1. Plant yield and chlorosis as in¯uenced by Zn supply
For plants grown in the compost±peat substrates, dry matter reduction was
observed at 635 mg Zn kg±1 plant dry matter. This is in the upper range of Zn toxicity
levels for other crops quoted in the literature. Zn-induced yield depression may
occur in a wide range of plant Zn contents, i.e. 150±500 mg Zn kg±1 plant d.m.
(Sauerbeck, 1989), depending on the species. For Zea mays and Sorghum bicolor, a
signi®cant yield reduction has been observed at 500 mg Zn kg±1 plant d.m., whereas
360 mg Zn kg±1 plant d.m. were tolerated without symptoms (Smilde et al., 1974).
Chlorosis occurred at much lower plant Zn contents in petunia dry matter, i.e.,
at 160 mg Zn kg±1 plant d.m. (Fig. 3) than yield reduction. The toxicity level
obtained in our trials is lower than the toxicity levels for Zn-induced chlorosis in
sugar beets of 200 mg Zn kg±1 plant d.m. (Delschen and Werner, 1989), and for
petunias (Alt et al., 1994) and cereals (Merkel and KoÈster, 1976) of 300 mg Zn kg±1
plant d.m. This might be because these toxicity levels were based on the occurrence
of chlorosis at the time of harvesting, whereas in our trials the appearance of chlorosis
at any time during the growth period was taken into account.
4.2. Chlorosis as in¯uenced by Zn supply and Fe fertilization
It was decided to use the occurrence of chlorosis at any time during the growth
period as the toxicity parameter since its appearance was a very sensitive
348
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
Fig. 4. Relationship between Zn content of the substrate (CaCl2 method) and Zn content of plants
and toxicity level for the occurrence of Zn-induced chlorosis in compost±peat substrates (data of all
compost±peat substrate trials I±IV): (a) all treatments, (b) treatments having less than 13 mg l±1
CaCl2-extractable Zn.
parameter, occurring at much lower Zn contents than yield reduction. The
observed transient chlorosis resembled Fe de®ciency chlorosis and could be
alleviated by Fe supply.
Chlorosis was more severe at lower pH, indicating that chlorosis was induced
by Zn, because absolute Fe de®ciency chlorosis increases with increasing pH
(reduced Fe availability). Indeed, a higher proportion of Zn was phytoavailable at
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
349
Fig. 5. In¯uence of Zn supply on plant dry matter yield (a), plant Zn content (b), chlorosis (c), and
plant Fe content (d) of petunias during the culture in Zn-enriched peat substrate (trial V).
lower pH, as shown by the higher amount of CaCl2-extractable Zn at pH 4 than
pH 6 (Figs. 1 and 2).
Morphological evidence of Zn-induced Fe de®ciency chlorosis is supported by
the ®ndings that the chloroplast ultrastructure of tomato and spinach showed
similar changes in cases of Fe de®ciency and Zn toxicity (Rufner and Barker,
1984).
In the leaves, Zn might alter the subcellular or cellular distribution or
availability of Fe (Rosen et al., 1977). Zn may also inhibit reduction of FeIII to
FeII, which is the physiologically active form (Olsen et al., 1982). Hence, Zn can
interfere with the utilization of Fe in the leaf, perhaps in chlorophyll biosynthesis
(Chaney, 1993). Plant Fe contents were not affected by the Zn treatments (Fig.
2(a)) and they were considered as suf®cient, being in the range of 60±300 mg
Fe kg±1 plant d.m. which is regarded as normal for plant growth (Vose, 1982).
350
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
It is supposed that the additional Fe±EDDHA supply reduced chlorosis (Fig.
1(a) and (b)) due to decreased plant Zn contents (Fig. 2(b)) since the total plant Fe
contents were not affected. In a similar manner, Chaney (1993) observed that Zninduced chlorosis was corrected by spraying FeSO4 or Fe chelates on the leaves.
Greipsson (1995) and Pich et al. (1994) observed similar results with increasing
Fe levels in the growth medium and Fe supply decreasing Zn concentrations in all
plant organs.
The time courses of plant Zn and Fe contents were examined in trial V, growing
petunias in peat with de®ned Zn contents, in order to explain, why Zn-induced
chlorosis was a transient symptom during the growth period.
