Available online at www.ehs.or.

kr

Physiological Response of Lemna Species to Herbicides

and Its Probable Use in Toxicity Testing
K. Suresh Kumar1 & Taejun Han1,2
1

Institute of Green Environmental Research Center, University of

Incheon, Mireaguan, 7-46 Songdo-dong, Yeonsu-gu,
Incheon 406-840, Korea
2
Department of Biology, University of Incheon, 12-1 Songdo-dong,
Yeonsu-gu, Incheon 406-840, Korea
Correspondence and requests for materials should be addressed
to T. Han (hanalgae@incheon.ac.kr)
Accepted 20 January 2010

Abstract
Herbicides such as atrazine, diuron, simazine and
glyphosate are of growing concern with respect to
ecotoxicity as they are often encountered in outdoor
water samples. The present study evaluates the potential damage caused by these xenobiotics on physiology of the aquatic plant Lemna sp., thereby proposing its use as a probable bioindicator. Comparative
study of a panel of classical endpoints (frond number
and relative growth rate) and chlorophyll fluorescence
parameters (Fo, Fm, NPQ, Fv/Fm and ETRmax) revealed
that diuron caused maximum inhibition of increase
in frond number. This coincided with the toxicity
ranking obtained on the basis of RGRarea and RGRFW
atrazine
simazine
glyphosate. The
i.e. diuron
chlorophyll (chl) a fluorescence parameters revealed
a concentration dependent decline in maximal fluorescence (Fm) in the plants exposed to diuron, atrazine
and simazine; this decline was negligible in presence
of glyphosate. Besides, an EC50 of 0.009 (0.008-0.010)
mg/L was recorded in case of Fv/Fm of the diuron
exposed Lemna sp.; furthermore, the glyphosate
16 mg/L. ETRmax
exposed plant demonstrated EC50s
0.001) declined in presof Lemna sp. significantly (p
ence of diuron, atrazine and simazine, whereas gly0.05) reducphosate did not cause any significant (p
tion. A steady concentration-dependent decline in
chl a fluorescence parameters of Lemna sp. (specifically Fv/Fm and ETRmax) as compared to the classical endpoints, demonstrated their superiority and
sensitivity in detection of herbicides in the aquatic
bodies. This study emphasizes on the probable use
of chlorophyll fluorescence of Lemna sp. as a tool or

bioindicator in evaluation of herbicides.

Keywords: Chlorophyll fluorescence, ETRmax, Frond number,
Relative growth rate, I-PAM, Herbicides, Lemna sp.

Introduction
Man has exponentially augmented huge quantities
of numerous herbicides to agricultural terrains of land
in order to reduce the labor and energy requirements
in crop production. As a result of this widespread
practice, large quantities of herbicides enter into soil
and inevitably reach the aquatic environments.
Herbicides have a tremendous impact on the biodiversity and productivity of aquatic communities1.
Adverse effects of herbicides on non-target organisms
of aquatic ecosystems are of special concern. These
herbicides cause rapid changes in the communities of
macrophytes, phytoplanktons and other photosynthetic
organisms. A common mechanism of herbicidal toxicity in aquatic plants and algae is inhibition of biological processes such as photosynthesis and mitochondrial electron transport2. This leads to inevitable changes
in plant cell physiology, growth and biomass yield3.
Recently herbicides such as diuron and Irgarol have
also been incorporated into antifouling paint formulations to prevent the growth of algae on boats, buoys
and marine structures4.
Diuron, atrazine, simazine and glyphosate are
amongst widely used herbicides in agricultural formulations as well as in antifouling paints. The toxicological impact of diuron on non-target species is poorly
understood, although it extensively used in selective
control of broad-leaved weeds and mosses. On the
other hand, atrazine is a selective systemic triazine
based herbicide absorbed through and roots and foliage,
and accumulates in the apical meristems and leaves.
Simazine is another triazine derivative with similar
mode of action and toxicity equivalent to atrazine,
used to control germinating annual grass and broadleaved weeds. Glyphosate is the most extensively used,
non-selective systemic herbicide in the world; its mode
of action is absorption through foliage and subsequent
translocation.
Amongst the techniques employed in detecting her-

