Herbicides can stimulate plant growth

Herbicides can stimulate plant growth

N CEDERGREENDepartment of Agricultural Sciences, Faculty of Life Science, University of Copenhagen, Denmark

Received 15 October 2007

Revised version accepted 13 March 2008

Summary

Low dose stimulations by toxicants have long been

observed. Great controversies exist concerning the

interpretation of these observations, spanning from

believing that they are a general stress response occur-

ring for all chemicals, to simply being an experimental

artefact resulting from poorly growing control plants or

from biomass allocation between plant parts. This study

investigates the growth response and biomass allocation

pattern of barley exposed to 10–15 doses of eight

different herbicides. The results show that the globally

most widely used herbicide, glyphosate, together with

the sulfonylurea, metsulfuron-methyl, can induce a real

stimulation in biomass growth of approximately 25%

when applied at doses corresponding to 5–10% field

rate. The other six herbicides tested did not induce

consistent hormesis, thereby undermining the theory of

hormesis being a general stress response. Biomass

allocations between plant parts did take place, but were

not the cause of the hormetic growth stimulations. The

results demonstrate that plant physiological responses to

low herbicide doses cannot be extrapolated from our

knowledge of effects of higher, commercially used,

doses. Other physiological mechanisms seem to be

triggered in the low dose-range, and the investigation

of these mechanisms poses new challenges for agrono-

mists, environmentalists and plant physiologists.

Keywords: hormesis, plant traits, trade-off between

traits, glyphosate, metsulfuron-methyl, growth stimu-

lation, herbicides, biphasic dose–response curves.

CEDERGREEN N (2008). Herbicides can stimulate plant growth. Weed Research 48, 429–438.

Introduction

It has been known for more than a century that some

substances at low doses can be beneficial, while they are

lethal at higher doses. This phenomenon was first called

the Arndt-Schulz law (Calabrese, 2005). Southam and

Erlich (1943) introduced the term hormesis to describe

the effect of an oak bark compound that enhanced

fungal growth at low doses but strongly inhibited it at

higher doses. The concept of hormesis has highly

controversial implications within the areas of environ-

mental and medical toxicology (Calabrese, 2005; Thayer

et al., 2005), as it questions the ways we set limit values

for pollutants and toxins. There are those who claim

hormesis is the rule rather than the exception and that it

represents an evolutionary-based adaptive response to

environmentally induced disruption in homeostasis

(Stebbing, 1998; Calabrese & Baldwin, 2001). Others

are more sceptical in terms of the generality of the

phenomenon, claiming that hormesis leading to an

increase in population fitness is not an evolutionary

expectation and that even though some traits might be

stimulated by low doses of stress, this stimulation will

occur at a cost (Forbes, 2000; Parsons, 2003). In fact,

very few studies have been performed trying to elucidate

the mechanisms behind the biphasic dose–response

relationships often encountered. Most reports on hor-

mesis come from studies designed to investigate some

other problem, most often high dose effects, and the

hormetic response is rarely more than noted (Streibig,

1980).

This is also the case for studies on the effect of

herbicides on plants (Streibig, 1980). Since herbicides are

one of the groups of chemicals we deliberately spread

into the environment in large quantities, understanding

how low doses affect the growth and physiology of

plants is of utmost importance, both to farmers and risk

assessors. Since the initiation of herbicide development

in the early years after the second world war, stimula-

tions of one trait or another in plants in response to low

Correspondence: Nina Cedergreen, Department of Agricultural Sciences, Faculty of Life Science, University of Copenhagen, Højbakkegard Alle 13,

2630 Tastrup, Denmark. Tel: (+45) 35 33 33 97; Fax: (+45) 35 28 34 78; E-mail: [email protected]

� 2008 The Author

Journal Compilation � 2008 European Weed Research Society Weed Research 48, 429–438

herbicide doses have been noted (references summarised

in Duke et al., 2006). Only a few studies have tried to

investigate the mechanisms behind the growth stimu-

lation; for those that did, the results were largely

inconclusive (Allender et al., 1997; Morre, 2000;

Appleby, 2001). Any sceptic could therefore, with good

reason, claim that the biphasic dose–response curves

reported in plant science are either a result of poorly

growing controls or of trade-offs between traits.

Regarding whether the occurrence of hormesis is real

or whether it is an experimental artefact, a recent

database study including 687 dose–response curves

divided between three plant- or algae species and 10

herbicides, showed treatment averages above the control

level in 25–76% of the dose–response curves, depending

on the species (Cedergreen et al., 2007). In some species,

hormesis therefore seems to happen more frequently

than would be predicted by chance. The study also

showed a rather large variance between the different

herbicides, in terms of which induced hormesis. Some

only did it in <20% of the curves analysed, while others

had significant biphasic dose–response curves in more

than 70% of the curves (Cedergreen et al., 2007). Hence,

these results indicate not only that (shoot) growth

stimulations do take place, but also that not all

herbicides are equally effective in inducing this growth

increase. The dose–response curves analysed were, as

most others reporting hormesis, not designed to inves-

tigate low dose effects. Hence, it cannot be excluded that

using more low doses could reveal a hormetic response

for all herbicides, confirming the theories of biphasic

dose–response curves being a general phenomenon

(Calabrese & Baldwin, 2001).