Chlorosis (Fig. 5(c)) displayed a course similar to those in the compost±peat
substrate trials (Fig. 1). The observed courses of Fe and Zn contents in petunias
during the growth period might be explained by the following: after the start of
Zn treatment the plant Zn content increased during the ®rst two weeks due to
increased availability of Zn. Plant Fe contents decreased in the third week,
leading to chlorosis in the third week. It is assumed that high plant Zn contents
resulted in chlorosis of the leaves at decreased plant Fe contents (three weeks
after the start of Zn treatment). The decrease in plant Zn and Fe contents with
increasing plant dry matter might be interpreted by dilution (Sauerbeck, 1989).
Since plant Zn concentration thus decreased until the end of the culture, chlorosis
was also reduced, again showing a delay of one week.
4.3. Toxic level of CaCl2-extractable Zn in compost±peat substrates
The toxic level is de®ned as the lowest Zn content in plants or the lowest
CaCl2-extractable Zn content in compost±peat substrates resulting in Zn-induced
chlorosis.
The toxic level of 160 mg kg±1 for Zn in petunia dry matter was related to a
level of about 6 mg CaCl2 extractable Zn l±1 substrate (Fig. 4(b)). 6 mg CaCl2extractable Zn l±1 substrate is in the lower range of toxic levels of CaCl2extractable Zn mentioned in the literature, which were also based on the
occurrence of chlorosis. To facilitate comparability, literature data in mg kg±1
soil were converted to mg l±1 soil, based on a bulk density of 1.5 kg l±1 soil. At
6 mg CaCl2-extractable Zn l±1 soil, chlorosis has been shown to occur in sugar
beet (Delschen and Werner, 1989). 30 % of the examined cereals showed
Zn-induced toxicity symptoms below 10.5 mg CaCl2-extractable Zn l±1 soil
(Merkel and KoÈster, 1976). Birke and Werner (1991) have mentioned a toxic level
of 7.5±22.5 mg CaCl2-extractable Zn l±1 soil for different crops. However, for
petunias, a toxic level, based on the occurrence of chlorosis, of 55 mg CaCl2extractable Zn l±1 substrate has been reported by Alt et al. (1994). The
corresponding toxic level of Zn in plants was also higher than the one determined
in our trials.
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
351
A tolerable level of CaCl2-extractable Zn was ®xed as a value which should not
be exceeded in order to avoid Zn-induced chlorosis. Thus, a safety factor of 25%
was considered, resulting in a tolerable level of 4.5 mg CaCl2-extractable Zn l±1
compost±peat substrate.
Acknowledgements
The authors thank the Deutsche Bundesstiftung Umwelt and the Deutsche
Kompost Handelsgesellschaft for ®nancial support. Composts were prepared by
PlanCoTec, Witzenhausen. In addition, we thank Dr. P. Seward (Hydro Agri
Deutschland GmbH, DuÈlmen) for valuable comments and correction of the
English.
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Toxicity level for phytoavailable zinc in
compost±peat substrates
Annette S. Bucher*, Manfred K. Schenk
Institute of Plant Nutrition, University of Hannover, HerrenhaÈuser Straûe 2,
30449 Hannover, Germany
Accepted 17 June 1999
Abstract
Petunias were grown in compost±peat substrates with different Zn contents (basic load, 400, 800,
and 1600 mg kg±1) in order to identify toxic levels of Zn in plant dry matter and of phytoavailable
(CaCl2-extractable) Zn in compost±peat substrates. Yield reduction of petunias was only observed
at an extremely high Zn content (635 mg Zn kg±1 plant d.m.), although chlorosis was evident at
lower Zn levels (160 mg Zn kg±1 plant d.m.). Thus, the occurrence of Zn-induced chlorosis during
the growth period was chosen as the toxicity parameter. Chlorosis and plant Zn content were
reduced by additional Fe supply, although the Fe content of the plants was not affected. During a six
week growth period, petunia Zn content and chlorosis increased in the ®rst two and three weeks,
respectively, and then decreased, whereas plant Fe content decreased sharply between the second
and third week after the start of Zn treatment. Chlorosis ®rst occurred with a plant Zn content of
160 mg kg±1, corresponding to a CaCl2-extractable Zn content in the substrate of 6 mg l±1, which
was identi®ed as the toxicity level for phytoavailable Zn in compost±peat substrates. Taking a safety
factor into account, a critical level of 4.5 mg l±1 was computed, which should not be exceeded in
order to avoid Zn-induced chlorosis. # 2000 Elsevier Science B.V. All rights reserved.