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Toxicol. Environ. Health. Sci. Vol. 2(1), 39-49, 2010

bicides, chemical analysis has several drawbacks such

as involvement of numerous cumbersome tests, use of
expensive chemicals or equipment, and cause secondary pollution. Moreover, they do not take into account
bioavailability, temporal changes in exposure or the
interactive effects of pollutants and do not ecological
significant information. To overcome these limitations,
bioassay techniques have been developed that involve
physiological or behavioural response of a living organism to assess overall water quality. They possess several key attributes such as being sufficiently sensitive
to detect low concentrations of toxic chemicals, are
inexpensive, easy and time-saving. Aquatic plant toxicity tests are frequently conducted to determine the
potential impact of contaminants on primary producers
and to assess their environmental risks. ATP-formation,
carbon dioxide fixation, and oxygen evolution have
been used as reliable indicators for testing the effect of
pollutants particularly herbicides5. In recent years,
there is growing interest in the practical application
of chlorophyll a (chl a) fluorescence as a rapid and
sensitive bioindicator of plant stress in response to different chemicals. Pulse-amplitude modulated (PAM)
chl a fluorescence permits in vivo non-destructive
determination of changes in the photosynthetic apparatus much earlier than the appearance of visible injury.
Hulsen et al.6 discussed the use of a biological monitoring system based on in vivo chl a fluorescence as a
cheap and time-saving alternative for detecting pollution. The advantage of this approach lies in its versatility and the possibility of using a portable instrument
with rapid data evolvement and advanced software
enabling statistical analysis7. In fact, Philippe et al.8
stated that chl a fluorescence offers higher sensitivity
as compared to routine biotests based on growth inhibition. Endo and Omasa9 suggested chl a fluorescence
measurement to be effective for analyzing herbicide
toxicity to aquatic plants and algae.
Aquatic plants, Lemna spp. (duckweeds) have worldwide distribution and serve numerous critical ecological roles (primary production, nutrient cycling and
habitat for other aquatic life)10. Their rapid predominant vegetative reproduction, small-size and ability to

form genetically uniform clones, make them valuable

research organisms in studies pertaining to plant physiology, genetics, ecology and environmental monitoring. Duckweeds are amongst the few popular organisms to be used as a tool to evaluate presence of pesticides11, but less attention has been paid to such investigations. In the present study a comparative sensitivity
of Lemna sp. to four herbicides (diuron, atrazine, simazine and glyphosate) were evaluated. Several classical
endpoints such as frond number and relative growth
rate (fresh weight and surface area) as well as chl a
fluorescence parameters such as (Fv/Fm and ETRmax)
was determined. Apart from emphasizing on the use of
chl a fluorescence as a more sensitive endpoint, this
investigation suggests the use of Lemna sp. as a quick
and accurate bioassay tool for the detection of herbicides in aqueous samples.

Results
Conventional Endpoints
Fronds of Lemna sp. in the control medium were
green and healthy throughout the test period. There
was a significant decrease in the frond number with
respect to the type and concentration of toxicant tested
(p0.001). The EC50s of RGRFN are shown in Table 1.
Based on the EC50s obtained, the toxicity rank of the
tested herbicides was: diuron (0.020)atrazine (0.062)
simazine (0.564)glyphosate (16 mg/L). Thus,
diuron was most toxic in terms of inhibition of frond
number multiplication, whereas glyphosate was least
inhibitory. Concentration dependent effect of the four
herbicides is demonstrated in Figures 1 and 2. Significant differences in growth rates in terms of area (p
0.001) were observed after exposure to toxicants. As
observed in frond number, statistical analysis revealed
that diuron was the most toxic (0.016 mg/L) whereas
glyphosate (16 mg/L) was less toxic to Lemna sp.
when EC50s of RGRarea were evaluated. It could be
further stated that presence of atrazine also caused a
significant inhibition in area of growth (0.067 mg/L).
The RGRFW of all the herbicide exposed plants were

Table 1. EC50s of RGRFN, RGRarea, RGRFW, Fv/Fm and ETRmax of herbicide exposed Lemna sp.
Herbicides (mg/L)
Diuron
Atrazine
Simazine
Glyphosate