The question remains whether the observed growth

increase is real or just a result of trade-offs between

traits? Studies on insects have shown that low doses of

chemicals can enhance egg production, but that the

enhanced production of offspring was counterbalanced

by lower offspring survival (Fujiwara et al., 2002;

Zanuncio et al., 2003). Similar trade-offs between traits

could be expected in plants, which can allocate their

resources in ways to optimise their growth under stress

conditions. It is well known that plants allocate root

biomass in soil patches where the environment is

favourable in terms of water and nutrients, while

avoiding more unfavourable soil patches (Kleijn &

Van Groenendael, 1999; Wijesinghe & Hutchings,

1999). Sub-lethal concentrations of herbicides in soils

might therefore induce an increased allocation of bio-

mass to roots to promote the search for a more

favourable root habitat. In fact, for seven soil applied

herbicides, Wiedman and Appleby (1972) showed root

growth of oat and cucumber was increased at low

herbicide doses, while shoot growth was unaffected. For

the remaining four herbicides tested, both root and

shoot growth were affected (Wiedman & Appleby,

1972). Other allocation patterns might be seen for

sprayed plants. In most studies showing hormesis, only

one trait is measured. This is most often shoot biomass

growth, leaf length or leaf area (Wiedman & Appleby,

1972; Allender et al., 1997; Schabenberger et al., 1999;

Davies et al., 2003; Cedergreen et al., 2007). Any kind of

resource allocation can therefore not be evaluated from

these studies. Hence, it cannot be excluded that whole

plant biomass growth is unaffected in the �hormetic�dose-range, but that a change in root:shoot allocation,

an allocation of biomass between leaves and stems or an

increase in specific leaf area are causing the apparent

growth stimulation.

We therefore tested the following hypotheses: (i)

hormesis in plants is not a result of a real growth

increase, but rather a result of (a) poorly growing

control plants or (b) a trade-off between traits; (ii)

hormesis is a general stress response across chemicals

with different physiological mechanisms of action; (iii)

hormesis is independent of whether the chemical is

absorbed through leaves or roots.

To test these hypotheses, barley was grown in

hydroponic cultures and exposed to 10–15 doses of

eight herbicides, either through the root media or by

spraying. Each herbicide represented chemicals affecting

different molecular pathways in the plants, ranging from

compounds affecting photosynthesis to compounds

affecting the synthesis of amino acids or fatty acids or

preventing microtubule assembly. The dry weight

increase of both roots and shoots were measured at

harvest, together with leaf and stalk dry weight and

length.

Material and methods

Plants

Barley (Hordeum vulgare, L.) seeds var. Barke

(Saatzucht Josef Breun, Harzogenawach, Germany)

were sown in moistened vermiculite (Sorbix-fine ⁄Damolin, Garta, Copenhagen, Denmark) and placed

in a climate chamber (1.2 m · 1.2 m · 1.2 m) at a

day ⁄night temperature of 21 ⁄ 15�C and a 16 ⁄ 8 h light ⁄dark cycle (300–340 lmol m)1 s)1 Photosynthetic

Active Radiation). One week after sowing, when the

plants had two true leaves, the plants were removed

from the vermiculite, the roots were gently washed and

each plant was placed in a foam stopper (Bostik

baggrundsprofil, 16 mm, Bauhaus, Tastrup, Denmark)

fitted to a hole in a plastic plate covering a 650 mL pot

(diameter: 11 cm) with nutrient media. The composition

of the media is described in Pedas et al. (2005) with the

430 N Cedergreen

� 2008 The Author


only modification of adding 0.15 lM MnSO4. Four

plants were placed in each pot. Ten plants were killed

and dried at 80�C for initial root and shoot dry weight

determination.

Herbicide treatment

The plants were exposed to herbicides in two ways:

either through the growth media or by spraying the

herbicides onto the leaves. Eight herbicides with

different molecular target sites were tested. Only

technical herbicides were used, apart from diquat

where the formulated compound Reglone (diquat

bromide) was used (Table 1). For the plants exposed

through the media, 15 doses with four plants in each

were used. Four herbicides were tested in the same

experiment sharing 16–32 control plants per experi-

ment. The herbicide concentrations increased with a

factor two between doses (dose-ranges are given in

Table 1). The media with herbicide was changed every

3 days. Growth conditions were as described above

and the plants were harvested 14 days after the

initiation of the herbicide exposure.