Keywords: CaCl2 extraction; Fe de®ciency chlorosis; Petuniahybrida; Zn toxicity
Abbreviations: AAS, atomic absorption spectroscopy; d.m., dry matter; EDDHA, ethylenediamine
di(o-hydroxyphenylacetic acid); ICP, inductively coupled plasma (spectroscopy); PTFE, polytetrafluorethylene (Teflon); SD, standard deviation
*
Corresponding author. Tel.: 49-511-2626; fax: 49-511-3611.
E-mail address: [email protected] (A.S. Bucher).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 8 6 - 2
340
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
1. Introduction
Composts prepared from green yard waste (grass and vegetative cuttings) and
organic waste are often used as a component of substrates for pot and container
plants. However, they often contain large amounts of heavy metals (Kehres,
1991), of which Zn is the one with the highest potential toxicity, because of its
high concentration and the comparatively high phytoavailability of Zn in the
compost. Zn phytoavailability depends on the total Zn content, pH, content of
other heavy metals, and the salt content of the substrate (Herms, 1989). With
decreasing pH, intensity and selectivity of Zn sorption decrease, resulting in a
higher phytoavailability (Herms, 1982). In compost±peat substrates, phytoavailable Zn contents can be characterized well by CaCl2 extraction (Bucher and
Schenk, 1997).
Zn-induced chlorosis is known to occur with a Zn content of 200 mg kg±1 in
sugar beet leaves (Delschen and Werner, 1989) and of 300 mg kg±1 for petunias
(Alt et al., 1994) and cereals (Merkel and KoÈster, 1976), indicating different
susceptibility between plant species. The toxicity level for CaCl2-extractable Zn
in soil and substrates, based on the occurrence of chlorosis, has been shown to
vary between 6 mg l±1 for sugar beet (calculated after Delschen and Werner,
1989) and 55 mg l±1 for petunias (Alt et al., 1994).
Zn toxicity-induced chlorosis resembles Fe de®ciency chlorosis (Rufner and
Barker, 1984), which can be corrected by Fe supply (Chaney, 1993). Zn may
interfere with Fe uptake, translocation or utilization in the leaves (Chaney, 1993).
The aim of this paper was to de®ne the toxicity levels for Zn in the plant dry
matter of petunias and for CaCl2-extractable Zn in compost±peat substrates.
Further emphasis was laid on the interaction of Zn and Fe with regard to the
occurrence of Zn-induced Fe de®ciency chlorosis.
2. Material and methods
The test plant used was the heavy metal-sensitive Petunia hybrida `Express
Orchid' (Bucher and Schenk, 1997).
2.1. Growing conditions
Plants were sown and pricked out in standard peat substrate. Seedlings were
potted in all trials in plastic pots of 250 ml volume and they were grown in a
greenhouse with 14±168C day and 12±148C night heating temperature, the
ventilation temperature being 28C higher. In autumn/winter (trial II and IV)
additional arti®cial light (about 165 mE m±2 s±2 PAR for 12 h) was provided.
Treatments held at pH 4 (CaCl2) received water without carbonate hardness, in
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
341
order to maintain the low pH during the growth period. Plants were harvested
after 38 (trial I), 56 (trial II), 45 (trial III), 50 (trial IV), and 42 (trial V) days,
respectively.
2.2. Treatments
1. Compost trials (trials I±IV). Mixtures of 40 vol% compost and 60 vol% peat
were used as substrates. Compost was prepared from green yard waste (grass and
vegetative cuttings). Zn sulfate was added before the waste was composted in
order to achieve similar Zn forms as in compost made from polluted raw material.
Zn addition was varied to match the contents occurring in commercial composts.