RGRFN

RGRarea

RGRFW

Fv/Fm

ETRmax

0.02
(0.015-0.028)
0.062
(0.041-0.092)
0.564
(0.374-0.848)
16

0.016
(0.010-0.022)
0.067
(0.056-0.089)
0.636
(0.449-0.827)
16

0.011
(0.005-0.015)
0.046
(0.038-0.059)
0.422
(0.355-0.594)
16

0.009
(0.008-0.010)
0.069
(0.066-0.074)
0.343
(0.223-0.389)
16

0.009
(0.006-0.010)
0.023
(0.018-0.039)
0.176
(0.144-0.217)
16

Physiological Response of Lemna Sp. to Herbicides

30

30

LSD: 4.52

LSD: 3.83
RGRFN (%/d)

25
RGRFN (%/d)

41

20
15
10

20

10

5
0

40

40

LSD: 4.00

LSD: 4.33
30
RGRarea (%/d)

20
10

20
10

30

30
LSD: 2.92

LSD: 3.69
20
RGRFW (%/d)

10

0.8

0.2

0.1

0.05

0.4

0.2

0.1

0.05

0.025

0.0125

Ctrl

0.00625

-10
Diuron (mg/L)

0.025

-10

10

Ctrl

RGRFW (%/d)

20

0.4

RGRarea (%/d)

30

Atrazine (mg/L)

Figure 1. Effect of diuron and atrazine on RGRFN, RGRarea and RGRFW of Lemna sp.

lower than the control, indicating the inhibitory effect

these toxicants. The EC50s for RGRFW after 7 d of
exposure to diuron, atrazine, simazine and glyphosate
were 0.011, 0.046, 0.422, and 16 mg/L respectively
(Table 1).

Chlorophyll a Fluorescence
Typical measurements of chl a fluorescence of the
herbicide exposed fronds (after 7 d) evaluated using
saturation pulse method are summarized in Figure 3
and 4. The Fo of the exposed plants differed significantly (p0.001) from the control plants. Lemna sp.
exhibited a relatively stable Fo in presence of 0.025 to
0.1 mg/L diuron, but further increase in diuron concentration caused a significant decline in the Fo (p
0.001). In presence of atrazine, the Fo value increased
as the concentration of this herbicide increased from 00.2 mg/L, after which a decline in initial fluorescence
yield was observed (0.2 mg/L). Contrary to the
above mentioned, the glyphosate treated plants did

not show a decrease in initial fluorescence yield. As

the concentration of diuron, atrazine and simazine increased, a notable decline in the maximal fluorescence
yield (Fm) was observed; however, this decline was
negligible in the presence of glyphosate.
NPQ decreased significantly at all external concentrations of herbicides (viz. diuron, atrazine and simazine) except glyphosate. Notable decline in NPQ was
found in presence of 0.0125 mg/L diuron (0.094), 0.05
mg/L atrazine (0.055) and 0.25 mg/L simazine (0.044).
The maximum quantum yield of PS II, as measured
by Fv/Fm after a 7 d exposure to herbicides are shown
in Figure 3 and 4. The Fv/Fm values dropped significantly with increasing concentrations (p0.001) of
most herbicides except glyphosate (p0.05). The
toxicity rank based on EC50s was: diuron (0.009 mg/L)
atrazine (0.069 mg/L)simazine (0.343 mg/L)
glyphosate (16 mg/L). Minimum EC50 was recorded
in case of diuron, while glyphosate did not affect the
Fv/Fm values. As observed in the other endpoints,

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Toxicol. Environ. Health. Sci. Vol. 2(1), 39-49, 2010

30

40
LSD: 3.71

LSD: 3.01
RGRFN (%/d)

RGRFN (%/d)

30
20

20

10

10
0

50

40

LSD: 3.26
40

30

RGRarea (%/d)

RGRarea (%/d)

LSD: 3.60

20
10

30
20
10
0

0
30

40
LSD: 2.76

LSD: 3.89
30
RGRFW (%/d)

RGRFW (%/d)

20

10

20

10

0
Ctrl

0.25

0.5

-10
Simazine (mg/L)

Ctrl

16

Glyphosate (mg/L)

Figure 2. Effect of simazine and glyphosate on RGRFN, RGRarea and RGRFW of Lemna sp.

apparent photosynthetic electron transport rates of the

exposed samples were significantly (p0.001) inhibited in the presence of diuron, atrazine and simazine,
whereas glyphosate did not cause any significant (p
0.05) reduction. EC50s of all the chl fluorescence
parameters confirmed diuron to be the most toxic
[0.009 (0.006-0.010) mg/L] amongst the toxicants tested (Table 1).