For the spray exposure experiments, 10 doses were

used with eight replicates of the four lowest doses and

four replicates of the six highest doses. Five or six

herbicides were tested in parallel sharing 32 control

plants. All herbicides were formulated by dissolving

them in 0.1% polyoxyethylene sorbitan monolaurate

(PSM) (Tween� 20, CAS: 9005–64–5, Bie & Berntsen,

Rødovre, Denmark). In addition, the relatively lipo-

philic herbicides: haloxyfop, pendimethalin and terbu-

thylazine, were first dissolved in 150 mL acetone

before adding 850 mL 0.1% PSM solution to be able

to dissolve them at high enough concentrations to

have an effect on growth. The plants were sprayed 1

or 2 days after having been placed in the pots. The

spraying was performed in a spray cabin using Hardi

LD-02–110 hydraulic nozzles, a pressure of 400 kPa

and a spraying volume of 150 L)1 ha)1. Half the

control plants were sprayed with 0.1% PSM and the

other half with a 15% acetone 0.1% PSM solution, to

test whether the addition of acetone at these concen-

trations had an effect on growth. The nutrient media

of all plants were changed every 3 days. Growth

conditions were as described previously and the plants

were harvested 14 days after spraying. All experiments

were duplicated in time.

Harvest

At harvest, all plants were separated into root and shoot

and the two plant parts were dried at 80�C for 48 h and

weighed. For the root exposure experiments, stalk Table

1Listoftheherbicides,theirprimary

moleculartarget

site,Kow,purity,thetotaldose-ranges

tested

intheexperim

ents

andthesuppliersoftheproducts

Herb

icid

eS

ite

of

action

Log

Kow

at

pH

7

Technic

al

purity

Dose-r

ange,

spra

y(g

a.i.

ha)

1)

Dose-r

ange,

media

(lg

L)

1)

Com

pany

Acifl

uorf

en-s

odiu

mP

roto

porp

hyrinogen

oxid

ase

inhib

itor

1.1

944%

0.0

13–1000

1.1

7–1200

BA

SF

Diq

uat

PS

I-ele

ctr

on

div

ers

ion

)4.6

0374

gL

)1a

0.0

12–1000

1.1

7–600

Syngenta

Gly

phosate

EP

SP

synth

ase

inhib

itor

)3.2

0m

in.

95%

0.0

46–760

2.2

3–1140

Chem

inova

Halo

xyfo

p-P

-meth

yl

AC

Case

inhib

itor

4.0

096.7

%0.0

07–137

0.2

0–100

Dow

Agro

Scie

nce

MC

PA

Auxin

agonis

ts0.7

193%

0.6

1–10000

2.1

9–1100

Kla

rsoe

Mets

ulfuro

n-m

eth

yl

ALS

-inhib

itor

)1.7

598.5

%0.0

30–1000

0.0

35–20

DuP

ont

Pendim

eth

alin

Mic

rotu

bule

assem

bly

inhib

itor

5.1

898%

0.0

02–300

0.9

8–500

BA

SF

Terb

uth

yla

zine

PS

II-inhib

itor

3.2

199%

0.0

12–1500

1.1

9–600

Syngenta

Theinform

ationisobtained

from

ThePesticideManual(Tomlin,2003).Followingacronymsare

used:EPSP(5-enoyl-pyruvylshikim

icacid3-phosphate),ALS(A

cetolactate

Synthase),ACCase

(acetylCoA

carboxylase),PSI(Photosystem

I)andPSII

(Photosystem

II).

aDiquatbromide.

Herbicides can stimulate plant growth 431

� 2008 The Author


length, total leaf length and dry weight of each were also

measured on each plant. Leaf length was used instead of

leaf area, as the leaves coiled, which made leaf area

determinations inaccurate. Dry weight increase of roots

and shoots were calculated subtracting the average root

and shoot biomass of plants on the day of treatment of

15.5 ± 3.26 mg root plant)1 and 9.78 ± 1.48 mg shoot

plant)1 (mean ± SEM, n = 10).

Statistics

All data on biomass increase were described with

a classic monotonous four parameter logistic

dose–response model and with a biphasic logistic

dose–response model including a term for hormesis

(Cedergreen et al., 2005):