The target Zn values of the compost±peat substrates (400, 800, 1600 mg kg±1)
were not met exactly, as shown in Table 1.
Composting until maturity of the compost lasted for 14.5±19 weeks. In trials I,
II, and III, pH was adjusted to 4, 5, 6, and 7 according to buffer curves by using
H2SO4 and CaO. Substrates in trial IV were adjusted to pH 4. The target pH
values were obtained on average with an accuracy of 0.22 and ±0.37 pH units.
During the trials, pH increased on average by about 0.42 units at the time of
harvest. Basic fertilization consisted of 200 mg N l±1 substrate given in the form
of NH4NO3. No further nutrients were necessary as basic fertilization, since they
were present in the compost. During the growth period, plants were fertigated
with a nutrient solution containing (in mg l±1) 75 N, 15.26 P, 91 K, 0.6 Fe, 0.15 B,
0.01 Cu, 0.25 Mn, 0.025 Mo, and 0.05 Zn. In addition to fertilization, 1 mg Fe per
plant as solution of Fe-EDDHA was applied once after one week of cultivation as
is usual in horticultural practice (Fe treatments). Some further treatments were
included which did not receive the additional Fe supply (±Fe treatments). In trial
III these ±Fe treatments were only conducted at pH 4 and 6.
2. Peat trial (trial V). The Zn contents of petunias during the growth period
were investigated by growing plants in peat with varied Zn supply, with adjusted
substrate pH to 5.2 by the addition of 2.5 g CaCO3 l±1 peat. Zinc sulfate was
added before potting the plants in order to achieve the target total Zn contents
(800, 1600 mg kg±1). In addition, the peat substrate was fertilized with (in mg lÿ1):
225 N, 150 P, 225 K, 30 Mg, 1.125 Fe, and other micronutrients. Fertigation
was the same as for the compost±peat substrates, but no additional Fe was
supplied.
2.3. Experimental design and statistics
Trials were conducted using a completely randomized block design with three
replicates of each treatment. Ten plants were used for each treatment. Analysis of
variance, Tukey test, and linear regressions (Pearson) were computed, using the
statistical package SAS for Windows (1995).
342
Target Zn contents
(mg kg±1 substrate d.m.)
Basic load
400
800
1600
a
b
Aqua regia soluble Zn contents (mg kg±1 substrate d.m.)a
Trial I
(compost±peat
substrate)
Trial II
(compost±peat
substrate)
Trial III
(compost±peat
substrate)
Trial IV
(compost±peat
substrate)
Trial V
(peat)
157
552
975
±b
±
390
695
±
76
420
700
±
92
±
675
1306
26
±
886
1911
Average of all treatments with different pH.
Treatment in this trial not available.
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
Table 1
Target and aqua-regia-soluble Zn contents in the compost±peat substrates and in peat substrate
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
343
2.4. Chemical analysis and observation of plants
The pH in 0.01 M CaCl2 solution was determined in all substrates at the start of
the culture and at harvesting. Substrate samples were taken before potting the
plants and after drying and grinding, 2.5 g substrate was digested in aqua regia
(10.5 ml HCl 3.5 ml HNO3). Zn in the digest was detected by atomic
absorption spectroscopy (Hoffmann, 1991). Extractable (phytoavailable) Zn
contents in substrates were determined by extracting 1 g dried, sieved (2 mm
mesh, stainless steel) substrate with 10 ml 0.1 M CaCl2, and shaking for 1 h
(KoÈster and Merkel, 1982).
Plant samples were oven-dried at 60±708C to determine plant dry matter. Dried,
ground plant matter was digested under pressure in PTFE vessels with nitric acid.
Zn and Fe were analyzed using AAS and ICP. Plants were harvested weekly for
chemical analysis for trial V and at the end of the culture for the other trials.
Chlorosis of the plants was monitored weekly in all trials and is reported as a
percentage of chlorotic plants, without consideration of the severity of chlorosis.