Discussion
Environmentally realistic concentrations of a range
of phytotoxicants impact aquatic ecosystems by exerting selective pressure and altering phototrophic species
assemblages. Herbicides targeting photosynthesis are
ubiquitous in human impacted aquatic environments.
They either affect the electron acceptors of PSI and
PSII (e.g. diuron, atrazine and simazine) or alter aromatic amino acid synthesis via the inhibition of the

shikimate pathway, interfering in protein synthesis as

well as molecules that require these amino acids as
precursors, such as auxins and polyphenols (e.g. glyphosate)14. Studies on physiological effects of potent
pollutants on non-target organisms require criteria to
distinguish their lethal or sublethal effects on plants15.
Kovach et al. (1992)16 summarized several reproductive, teratogenic, mutagenic, oncogenic and toxic effects of pesticides and stated that the Environmental
Impact Quotient (EIQ) value of diuron and glyphosate
was 15, while that of simazine and atrazine was 3 and
9 respectively.
Diuron [3-(3-4-dichlorophenyl)-1,1-dimethylurea
(DCMU)], a widely used non-selective phenyl urea
herbicide, inhibits photoreduction sites of PS II. DCMU
strongly blocks the reoxidation of the primary electron
acceptor (QA-) by forward electron transfer 17. It is
commonly sprayed to control weeds in agriculture
and in landscape maintenance. Its relatively weak
soil-adsorption coefficient, high water-solubility, and

Physiological Response of Lemna Sp. to Herbicides

0.5

43

0.5
LSD: 0.10

LSD: 0.07
0.4

0.3

0.3
FO

FO

0.4

0.2

0.2

0.1

0.1

0
0.8

0
1
LSD: 0.16

LSD: 0.11
0.8

0.6
Fm

Fm

0.6
0.4

0.4
0.2

0.2
0
0.6

0
1
LSD: 0.09

0.4

0.6

NPQ

NPQ

LSD: 0.06

0.5

0.8

0.4

0.3
0.2

0.2

0.1

0
1

0
1
LSD: 0.04
0.8

0.6

0.6

Fv/Fm

Fv/Fm

LSD: 0.04
0.8

0.4

0.4

0.2

0.2

0
60

60

40

LSD: 6.09

ETRmax

40

0.05

0.1

0.2

0.4

Diuron (mg/L)

*
0.8

0.4

0.1

0.05

0.025

*
0.025

0.0125

0.00625

Ctrl

0.2

20

20

Ctrl

ETRmax

LSD: 5.99

Atrazine (mg/L)

Figure 3. Effect of diuron and atrazine on chlorophyll fluorescence (Fo, Fm, NPQ and Fv/Fm) of Lemna sp.

moderate stability make it highly mobile and persistent. Despite the heavy prevalent use of diuron in
watersheds, very few studies have addressed its potential effects on estuarine and marine plants. Atrazine

affects photosynthesis by binding to the second electron acceptor (QB) protein, inhibiting electron transport and this causes an increase in maximum fluorescence (Fm), and a decline in the maximum quantum

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Toxicol. Environ. Health. Sci. Vol. 2(1), 39-49, 2010

0.2

0.6
LSD: 0.13

0.5

LSD: 0.03
0.15
FO

FO

0.4
0.3

0.1

0.2
0.05
0.1
0
1

0
1
LSD: 0.20

LSD: 0.18
0.8

0.6

0.6

Fm

Fm

0.8

0.4

0.4

0.2

0.2

0
0.6

0
1
LSD: 0.17

LSD: 0.06
0.8
NPQ

NPQ

0.4

0.2

0.6
0.4
0.2
0

0
1

1
LSD: 0.03

0.8

0.8

0.6

0.6

Fv/Fm

Fv/Fm

LSD: 0.06

0.4

0.4

0.2

0.2

0
80

60
LSD: 6.27

LSD: 14.40

40
ETRmax

ETRmax

60
40

20
20
0
Ctrl

0.25

0.5

Simazine (mg/L)