y ¼ cþ d � cþ f expð�1=xaÞ1þ ðx=eÞb

; ð1Þ

where y is the measured response, c is the lower limit at

infinite herbicide concentration ⁄dose, d is the upper

limit of the curve corresponding approximately to the

average of the control plants, f is the parameter

describing the degree of hormetic increase, a is a

parameter determining the slope of the hormetic

increase, e determines the lower limit of the concentra-

tion ⁄dose of 50% response decrease (EC ⁄ED50) and b is

proportional to the slope of the dose–response curve

around e (Cedergreen et al., 2005). As there are rarely

enough data to estimate a, its value was fixed to either

0.25, 0.5 or 1, which experience has shown to cover the

range of slope increase in most hormetic dose–response

curves in plants, and the value allowing the best fitting

curve of the three was chosen. Setting f to zero removes

the hormetic term f expð�1=xaÞ from the equation and

reduces it to the classic four parameter log-logistic

model with e equal to EC ⁄ED50. To minimise variance

inhomogeneity, an optimal Box-Cox transformation was

applied (Streibig et al., 1993). All data but one dataset

were described setting the lower limit (c) to zero,

assuming no dry weight increase of plants exposed to

infinite high herbicide concentrations. The only excep-

tion was the data on metsulfuron-methyl, which is a

slowly acting herbicide (Cobb, 1992a) and therefore has

a lower limit equal to the dry weight gained before the

herbicide affects photosynthesis. The two non-linear

models, with and without the hormetic term, were

compared by an F-test to determine which of the models

best described the data (Bates & Watts, 1988). If the

model including hormesis did not fit data better than

the three-parameter logistic model (P > 0.05), the

logistic model was chosen. For the data on acifluorfen,

MCPA, metsulfuron-methyl and pendimethalin-treated

plants in the spray experiments, the doses did not give an

adverse effect of more than approximately 30%. Hence,

these data could not be fitted to a sigmoid model.

Instead, each rate was compared to the untreated

control with a students t-test to detect significant growth

increases.

To test whether the allocation of biomass to roots

and shoots, leaves and stems in the plants treated with

low herbicide doses differed from the untreated control,

root:shoot ratio, leaf:stalk ratio, length-specific leaf dry

weight (DW cm)1) and length-specific stem weight

(DW cm)1) from treatments with an average dry weight

>90% of control plants were compared with the

controls with a students t-test. All analyses were made

using the language and environment R (R Development

Core Team, 2004) with the add-on package drc (http://

www.bioassay.dk) (Ritz & Streibig, 2005).

Results

Dry weight growth

The dry weight increases during the exposure period of

the herbicide-treated barley plants are given in Figs 1

and 2 and the parameters of the models fitted to the data

are given in Table 2. Plant growth did increase in some

experiments in response to low herbicide doses when

compared with control plant values. The growth of the

control plants did, however, also vary between experi-

mental times (Figs 1 and 2). For three of the four

experimental times with media exposure, the control

plants were not significantly different from each other

(ANOVA: P = 0.73), but for the fourth experiment the

control plant dry weight increased from the average of

0.184 ± 0.056 g dry weight plant)1 (n = 56) to

0.292 ± 0.045 g dry weight plant)1 (n = 16). A similar

observation was made for the spray experiments, where

control plants of the two first experiments were the same

(t-test: P = 0.34), but increased from 0.203 ± 0.050 g

dry weight plant)1 (n = 58) to 0.269 ± 0.041 g dry

weight plant)1 (n = 31) in the third experiment. There

was no difference in growth of control plants sprayed

with and without acetone (t-test, P > 0.05). As a

hormetic growth increase is evaluated in relation to the

control plants, the variation in the control plant growth

also affected the reproducibility of the herbicide induced

growth increase. Hence, when data were evaluated using

the control plants grown in the same growth chamber as

the treated plants, all herbicides except acifluorfen

induced a growth increase in at least one of the

experiments, but none induced it in all (Figs 1 and 2,

Table 2).

Evaluating all data in relation to the pooled control

plants for either the experiments with media exposure

(n = 72) or those with spray exposure (n = 89) gave

432 N Cedergreen

� 2008 The Author


Fig. 1 The dry weight increase of barley plants exposed through the root media to increasing concentrations of eight different herbicides.

The experiments were repeated and data from each experiment were described with the best fitting model (Table 2, common controls).

Data from experiment one (black symbols, full curve) and experiment two (grey symbols, broken curve) are given as mean ± SE. The

average control across experiments was 0.21 ± 0.07 g dry weight plant)1 and is given by the horizontal line.

Fig. 2 The dry weight increase in barley plants sprayed with increasing concentrations of eight different herbicides. The experiments

were repeated and data from each experiment were described with the best fitting model (Table 2, common controls). For experiments that

could not be described with a dose–response model, data from each dose was compared by a t-test with either the controls of the experiments

(significant increase is marked with x) or the average control across all spray experiments (significant increase is marked with *). Data

from experiment one (black symbols, full curve) and experiment two (grey symbols, broken curve) are given as mean ± SE. The average

control across experiments was 0.23 ± 0.06 g dry weight plant)1 and is given by the horizontal line.