3. Results
No signi®cant effect of Zn supply on plant dry matter yield was found in most
trials (see Table 2), except trial IV. In this case, plant dry matter decreased when
treated with the highest Zn supply. This occurred with a plant Zn content of
635 mg Zn kg±1 d.m. and a CaCl2-extractable Zn content of 69 mg Zn l±1
compost±peat substrate. The highest Zn contents without yield reduction were
471 mg Zn kg±1 plant d.m. for the plants grown in the compost±peat substrates
and 550 mg Zn kg±1 plant d.m. for the plants grown in the peat-only substrate at
the end of the experiment.
In all trials, Zn toxicity was visible as interveinal chlorosis of the leaves, often
as a transient symptom. Fig. 1(a) and (b) show a typical course of chlorosis during
the growth period, using trial III as an example. During the ®rst three weeks after
the start of Zn treatment, the percentage of chlorotic plants increased drastically.
In the second half of the study, chlorosis decreased, resulting in mainly nonchlorotic plants at the end of the culture. Chlorosis was much more severe at pH 4
(Fig. 1(a)) compared to pH 6 (Fig. 1(b)), since the phytoavailable (CaCl2extractable) Zn content of the compost±peat substrate was almost ®ve times
higher at pH 4. Supply of 1 mg Fe as Fe±EDDHA per plant, as is usual in
horticultural practice, reduced chlorosis appreciably at both pH 4 and 6.
Although chlorosis was reduced by additional Fe supply, no signi®cant effect of
Fe supply and pH on plant Fe content was observed (Fig. 2(a)). On the other
hand, the Zn content in shoots of petunias was signi®cantly (p 0.01) reduced by
additional Fe supply at all pH levels (Fig. 2(b)). Plant Zn content decreased with
344
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
Table 2
Dry matter yield of petunias as in¯uenced by Zn supply (trials I±V)
Zn supply (mg kg±1
substrate d.m.)
Dry matter yield (g plant±1)
pH 4
pH 5
pH 6
pH 7
Trial I
Basic load
400
800
2.59aa
2.54a
2.68a
2.61a
2.62a
2.62a
2.46a
2.40a
2.32a
2.47a
2.74a
2.58a
Trial II
400
800
2.18a
2.11a
2.31a
2.16a
1.80a
2.19a
1.39a
1.41a
Trial III
Basic load
400
800
3.11a
3.09a
3.35a
3.30a
3.23a
3.31a
3.07a
3.04a
2.97a
2.99a
2.87a
3.29a
Trial IV
Basic load
800
1600
2.27a
2.31a
1.78b
Trial V
Basic load
800
1600
a
3.02a
2.84a
2.81a
Within one pH degree means with the same letter are not signi®cantly different.
increasing pH, since the CaCl2-extractable Zn content of the compost±peat
substrates decreased. No correlation between plant Zn and plant Fe content was
observed in the investigated plants.
The occurrence of chlorosis at any time during the growth period was chosen as
the parameter for the de®nition of toxicity levels for Zn contents in petunia dry
matter and for CaCl2-extractable Zn in compost±peat substrates, since it was the
most sensitive parameter. Summarizing the results of all four compost±peat
substrate trials (trials I±IV), chlorosis ®rst occurred at a plant Zn content of
160 mg Zn kg±1 d.m., although non-chlorotic plants were observed at higher plant
Zn contents (Fig. 3).
For all trials, the plant Zn content of petunias was closely correlated (r2 0.94)
to the CaCl2-extractable Zn content of compost±peat substrates (Fig. 4(a)),
whereas the correlation was not as good (r2 0.67) in the range below 13 mg
CaCl2-extractable Zn l±1 (Fig. 4(b)). Chlorosis ®rst occurred at approximately
6 mg CaCl2-extractable Zn l±1 compost±peat substrate.
In trial V, plant Zn and Fe contents were analyzed and chlorosis estimated at
weekly intervals during the growth period. The increase in petunia dry matter
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
345
Fig. 1. Occurrence of chlorosis in petunias during the culture period as in¯uenced by the Zn
content (76 and 700 mg kg±1, aqua regia soluble) of compost±peat substrates and Fe supply at pH 4
(a), and pH 6 (b) (trial III).
grown in peat with Zn supply was almost linear during the observed period and
was unaffected by the Zn level (Fig. 5(a)). Chlorosis did not occur in the basic
load treatment but increased with Zn supply to a level where 90% of the plants
were chlorotic (Fig. 5(c)). Chlorosis increased during the ®rst three weeks of the
growth period and then decreased until the end of the experiment.
