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Effects of the Phenylurea Herbicide Diuron on the Physiology

of Saccharina japonica Aresch
K. Suresh Kumar1, Kyung-sil Choo1,
Sung Su Yea2, Youngwan Seo3 & Taejun Han4
1

Institute of Green Environmental Research Center,

University of Incheon, Mireaguan, 7-46 Songdo-dong,
Yeonsu-gu, Incheon 406-840, Korea
2
Department of Biochemistry, College of Medicine, Inje University,
Busan 614-735, Korea
3
Division of Marine Environment & Bioscience,
Korea Maritime University, Busan 606-791, Korea
4
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 31 April 2010

Abstract
Diuron (N-[3,4-dichlorophenyl]-N,N-dimethylurea), a
herbicide belonging to the phenylurea family, is not
only widely used to destroy unwanted weeds but is
also used as a antifouling agent in paints. This toxicant has a dramatic effect on the aquatic dwellers
including seaweeds. This study investigates the effect of diuron on growth, pigment content, chlorophyll fluorescence, and antioxidant capacity of Saccharina japonica, a brown seaweed occurring in South
0.001) reduction in fresh weight
Korea. Significant (p
and area were observed after a fortnight of exposure
to diuron (0.1-0.4 mg/L diuron). Increasing concentrations of diuron caused reduction in the carotenoid
content, whilst comparatively lower reduction in chlorophyll a content were observed within the concentrations tested i.e. 0.1-0.4 mg/L diuron. Chlorophyll a
fluorescence parameters revealed a slight increase
in minimal (Fo) and maximal (Fm) fluorescence yield
up to 0.025 mg/L; further increase in diuron concentration caused significant reduction in the mentioned
parameters. A concentration dependent decrease in
the optimal quantum yield (Fv/Fm) as well as the maximum electron transport rate (ETRmax) was recorded
with increasing diuron concentrations; except for the
fact that, no electron transport was evidenced at 0.4
mg/L. Concentration dependent decline in the DPPH
radical scavenging activity was also observed. The

generation of high levels of reactive oxygen species

probably decreased the non-enzymatic antioxidant
defense system in S. japonica. Based on this investigation, it could be stated that diuron dramatically
influenced the physiology and growth of the seaweed
S. japonica. Further, this study ascertains the negative impact of diuron contamination, from terrestrial
(agricultural) and marine (antifouling paints) sources,
on the marine dwellers. As S. japonica is highly sensitive to diuron, it demonstrates lower EC50s considering chlorophyll fluorescence as an end-point;
thus this parameter could be used as a biomarker in
toxicity testing. Based on this study which elucidates
the harm that this antifouling herbicide causes to S.
japonica, one can surely ascertain that this weed killer and antifouling agent can also tremendously influence other marine communities too.
Keywords: Chlorophyll fluorescence, Diuron, I-PAM, Photosynthesis, Saccharina japonica, Seaweed

Introduction
Diuron or DCMU {3-(3,4-dichlorophenyl)-1,1dimethylurea)}, a phenylurea herbicide, is a potent
inhibitor of photosynthesis1. It has been used in agriculture for more than 50 years to inhibit the growth of
wide variety of annual and perennial weeds, mosses
and agricultural crops1. It is also applied to non-crop
areas such as roads, garden paths and railway to control weeds. After dispersion and subsequent soil leaching or runoffs, this compound finds its way to water
bodies and poses a significant threat to the aquatic
dwellers. Apart from this, the organic biocide diuron
has gained popularity as an antifouling agent in paints,
especially to prevent fouling of ship hulls and boats.
To function as antifoulants, herbicides are added to
the paint matrix and create a protective boundary layer
that prevents or reduces attachment and growth of
algae. Several researchers have surveyed the utility of
diuron as a representative of organic booster biocides
in replacing tributyltin-based antifouling products,
which have been banned due to their negative impact
on the marine environment2,3. Diuron has been used
in a number of countries such as United Kingdom,
Sweden, Spain, Netherlands, Portugal and Japan4. High