0
Ctrl

16

Glyphosate (mg/L)

Figure 4. Effect of simazine and glyphosate on chlorophyll fluorescence (Fo, Fm, NPQ and Fv/Fm) of Lemna sp.

yield (Fv/Fm ratio)18. Atrazine has significant effects

even at micromolar (M) concentration on photosynthesis, while growth is only affected at millimolar
(mM) concentrations18. Simazine is known to effect

chlorophyll a fluorescence by showing a strong increase in minimum fluorescence (Fo), and a decrease in
maximum quantum yield, as well as the effective quantum yield. Glyphosate acts on various enzyme sys-

Physiological Response of Lemna Sp. to Herbicides

tems throughout the plant, and interferes with amino

acid formation18.
The frond number of duckweed is a superior indicator of potential toxicity19. Fairchild et al. (1997)20 considered frond number as an endpoint and reported
Lemna minor to be more sensitive to atrazine (EC50
0.153 mg/L) and simazine (EC50 0.166 mg/L) as compared to Selenastrum capricornutum (EC50 value 0.235
and 1.240 mg/L for atrazine and simazine respectively).
The present investigation revealed EC50 values of 0.062
and 0.564 mg/L in the presence of atrazine and simazine respectively, indicating that the Lemna sp. tested
had a higher sensitivity to atrazine as compared to
both L. minor and Selenastrum capricornutum. Lemna
sp. demonstrated EC50s (frond number) of 0.564 mg/L
after 7 d of exposure to simazine which lay within the
range of EC50s of frond number in Lemna gibba after
a 14 d exposure21. The EC50s of frond number showed
glyphosate to be least inhibitory amongst the four herbicides tested, while diuron ranked as the most toxic
herbicide for Lemna sp.
According to the EC50s calculated for fresh weight
of Lemna sp., diuron was the most toxic followed by
atrazine. EC50s recorded for the glyphosate exposed
plant was 16 mg/L which ranged parallel to the IC50
for growth rate inhibition of Lemna gibba (20.5 mg/
L)14.
The EC50 for frond area of Lemna sp. in presence of
the phenyl urea herbicide diuron was 0.016 (0.0100.022) mg/L, which was lower than IC50 of L. minor
(25 mg/L)22. Michel et al. (2004)23 reported EC50 value
of leaf growth inhibition in L. paucicostata to be 0.929
0.144 M for atrazine; contrarily, the present study
revealed EC50 0.070 (0.054-0.090) mg/L i.e. approximately 0.324 M for atrazine. Thus this study indicates
a lower EC50; besides it is important to emphasize the
accuracy in testing in this study which is indicated by
the marginal range of deviation. Mazzeo et al. (1998)21
reported EC50s of 0.33-0.53 mg/L in case of surface
area of Lemna gibba after 14 d of exposure to simazine.
In this study, however, Lemna sp. demonstrated EC50s
of 0.636 mg/L after exposure to the same toxicant for
7 d. Though the EC50s are slightly higher than that
reported by Mazzeo et al. (1998)21, it is notable to highlight that the same was obtained after a shorter period
of exposure.
Considering area and fresh weight as endpoints,
diuron ranked the most toxic while, glyphosate proved least toxic and ranked at the bottom cadre. The differences in response to various herbicides can be attributed to the physiological state of the plant, physicochemical properties, uptake, metabolic degradation,
and molecular target sites23. Lemna sp. has been the
most prevalent, preferred and popular toxicity detector