The Zn contents of petunias followed a similar course during the growth period
(Fig. 5(b)), but the highest plant Zn contents were determined one week before
346
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
Fig. 2. In¯uence of Fe supply on Fe (a), and Zn (b) content of petunias after six weeks treatment
with a Zn supply of 695 mg kg±1 substrate d.m. (aqua regia soluble) at varied pH (trial II).
maximum chlorosis of plants. Plant Zn content then decreased until the end of the
experiment, followed one week later by the decrease in chlorosis. Although plant
Zn contents were high during the growth period, no dry matter yield reduction
was observed in any of the treatments.
Plant Fe content decreased between the second and third week after the start of
Zn treatment to one third of the initial content. The Fe content was at a similar
level in all Zn treatments (Fig. 5(d)).
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
347
Fig. 3. Occurrence of chlorosis of petunias depending on plant Zn content and toxicity level for Zn
in petunias (trials I±IV).
4. Discussion and conclusions
4.1. Plant yield and chlorosis as in¯uenced by Zn supply
For plants grown in the compost±peat substrates, dry matter reduction was
observed at 635 mg Zn kg±1 plant dry matter. This is in the upper range of Zn toxicity
levels for other crops quoted in the literature. Zn-induced yield depression may
occur in a wide range of plant Zn contents, i.e. 150±500 mg Zn kg±1 plant d.m.
(Sauerbeck, 1989), depending on the species. For Zea mays and Sorghum bicolor, a
signi®cant yield reduction has been observed at 500 mg Zn kg±1 plant d.m., whereas
360 mg Zn kg±1 plant d.m. were tolerated without symptoms (Smilde et al., 1974).
Chlorosis occurred at much lower plant Zn contents in petunia dry matter, i.e.,
at 160 mg Zn kg±1 plant d.m. (Fig. 3) than yield reduction. The toxicity level
obtained in our trials is lower than the toxicity levels for Zn-induced chlorosis in
sugar beets of 200 mg Zn kg±1 plant d.m. (Delschen and Werner, 1989), and for
petunias (Alt et al., 1994) and cereals (Merkel and KoÈster, 1976) of 300 mg Zn kg±1
plant d.m. This might be because these toxicity levels were based on the occurrence
of chlorosis at the time of harvesting, whereas in our trials the appearance of chlorosis
at any time during the growth period was taken into account.
4.2. Chlorosis as in¯uenced by Zn supply and Fe fertilization
It was decided to use the occurrence of chlorosis at any time during the growth
period as the toxicity parameter since its appearance was a very sensitive
348
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
Fig. 4. Relationship between Zn content of the substrate (CaCl2 method) and Zn content of plants
and toxicity level for the occurrence of Zn-induced chlorosis in compost±peat substrates (data of all
compost±peat substrate trials I±IV): (a) all treatments, (b) treatments having less than 13 mg l±1
CaCl2-extractable Zn.
parameter, occurring at much lower Zn contents than yield reduction. The
observed transient chlorosis resembled Fe de®ciency chlorosis and could be
alleviated by Fe supply.
Chlorosis was more severe at lower pH, indicating that chlorosis was induced
by Zn, because absolute Fe de®ciency chlorosis increases with increasing pH
(reduced Fe availability). Indeed, a higher proportion of Zn was phytoavailable at
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
349
Fig. 5. In¯uence of Zn supply on plant dry matter yield (a), plant Zn content (b), chlorosis (c), and
plant Fe content (d) of petunias during the culture in Zn-enriched peat substrate (trial V).
lower pH, as shown by the higher amount of CaCl2-extractable Zn at pH 4 than
pH 6 (Figs. 1 and 2).
Morphological evidence of Zn-induced Fe de®ciency chlorosis is supported by
the ®ndings that the chloroplast ultrastructure of tomato and spinach showed
similar changes in cases of Fe de®ciency and Zn toxicity (Rufner and Barker,
1984).