Effects of the Diuron on Physiology of S. japonica

levels of diuron were reported in UK (up to 6,742 ng/

L), Spain (up to 2,000 ng/L), and Japan (up to 3,054
ng/L). High diuron concentrations have also been
reported for French5 (10 g/L) and North American6
(30 g/L) surface waters7. Lamoree et al.8 also evidenced higher than maximum permitted levels (430
ng/L) of diuron in the Dutch coastal waters and marinas during the yachting season. Diuron is categorized
as a Priority Hazardous Substance by the European
Commission1. Due to a growing awareness of the
environmental issues associated with antifouling paints,
the UK Health and Safety Executive (HSE) imposed
a ban on the use of diuron as antifouling agent in
November 2002. Unfortunately, the ban is not applicable to Europe and the persistence of diuron suggests
that herbicides may still pose a threat to the marine
environment2,9. Mukherjee et al.3 described that the
antifouling paint booster biocides such as irgarol, diuron, dichlofluanid, seanine, tolylfluanid, and zinc
pyrithione are considered somewhat safer than TBT,
but their chronic effect on the aquatic environment is
yet to be determined.
Most commercial preparations containing diuron
are classified under the category of harmful chemicals,
and are generally considered dangerous for aquatic
life and flora (LC50 5.6 mg/L in the case of trout) as
well as for mankind10. Extensive research over the
past few years have focused on the effects of commercial herbicides on the photosynthesis, reproduction,
morphology and leaf/stem development of non-target
macrophytes. The toxicological impact of diuron on
non-target species is poorly understood although it is
extensively used in the selective control of broad-leaved weeds and mosses11. Increasing evidence suggest
that diuron concentrations in seawater can have a potentially deleterious impact on seagrasses and limit
the photosynthetic activity of micro- and macroalgae4.
Lambert et al.12 also clearly state that the presence of
booster biocides in water bodies cause a wide ranges
of calculable risks to the aquatic plants. This in turn
also determines the primary productivity and biodiversity of an aquatic ecosystem. Plants and algae have
been considered as the green liver of ecosystems13,
hence it is important to study the impact of this xenobiotic on the physiology of these life forms.
The potential environmental significance of herbicidal contamination of marine environments has not
gone unnoticed14. Alarming concentrations of diuron
(6.74-8 g/L) in the aquatic environments have been
reported globally by several researchers15-18. Apart
from these, amongst the studies carried out in Asia,
Okamura et al.19 demonstrated 86% of 142 coastal
water samples of western Japan to contain ~3.05 g/L
diuron. Although diuron and its degradation products

189

are found mainly in water, it has been shown that they

can also be detected in sediments16; besides, an increase in suspended particulate matter enhances the transfer of diuron to sediments20.
Diuron has a tremendous impact on aquatic ecosystem, exerts selective pressure and alters phototrophic
species assemblages4. It is only recently that there is
an increased awareness of the potential environmental problems of this toxicant on the marine flora and
fauna17. Nowadays, much is written on the function
of diuron and its photosynthesis inhibiting capacity21-25.
As seaweeds (marine macroalgae) represent a high
biomass in the marine environment26-28 and contribute
ecologically to the marine productivity, it is essential
to study the impact of diuron on them. Lambert et
al.12 reported EC50 for relative growth rate (dry mass
per day) of diuron exposed Chara vulgaris, Myriophyllum spicatum and Apium nodiflorum as 350,
5,000 and 2,808 ng/L after 14 days of exposure.
Fluorescence measurements as an indication of PSII
photochemistry and electron transport activity provide
information of herbicide interaction with photosynthesis at molecular level29. Fluorescence measurements
of photosynthetic processes also indicate the toxic
effects of harmful chemicals on the physiological state
of a plant and its biomass growth. Presently measurements of chlorophyll (chl) a florescence by Pulse
Amplitude Modulated (PAM) fluorometer are used as
non-destructive, rapid and efficient methods for in
vivo study of photosynthetic activity when the plant
has been exposed to different environmental stress.
Recent studies utilizing pulse amplitude modulated
(PAM) fluorometry have demonstrated the utility of
this technique to rapidly measure stress response in
marine angiosperms. A proportion of the absorbed
light energy in PSII can not be used to drive electron
transport and is dissipated as heat or chlorophyll fluorescence, which can be estimated using PAM fluorometry25,30. Numerous photosynthetic parameters are
used in photosynthetic research, but we are still missing their validation for toxicological studies. It should
be admitted that some photosynthetic fluorescence
parameters are not indicative and useful for toxicity
tests probably resulting by complexity of interaction
between the pollutant and the cellular system. Apart
from being a non-invasive method, analysis of chlorophyll fluorescence could be used as a sensitive indicator for detecting damage to the photosynthetic apparatus. Further knowledge on the correlation between
growth and photosynthetic fluorescence parameters
could facilitate validation of photosynthetic fluorescence parameters as convenient toxicity biomarkers29.
This study involves physiological evaluation of various photosynthetic parameters using chlorophyll fluo-

Toxicol. Environ. Health. Sci. Vol. 2(3), 188-199, 2010

Results and Discussion

Evident differences in the response of the various
growth and physiological parameters of S. japonica
after exposure to diuron was elucidated in this study.