45

since many decades, but most toxicity tests commercialized as well as proposed till date using this genus
involves the use of various individual classical parameters or endpoints like growth rate, pigment content,
frond number, fresh weight, chlorosis, necrosis, dry
weight, etc.
On the other hand, PAM fluorometry is of great relevance to determine the toxicity of herbicides.
The development of Imaging-PAM (I-PAM), suited
for assessment of samples in multiwell-plates facilitates
recording of concentration-effect curves with single
measurements, and hence it is a boon to toxicity testing. Additionally, continuous recording of the timedependent fluorescence responses of toxicants can also
be visualized using this promising tool, which allows
fast chemical effect-screening of numerous samples
within a short time using little material (toxicant containing samples). This offers scope for high-throughput
biotesting using aquatic macrophyte species. Despite
being suitable for deriving concentration-effect curves
for toxicants inhibiting PSII inhibiting particularly
herbicides, I-PAM is particularly useful in short-term
chemical risk assessment using unicellular algae as
well as for aquatic macrophytes toxicity testing3.
Despite this, only few researchers have investigated
toxicity of herbicides using chl a fluorescence of Lemna
sp.3,6,24,25. Herbicides either have a direct or indirect
effect on the photosynthetic apparatus, or interfere with
some other metabolic pathways in the plant system
and thus induce related changes in the growth rate and
fluorescence parameters. The triazine herbicides
(atrazine and simazine), as well as DCMU significantly increase the Fo and Fm signals. This response would
be associated with blocking of the electron transport
from the primary to secondary plastiquinone (QA to
QB), resulting in an increase in fluorescence emission.
Since these herbicides block the reoxidation of QA,
absorbed energy cannot be used in photochemistry26.
The phosphanoglycine herbicide, i.e. glyphosate,
inhibits aromatic amino acid biosynthesis at the site
of chorismate mutase or prephenate dehydratase27, thus
it clearly means it is not interfering with the Z-scheme
of photosynthesis.
Fv/Fm has been widely used to detect stress-induced
perturbations in the photosynthetic apparatus. In aquatic pollution, Fv/Fm has been used to investigate the toxicological behaviour of pesticides on the photosynthetic apparatus of plants as well as algae. For PSII herbicides, studies have demonstrated that lower EC50s are
usually obtained when F v/F m is considered as the
response parameter, in contrast to the EC50s obtained
through algal growth assays28. In the present study,
Lemna sp. showed Fv/Fm of 0.25440.028 mg/L after
exposure to atrazine. Frankart et al.15 reported Fv/Fm

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Toxicol. Environ. Health. Sci. Vol. 2(1), 39-49, 2010

of 0.6990.02 for 100 g/L in atrazine-exposed L.

minor. A decrease in Fv/Fm was also observed in the
presence of diuron and simazine.
Magnusson et al. (2008)29 studied EC50s for Fv/Fm
in two tropical estuarine microalgae, Navicula sp. and
Nephroselmis pyriformis, and suggested diuron (1633 nM) to be more toxic than atrazine (130-620 nM).
Muller et al. (2008)30 reported EC50s of Fv/Fm to be
18, 45 and 400 g/L for Phaeodactylum tircornutum
and 20, 103 and 76 g/L for Chlorella vulgaris in
presence of diuron, atrazine and simazine respectively;
furthermore they found relative potencies of these PS
II impacting herbicides in the order of diuronatrazine
simazine. Symbiotic dinoflagellate Seriatopora
hystrix revealed the following toxicity ranking: diuron
(2.30.04 g/L)atrazine (453 g/L)simazine
(1507 g/L) based on the EC50s of Fv/Fm31.The above
mentioned reports coincide with the toxicity rank obtained herein using Fv/Fm of Lemna sp. (diruonatrazine
simazine, wherein EC50s of 0.009, 0.069 and 0.343
mg L-1 were obtained respectively). However it is
also important to emphasize that the above mentioned
are microalgae while Lemna sp. is a higher plant. Perhaps structural differences may be the reason for the
difference in the sensitivity between microalgae and
aquatic plants. However, Lemna sp. showed a higher
degree of sensitivity to diuron than symbiotic dinoflagellate Seriatopora hystrix (2.30.04 g/L) and Acropora formosa (EC50 2.70.07 g/L). Gausman32 observed Lemna minor to demonstrate EC50s (Fv/Fm) of
0.092-0.18, 0.0025-0.041 mg/L and 0.14-0.57 mg/L for
atrazine, diuron and simazine respectively; while he
reported that the EC50s of Pseudokirchnirella subcapitata (algae) ranged from 0.0918-0.147, 0.0024-0.0027
and 0.15-1.24 mg/L respectively. The values obtained
in the present study, were either comparable or lower
than that of L. minor in case of atrazine and simazine.
The electron transport rate provides an empirical
estimate of the rate of flow of electrons through the
electron flow pathway. Pesticides bind and inhibit the
electron transport chain, or act as uncouplers to prevent oxidative phosphorylation and formation of the
proton gradient33. The steps in electron transport are
sufficiently conserved from organism to organism, and
hence it is difficult to achieve large degrees of selectivity for inhibitors33. Hardly any reports are available
on the use of ETR as an endpoint in testing toxicity
of herbicides. In PAM instruments, calculations of
ETR based on chl fluorescence measurements are simple and reliable. In the present study, a clear concentration-dependent decline in ETRmax was observed in
presence of most of the herbicides; hence this could
be considered as the most sensitive endpoint.
Based on the EC50s it could be concluded that almost