� 2008 The Author


Table

2Model

parametersofthebestfittingmodel

forwhole

plantdry

weightasafunctionofherbicideconcentrationordose

Herb

icid

eE

xposure

Experim

ent

contr

ol

Poole

dcontr

ol

bd

ef

am

ax

bd

ef

am

ax

Acifl

uorf

en

Media

11.5

6±

0.2

20.1

8±

0.0

157

±8

1.4

5±

0.2

60.1

9±

0.0

149

±10

Media

21.0

7±

0.2

50.2

0±

0.0

119

±5

0.9

8±

0.2

70.2

1±

0.0

1159

±48

Diq

uat

Media

11.2

3±

0.2

30.2

0±

0.0

190

±15

1.0

0±

0.2

70.2

1±

0.0

155

±14

Media

21.4

4±

0.2

40.1

9±

0.0

121

±3

0.0

74

±0.0

16

1–

1.4

3±

0.4

80.2

1±

0.0

119

±5

0.0

99

±0.0

34

0.2

50.2

6

Spra

y1

2.5

3±

0.5

50.2

5±

0.0

1170

±23

3.1

1±

0.6

70.2

3±

0.0

1201

±24

Spra

y2

0.9

8±

0.1

70.1

9±

0.0

169

±20

0.0

70

±0.0

27

10.2

31.1

4±

0.1

60.2

2±

0.0

197

±16

Gly

phosate

Media

11.3

8±

0.1

90.1

6±

0.0

113

±4

0.2

23

±0.0

67

0.5

0.2

61.5

8±

0.6

40.2

1±

0.0

118

±6

0.1

00

±0.0

60

0.5

0.2

5

Media

21.4

3±

0.1

50.2

6±

0.0

1113

±14

1.6

7±

0.4

40.2

1±

0.0

1100

±20

0.1

20

±0.0

32

0.2

50.2

7

Spra

y1

2.7

0±

0.5

60.2

8±

0.0

1354

±38

2.3

3±

0.4

80.2

3±

0.0

1310

±36

0.1

16

±0.0

19

0.2

50.3

0

Spra

y2

1.2

2±

0.2

60.1

9±

0.0

1499

±87

0.0

70

±0.0

17

10.2

51.2

0±

0.3

20.2

2±

0.0

1513

±103

0.0

38

±0.0

16

10.2

6

Halo

xyfo

pM

edia

11.2

6±

0.1

40.1

9±

0.0

113

±2

1.1

3±

0.3

30.2

1±

0.0

111

±3

Media

22.2

2±

0.5

00.1

8±

0.0

125

±4

0.7

2±

0.4

00.2

1±

0.0

111

±5

Spra

y1

0.8

9±

0.1

60.2

1±

0.0

16.7

±5.1

0.1

68

±0.1

28

10.2

31.1

9±

0.1

80.2

2±

0.0

119

±3

Spra

y2

6.2

4±

5.3

30.2

1±

0.0

141

±6

1.5

5±

0.5

00.2

3±

0.0

133

±6

MC

PA

Media

10.5

8±

0.1

00.1

6±

0.0

174

±60

0.1

28

±0.0

74

0.2

50.1

90.6

4±

0.1

40.2

1±

0.0

1139

±50

Media

20.7

8±

0.1

30.2

5±

0.0

1109

±30

1.1

5±

0.3

20.2

1±

0.0

1149

±40

Mets

ulfuro

nM

edia

1a

0.5

6±

0.1

20.1

6±

0.0

11.0

±0.9

0.2

82

±0.1

31

0.2

50.2

10.6

6±

0.2

30.1

9±

0.0

10.8

±1.3

0.4

51

±0.4

92

0.2

50.2

2

Media

2a

1.4

6±

0.2

50.3

0±

0.0

14.3

±5.7

1.3

8±

0.4

80.2

1±

0.0

12.9

±0.8

0.2

60

±0.0

55

0.2

50.3

2

Pendim

eth

alin

Media

10.8

8±

0.1

20.2

0±

0.0

134

±6

0.7

3±

0.1

80.2

1±

0.0

122

±7

Media

20.2

9±

0.1

00.2

0±

0.0

15.0

±0.2

0.2

9±

0.1

20.2

1±

0.0

111

±31

Terb

uth

yla

zine

Media

10.9

1±

0.1

60.1

5±

0.0

116

±6

0.4

0±

0.0

80.2

1±

0.0

12.5

±1.2

Media

22.3

1±

0.4

50.2

6±

0.0

145

±6

1.5

2±

0.5

10.2

1±

0.0

133

±9

0.1

71

±0.0

54

0.2

50.2

8

Spra

y1

1.9

3±

0.4

90.2

1±

0.0

1204

±32

0.0

3±

0.0

21

0.2

39

2.2

2±

0.5

50.2

3±

0.0

1214

±31

Spra

y2

2.0

5±

0.4

70.2

1±

0.0

1209

±30

1.7

6±

0.4

00.2

2±

0.0

0186

±27

Thecalculationsare

madeboth

usingexperim

entalcontrolsandusingpooledcontrols.Thesignificance

oftheparametersisgiven

inEqn(1).Forcurves

withhorm

esisthemaxim

um

response

isgiven

under

max(g

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weightincrease).Allparametersexcept

a,whichisfixed,andmaxwhichiscalculated,are

given

±SE.

aThecurves

werefitted

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limitswhichwere0.037±

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0.004fordata

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withexperim

entalcontrols,respectively,and0.045±

0.016and0.057±

0.016fordata

fitted

withpooledcontrols.