In the leaves, Zn might alter the subcellular or cellular distribution or
availability of Fe (Rosen et al., 1977). Zn may also inhibit reduction of FeIII to
FeII, which is the physiologically active form (Olsen et al., 1982). Hence, Zn can
interfere with the utilization of Fe in the leaf, perhaps in chlorophyll biosynthesis
(Chaney, 1993). Plant Fe contents were not affected by the Zn treatments (Fig.
2(a)) and they were considered as suf®cient, being in the range of 60±300 mg
Fe kg±1 plant d.m. which is regarded as normal for plant growth (Vose, 1982).
350
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
It is supposed that the additional Fe±EDDHA supply reduced chlorosis (Fig.
1(a) and (b)) due to decreased plant Zn contents (Fig. 2(b)) since the total plant Fe
contents were not affected. In a similar manner, Chaney (1993) observed that Zninduced chlorosis was corrected by spraying FeSO4 or Fe chelates on the leaves.
Greipsson (1995) and Pich et al. (1994) observed similar results with increasing
Fe levels in the growth medium and Fe supply decreasing Zn concentrations in all
plant organs.
The time courses of plant Zn and Fe contents were examined in trial V, growing
petunias in peat with de®ned Zn contents, in order to explain, why Zn-induced
chlorosis was a transient symptom during the growth period.
Chlorosis (Fig. 5(c)) displayed a course similar to those in the compost±peat
substrate trials (Fig. 1). The observed courses of Fe and Zn contents in petunias
during the growth period might be explained by the following: after the start of
Zn treatment the plant Zn content increased during the ®rst two weeks due to
increased availability of Zn. Plant Fe contents decreased in the third week,
leading to chlorosis in the third week. It is assumed that high plant Zn contents
resulted in chlorosis of the leaves at decreased plant Fe contents (three weeks
after the start of Zn treatment). The decrease in plant Zn and Fe contents with
increasing plant dry matter might be interpreted by dilution (Sauerbeck, 1989).
Since plant Zn concentration thus decreased until the end of the culture, chlorosis
was also reduced, again showing a delay of one week.
4.3. Toxic level of CaCl2-extractable Zn in compost±peat substrates
The toxic level is de®ned as the lowest Zn content in plants or the lowest
CaCl2-extractable Zn content in compost±peat substrates resulting in Zn-induced
chlorosis.
The toxic level of 160 mg kg±1 for Zn in petunia dry matter was related to a
level of about 6 mg CaCl2 extractable Zn l±1 substrate (Fig. 4(b)). 6 mg CaCl2extractable Zn l±1 substrate is in the lower range of toxic levels of CaCl2extractable Zn mentioned in the literature, which were also based on the
occurrence of chlorosis. To facilitate comparability, literature data in mg kg±1
soil were converted to mg l±1 soil, based on a bulk density of 1.5 kg l±1 soil. At
6 mg CaCl2-extractable Zn l±1 soil, chlorosis has been shown to occur in sugar
beet (Delschen and Werner, 1989). 30 % of the examined cereals showed
Zn-induced toxicity symptoms below 10.5 mg CaCl2-extractable Zn l±1 soil
(Merkel and KoÈster, 1976). Birke and Werner (1991) have mentioned a toxic level
of 7.5±22.5 mg CaCl2-extractable Zn l±1 soil for different crops. However, for
petunias, a toxic level, based on the occurrence of chlorosis, of 55 mg CaCl2extractable Zn l±1 substrate has been reported by Alt et al. (1994). The
corresponding toxic level of Zn in plants was also higher than the one determined
in our trials.
A.S. Bucher, M.K. Schenk / Scientia Horticulturae 83 (2000) 339±352
351
A tolerable level of CaCl2-extractable Zn was ®xed as a value which should not
be exceeded in order to avoid Zn-induced chlorosis. Thus, a safety factor of 25%
was considered, resulting in a tolerable level of 4.5 mg CaCl2-extractable Zn l±1
compost±peat substrate.
Acknowledgements
The authors thank the Deutsche Bundesstiftung Umwelt and the Deutsche
Kompost Handelsgesellschaft for ®nancial support. Composts were prepared by
PlanCoTec, Witzenhausen. In addition, we thank Dr. P. Seward (Hydro Agri
Deutschland GmbH, DuÈlmen) for valuable comments and correction of the
English.
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