equivalent results were obtained from both ETRmax

and Fv/Fm, and that Lemna sp. was more sensitive to
atrazine and diuron as compared with simazine and
glyphosate. This plant could be efficiently used for
detection of diuron in aquatic samples as they fell
within the range of various water quality criteria (US
EPA DWEL and Health advisory lifetime criteria;
Australian, Canadian and New Zealand drinking water
criteria) stated by Hamilton et al.34. In addition, the
ETRmax of Lemna sp. fell within the range of water
standards to protect aquatic ecosystems in South Africa
and nearly matched the Australian standards, while
the yield (Fv/Fm) fulfilled the US EPA limits for pesticides in drinking water.
It is noteworthy to emphasize that if these endpoints
are able to detect low levels of diuron and atrazine in
drinking water samples, they would surely be useful
in detecting the same in effluents or waste water. Moreover, the results obtained herein confirm the observation of Ralph18, who described glyposate to be nontoxic to aquatic plants. One can hence foresee the probable use of Lemna sp. as tool for toxicity testing and
detection of these types of toxicants in various water
samples including dilute industrial wastes.
Apart from fulfilling the water standards, the ETRmax
and yield (Fv/Fm) complemented with each other and
confirmed the lethal toxicity of diuron and atrazine.
Thus based on the EC50 values obtained these two endpoints could be used for detection of these the mentioned toxicants in aquatic systems. The main benefit of
using these two endpoints is the speed of assessment.
Evaluation of endpoints involving growth parameters
such as RGRFN, RGRarea and RGRFW using Lemna sp.
bioassay would require 8 h and slight precision and
skill, whereas evaluation of ETRmax and Fv/Fm as endpoints in toxicity testing would just require 1 h and a
PAM fluorometer, which is very easy to handle and
requires minimum space. PAM fluorometery is an
easy and accurate technique, and the plant Lemna sp.
is easy to obtain and handle. Hence the use of ETRmax
and Fv/Fm of Lemna sp. as endpoints in toxicant detection is recommended. Typically, aquatic macrophyte
testing is very time consuming and relies on laborious
experimental set-ups, while measurements using I-PAM
enable quicker effect-screening for the listed chemical
toxicants. Thus, the fluorescence-based bioassay with
the I-PAM offers a promising approach for the miniaturization and high-throughput testing of chemicals
with aquatic macrophytes.

Conclusions
Traditional chemical methods of pollution monitor-

Physiological Response of Lemna Sp. to Herbicides

ing such as gas and high-performance liquid chromatography, atomic absorption and mass spectrometry are
sensitive and effective, but they are tiresome, expensive and are not target-oriented. Practical monitoring
programs require rapid, simple, and low-cost screening procedures for the detection of harmful chemicals
in aquatic and soil environments. Biosensors better
reflect the real physiological impact of active compounds present in the sample, because even low concentrations of pollutants can affect living organisms
by disturbing physiological processes. Fluorescence
based techniques can provide a means for rapid exposure assessment of pollutants. ETRmax and Fv/Fm of
Lemna sp. are excellent endpoints in testing herbicides,
specifically diuron and atrazine. It could be noted that
no ETRmax was observed in the presence of high herbicide concentration, while a gradual decline in the
Fv/Fm values was recorded. This proves that Fv/Fm is
a superior endpoint as compared to ETRmax. Lemna
sp. are ecologically important, easily grown, have wide
distribution and can be readily and reliably manipulated. Lemna sp. can adapt to a wide variety of geographic and climatic zones. Thus the Lemna method
studied herein based on chlorophyll a fluorescence
measurements is a valuable tool for aquatic risk assessment particularly for herbicides.