434 N Cedergreen

� 2008 The Author


more consistent results (Tables 2 and 3). Using the

pooled control plants showed no hormesis for haloxyfop

and pendimethalin, hormesis in one out of four exper-

iments for acifluorfen, diquat and terbuthylazine and

significant hormesis in four out of four experiments for

glyphosate and metsulfuron-methyl (Tables 2 and 3). In

Table 3, the hormetic growth increase is calculated from

the difference between the control plant average, either

experimental or pooled, and the corresponding fitted

maximum value of the hormetic dose–response curves

(Table 2). The results are given as percentage of control

plant growth. For the spray experiments, which could

not be described with a dose–response model, the

maximum value was calculated as either a single

treatment or an average of treatments within a range

bordered by treatments with growth rates significantly

higher than the untreated control. These treatment doses

or dose-ranges were: 0.28 lg L)1 for the second metsul-

furon-methyl experiment and 1.95–15.6 lg L)1 for the

second pendimethalin experiment when compared with

experimental control plants. When compared to the

pooled control plants, it was 1.17–75 g a.i. ha)1 and

1.3–42 g a.i. ha)1 for the first acifluorfen and MCPA

experiments and 0.16–0.31 g a.i. ha)1 and 0.28 g

a.i. ha)1 for the first and second metsulfuron-methyl

experiments respectively.

Trade-off between traits

The root:shoot and leaf:stalk ratios, length-specific leaf

weight and stalk weights and total leaf and stalk length

of plants exposed to herbicide concentrations or doses

giving <10% adverse effect were compared with the

control plants, using a two-tailed t-test (Table 4). There

were inter-experimental variations between control

plants in the morphological endpoints (ANOVA:

P < 0.05), so comparisons were made between treated

plants and experimental control plants and pooled

control plants. The comparisons with pooled control

plants are shown. Using experimental control plants did

not change any of the over-all conclusions, but showed,

as for the growth endpoint, more variance among

replications (not shown).

The MCPA was the only herbicide which increased

the root:shoot ratio of the plants in all treatments

(Table 4). Terbuthylazine showed a similar trend, but

not significantly in all experiments. For glyphosate-

treated plants, the root:shoot ratio increased in sprayed

plants but not in plants exposed through the media.

Plants treated with acifluorfen, diquat, haloxyfop and

metsulfuron-methyl did not show any consistent trend in

root:shoot allocation when sprayed, but they all had a

tendency towards decreasing root:shoot ratio when

exposed to the herbicides through the media. Pendi-

methalin was the only herbicide inducing a significant

decrease in root:shoot ratio, though not significantly for

all experiments.

The shoot morphology was only measured on plants

exposed to herbicides through the media. The results

showed an increased leaf:stalk DW ratio for haloxyfop-

treated plants (Table 4). For the remaining herbicides

and endpoints, there were no consistent trends in terms

of significant changes in shoot resource allocation

patterns. It was notable, however, that specific stalk

length was constant and equal to the untreated controls

across all treatment.

Discussion

The study showed that certain herbicides can increase

barley biomass growth and that the growth stimulation

is not a consequence of trade-off between morphological

traits. Also, the hormesis was independent of whether

the plants were exposed to the herbicide through the

media or by spraying. Though all of the eight herbicides

showed tendencies towards hormesis in at least one of

the four experiments, the two herbicides glyphosate and

metsulfuron-methyl gave the most consistent responses,

with an approximate 25% increased in biomass at

harvest when compared with the pooled control plants.

These results confirm the database study, which included

both terrestrial and aquatic plants and algae, where

growth stimulations were observed for more than

70% of the dose–response curves for these two herbi-

cides (Cedergreen et al., 2007). Studies on both barn-

yard grass (Echinochloa crus-galli, L.) and Eucalyptus

Table 3 Size of the growth stimulation given as percentage of

control for the data which were best described with a model

including hormesis

Herbicide

Experiment control Pooled control

Media

exp.

Spray

exp.

Media

exp.

Spray

exp.

1 2 1 2 1 2 1 2

Acifluorfen – – – – – – 17 –

Diquat – 17 – 4 – 26 – –

Glyphosate 33 – – 15 20 31 33 13

Haloxyfop – – 0.3 – – – – –

MCPA 1 – – – – – 17a –

Metsulfuron 11 – – 39a 6 54 21a 29a

Pendimethalin – – – 19a – – – –

Terbuthylazine – – 2 – – 36 – –

The calculations are made both in relation to the average

experimental controls and in relation to the average pooled

controls.aFor data which could not be fitted to a dose-response model,

growth increase was determined from the dose-range containing

treatments significantly higher than controls (Two-tailed t-test,

P < 0.05).