Materials and Methods

Culture and Maintenance
Lemna sp. was obtained locally and maintained in
an environmental chamber at 20
C, pH 6.5, under cool
white fluorescent light 80 mol photon m-2s-1 (FL
400, Kum-Ho, Seoul, South Korea); 6 : 18 L : D regime
in Hunters half strength growth medium under static
condition.
Exposure
Before commencement of the assay, the Lemna
plants (each with two fronds) were cleansed with fresh
deionized water followed by immersion in 0.5% (v/v)
sodium hypochlorite solution (1 min). The fronds were
re-rinsed twice with sterile deionized water and then
introduced to the test vessel using sterilize forceps.
The tests were performed under static conditions using
30 mL polystyrene culture plates (50 mm, Barloworld
Scientific Ltd., Stone, UK) containing 25 mL Hunters
half strength growth medium (control) or test solution
(different concentrations of toxicants). Six diminutive
pores were made in the lid of the plates to facilitate
aeration. Conditions similar to maintenance were retained throughout the test. A control (devoid of toxicant) was maintained throughout the test to ascertain

47

the effect of the toxicants. After a 7 d exposure period,

the fronds were collected for evaluation of endpoints.

Test Chemicals
Analytical grade reagents were used throughout the
test. Toxicant solutions of four herbicides were prepared (stock) and serial dilutions were consequently
accomplished using plant test medium to obtain the
final required concentration. The concentrations tested
included: diuron (CAS 330541, Sigma) 0.00625 to 0.4
mg/L, atrazine (CAS 1912249, Supelco) 0.025 to 0.8
mg/L, simazine (CAS 49089, Supelco) 0.25 to 4 mg/L,
and glyphosate (CAS 1071836, Sigma) 1 to 16 mg/L.
Relative Growth Rate (RGR)
Following exposure to experimental treatments for
7 d, the samples were harvested to determine changes
in frond number, fresh weight and surface area (Image
analyzer, MV200, Samsung, Seoul, Korea). Relative
growth rate (RGR) in surface area and fresh weight
were determined using similar formula as that of frond
number, i.e.
ln Nn-ln N0
RGR (%/d) = mmmmmmmmmmmm100
tn
Where,
N0=initial frond number at the beginning of the test
Nn=frond number at the end of the test, and
tn=the test duration

Chlorophyll a Fluorescence
Chl a fluorescence was measured using Imaging
Pulse Amplitude Modulated (I-PAM, Walz, Effeltrich,
Germany) fluorometer. Samples were initially darkadapted for 15 min before chl fluorescence was measured. Fm, the maximum fluorescence yield of darkadapted samples and Fo, the initial fluorescence yield,
and the Non-Photochemical Quenching (NPQ) were recorded. The maximum quantum yield of photosystem
II (PSII) in the dark-adapted state was expressed as
the ratio of variable to maximal chlorophyll fluorescence (Fv/Fm), derived from (Fm-Fo)/Fm. Rapid light
curves were measured using 10 s pulses of actinic light
increased stepwise from 0 to 1,517 mol photon m-2
s-1 12. Maximum electron transport rate (ETRmax) was
derived from the hyperbolic tangent formulation, ETR
=[1-exp (-*I/Pt)]*exp (-*I/Pt) where indicates
electron transport rate under light-limited conditions,
adapted from Platt et al. (1980)13.
Statistical Analysis
Analysis of variance (ANOVA) was performed to
confirm significant differences in response. Multiple

48

Toxicol. Environ. Health. Sci. Vol. 2(1), 39-49, 2010

comparison tests by the least significance difference

(LSD) were then carried out to find out significant differences in response from controls. Results are reported as EC50s (effective concentration at which 50%
inhibition occurs) with 95% confidence intervals estimated by the linear interpolation method (ToxCalc
5.0, Tidepool Science, California, USA).

Acknowledgements
This work was financially supported by Ministry of
Environment (091-091-088) and University of Incheon,
Korea. The authors are grateful to the anonymous
reviewers for assisting in publication of this manuscript.

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