� 2008 The Author


(Eucalyptus grandis, L.) also show growth stimulatory

effects of glyphosate (Schabenberger et al., 1999; Duke

et al., 2006) and another sulfonylurea, sulfosulfuron,

has initiated hormesis in several other species (Davies

et al., 2003). Hence, it seems that the globally most

widely used herbicide glyphosate and the large group of

sulfonylurea herbicides have the ability to stimulate

growth in a large range of species.

Low dose growth stimulations might have implica-

tions not only for non-target species, receiving spray

drift from herbicide-treated fields, but also for the weeds

growing under glyphosate-resistant genetically modified

crops, which will receive reduced and potentially growth

stimulating doses of glyphosate. Too little is yet known

about the cause and duration of the hormetic growth

stimulation to say whether it can be used to increase

crop production and quality. Presently, low glyphosate

doses are used to increase sucrose yield in sugarcane

(Osgood et al., 1981; Su et al., 1992; Robertson &

Donaldson, 1998) and there might be other situations

where its beneficial effects on plant physiology could be

commercially utilised. To the best of our knowledge,

low dose effects of sulfonylurea herbicides have not

been investigated; but it is clear that plant

physiological responses to low doses of both types of

herbicides cannot be extrapolated from our knowledge

of effects of higher, commercially used, doses. Other

physiological mechanisms are triggered in the low dose-

range and the investigation of these poses new chal-

lenges for agronomists, environmentalists and plant

physiologists.

The fact that some herbicides have the potential of

stimulating plant growth while others have not, under-

mines the theory of hormesis being a general stress

response (Stebbing, 1998; Calabrese & Baldwin, 2001),

at least for barley growing under the presented exper-

imental conditions. It is more likely that hormesis is

compound-specific and that the key should be found in

some physiological mechanisms that are only triggered

by some types of stress. Low production of reactive

oxygen species (ROS) have been proposed as a mech-

anism to enhance plant growth, possibly through

associated effect on Ca+ membrane transport (Appen-

roth et al., 1993; Allender et al., 1997; Kovalchuk et al.,

2003). But this theory was not confirmed in this study

where diquat, a known catalyst of ROS production, and

terbuthylazine, which increase ROS production through

the blocking of the electron transport of photosystem II,

did not show reproducible hormesis.

One could argue that since all herbicides, in at least

one experiment, could induce hormesis, it must be a

general phenomenon. But since this hormesis could not

be reproduced in the present test-system, it is believed to

be erratic. Even the most pronounced hormetic effects

Table 4 Morphological data of control and herbicide-treated plants within the potential hormetic zone, defined as concentrations ⁄ doseswhere plants have an average harvest biomass >90% of controls

Herbicide Exp

Media exposure Spray exposure

n

Max.

conc.

(lg L)1)

Root:shoot

DW ratio

Leaf:stalk

DW ratio

LSLW

(mg cm)1)

LSSW

(mg cm)1)

LL

(cm)

SL

(cm) n

Max. dose

(g a.i.

ha)1)

Root:shoot

DW ratio

Pooled control 71 0.48 ± 0.10 1.90 ± 0.38 1.39 ± 0.35 3.46 ± 0.84 75 ± 16 16 ± 5 89 0.40 ± .04

Acifluorfen 1 36 31 0.39 ± 0.07 2.25 ± 0.33 1.56 ± 0.31 3.78 ± 0.94 66 ± 8 12 ± 3 44 75 0.46 ± 0.05

2 36 3 0.45 ± 0.05 1.82 ± 0.32 1.35 ± 0.17 3.36 ± 0.93 74 ± 12 17 ± 5 32 75 0.35 ± 0.03

Diquat 1 35 16 0.38 ± 0.07 2.05 ± 0.32 1.50 ± 0.22 3.57 ± 0.91 72 ± 9 16 ± 3 32 9 0.40 ± 0.06

2 44 13 0.42 ± 0.12 2.04 ± 0.30 1.43 ± 0.19 3.59 ± 0.99 90 ± 13 19 ± 5 24 9 0.44 ± 0.06

Glyphosate 1 23 23 0.52 ± 0.14 3.53 ± 1.06 1.76 ± 0.58 3.62 ± 1.47 69 ± 26 15 ± 6 44 143 0.52 ± 0.06

2 36 156 0.45 ± 0.14 1.88 ± 0.39 1.47 ± 0.16 3.87 ± 0.92 84 ± 14 18 ± 4 36 143 0.44 ± 0.05

Haloxyfop 1 39 4 0.37 ± 0.07 2.90 ± 1.25 1.79 ± 0.77 3.44 ± 0.95 65 ± 9 13 ± 3 39 6 0.50 ± 0.17

2 35 2 0.46 ± 0.07 2.35 ± 0.73 1.33 ± 0.18 3.09 ± 0.97 76 ± 14 16 ± 5 32 6 0.36 ± 0.06

MCPA 1 35 156 0.58 ± 0.14 2.14 ± 0.34 1.39 ± 0.17 3.58 ± 1.25 62 ± 19 12 ± 4 43 140 0.43 ± 0.05

2 36 16 0.56 ± 0.10 1.93 ± 0.33 1.39 ± 0.15 3.51 ± 0.59 74 ± 14 16 ± 5 39 70 0.44 ± 0.04

Metsulfuron 1 20 1.0 0.46 ± 0.08 1.98 ± 0.51 1.43 ± 0.25 3.60 ± 0.87 74 ± 16 15 ± 4 40 1.3 0.45 ± 0.12

2 24 1.0 0.37 ± 0.09 2.03 ± 0.34 1.65 ± 0.25 4.28 ± 1.40 96 ± 16 19 ± 4 39 1.3 0.40 ± 0.05

Pendimethalin 1 26 1.7 0.40 ± 0.05 2.08 ± 0.44 1.52 ± 0.20 3.81 ± 0.63 71 ± 7 14 ± 2 40 31 0.38 ± 0.04

2 36 0.6 0.49 ± 0.20 2.10 ± 0.40 1.30 ± 0.29 3.53 ± 1.25 81 ± 18 15 ± 5 32 31 0.36 ± 0.03

Terbuthylazine 1 12 0.4 0.87 ± 0.29 0.83 ± 0.39 0.67 ± 0.41 3.62 ± 1.81 67 ± 12 17 ± 5 44 76 0.46 ± 0.08

2 26 0.8 0.51 ± 0.07 1.99 ± 0.43 1.50 ± 0.15 3.61 ± 0.82 84 ± 10 19 ± 4 36 76 0.40 ± 0.07

The maximal concentration ⁄ dose (Max. conc.) defining the hormetic zone is given together with the number of plants within the zone (n).

The morphological parameters measured are: root:shoot dry weight (DW) ratio, leaf:stalk dry weight ratio, length-specific leaf weight

(LSLW), length-specific stalk weight (LSSW), total leaf length (LL) and total stalk length (SL) given as mean ± SEM. Significant

differences were tested with a two-tailed t-test (P < 0.05); significant increases are denoted in bold, while significant decreases are given in

italics.

436 N Cedergreen

� 2008 The Author


produced by glyphosate and metsulfuron-methyl, were

not consistently repeated, because of variations in

experimental control plants, demonstrating the impor-

tance of a large number of untreated controls in these

types of studies. All growth conditions were kept

constant for all experiments, except the ambient air

surrounding the climate chambers, where varying levels

of human activities might have affected the CO2

concentrations. The climate chambers were connected

to the ambient air of the climate chamber room by a

2 cm diameter hole, but it is uncertain whether this was

enough to keep CO2 concentrations sufficiently high.

Measurements of CO2 concentrations in greenhouses

with vigorous plant growth have shown values down

to 100 ppm (Personal observations). This could

have occurred in the climate chambers as well.

Therefore, it cannot be excluded that varying CO2

concentrations could have caused some of the

variation between experimental controls. Also the

effects of ethylene, which is produced by stressed

plants and sensed by neighbouring plants (Crozier

et al., 2000), might have affected the growth of control

plants.

The experiment showed few consistent biomass

allocation patterns and none related to the glyphosate

and metsulfuron-methyl-induced hormesis, apart from

the increased root:shoot ratio for glyphosate sprayed

plants. The most notable observation from the morpho-

logical data was the lack of response of MCPA, a

synthetic auxin, on leaf and stalk length and length-

specific leaf and stalk weight. Synthetic auxins are

known to increase cell elongation in both roots and

shoots, though at different concentrations (Cobb,

1992b), and have been used to study the mechanisms

behind auxin-induced hormesis (Allender et al., 1997;

Morre, 2000). The increased root:shoot ratio in MCPA-

treated plants indicate that both root and leaf MCPA

exposure primarily stimulated root growth. The con-

centration range in the media inducing the shift in

root:shoot ratio corresponds to those found to stimulate

root elongation in studies on other plant species (Cobb,

1992b).

Conclusion

The study shows that some herbicides, but not all, can

induce a real stimulation in biomass growth when

applied at doses corresponding to 5–10% field rate.

The growth stimulations are relatively small (c. 25% dry

mass increase) and their significance is therefore very

dependent on variations in control plant growth.

Biomass allocations between plant parts did take place

in plants treated with all herbicides, but these were not

the cause of the hormetic growth stimulations.

Acknowledgements

I greatly appreciate the help of BASF, Cheminova, Dow

AgroScience, DuPont, Klarsoe and Syngenta, for

providing technical herbicides and Saatzucht Josef

Breun for providing the Barke barley seeds. I am also

grateful to Jens C. Streibig for commenting on an earlier

version of the manuscript. This work was funded by the

Danish Research Agency, project 272–05–0022.

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Herbicides can stimulate plant growth