Effect of Diuron on Growth

A strong concentration dependent toxicity of diuron
was observed for all the growth parameters tested.
The RGRFW of diuron exposed S. japonica significantly (p0.001) varied from the control. Nearly similar
RGRFW was recorded when the concentrations of diuron were 0.00625 and 0.025 mg/L, further increase in
diuron concentration (0.4 mg/L) caused a 72.91%
reduction in the fresh weight which was also indicated
by a slight shrinkage of disks (Figure 1). After a fortnight of continuous exposure to diuron, an EC10 and
EC50 of 0.0023 and 0.0878 mg/L respectively could
be observed in case of the fresh weight.
Though a concentration dependent decline was observed in case of RGRarea, this decline was less visible
than the RGRFW (Figure 1). Discernable reduction in
RGRarea (p0.001) with increasing diuron concentra-

6
LSD: 0.65
5
RGRFW (% d-1)

rescence technique.
Moreover, few studies have been undertaken on the
effect of phenyl urea-based herbicides on antioxidant
~
systems in weeds31. Apart from Cairr ao
et al.13 who
reported increased Glutathione S-transferases (GST)
activity of some species of algae (e.g. Scenedesmus
obliquus, Ulva lactuca, Saccharina japonica and Cyclotella meneghiniana) and plants (e.g. Lemna minor
and Nuphar lutea) in the presence of herbicides such
as atrazine, oxyfluorfen and diuron; hardly any vivid
herbicide concentration-dependent antioxidant studies
have been undertaken on seaweeds.
Saccharina japonica is a macroalgae seaweed native
to temperate regions, particularly South Korea. The
objective of this work was to assess the physiological
response of a range of environmentally relevant diuron
concentrations on S. japonica using chl a fluorescence
to measure photosynthetic efficiency; moreover, effect
of diuron on scavenging activity of stable DPPH free
radicals was also evaluated. Rather than using a single
physiological parameter to assess the effect of an environmental stressor, in this study, relative sensitivities
of several end-points of S. japonica to diuron were assessed. Such an approach allows a more detailed evaluation of the response, can provide information on the
mechanism (s) of sensitivity, and also highlights which
of the parameter is most inhibited. The four parameters
were selected included: growth, pigmentation, chlorophyll fluorescence and antioxidant activity.

4
3
2
1
0
6
LSD: 0.32
5

RGRarea (% d-1)

190

4
3
2
1
0
Ctrl

0.00625

0.025

0.1

0.4

Diuron (mg/L)

Figure 1. Relative growth rate of S. japonica after a fortnight

of exposure to a range of diuron concentrations. Mean95%
=4) are shown.
confidence interval (n=

tion was more evident by EC10 [0.0039 (0.0027-0.0091)

mg/L] and EC50 [0.4 mg/L]. A distinct reduction in
the RGRarea (EC50s0.4 mg/L) was evident after a
fortnight of diuron exposure in this study.
The concentration that resulted in a significant reduction in relative growth rate (RGR) was 0.025 mg/L
for fresh weight and 0.00625 mg/L for area; significant reduction in growth occurred at concentrations
greater than this value. The EC50s obtained for fresh
weight (0.06 mg/L) after 15 days of exposure to diuron was higher than the EC50s reported for relative
growth rate of marine diatom Fragilaria pinnata 4
(0.00009 mg/L) and the green algae Raphidocelis
subcapitata32 (0.0007 mg/L; 96 h); these higher EC50s
could be justified by the fact that these are microorganisms. Further the EC50s for RGR were also lower than
that reported for macrophytes such as Lemna gibba G3
(EC50s 0.029 mg/L, frond number), Lemna minor 1769
(EC50s 0.030 mg/L, frond number) and Latuca sativa
(EC50s 9.5 mg/L, root length)33. Based on the EC50s
obtained it could be stated that S. japonica was more
sensitive to diuron as compared to the mentioned macrophytic plants.

Effects of the Diuron on Physiology of S. japonica

0.3

Pigment (mg/FW)

Chl a
Carotenoid

LSD: 0.06
LSD: 0.02

0.2

0.1

0
Ctrl

0.00625

0.025

0.1

0.4

Diuron (mg/L)

Figure 2. Pigment content of S. japonica exposed to a range

of diuron concentrations. Mean95% confidence interval
=4) are shown.
(n=

Effect of Diuron on Pigment Content

Changes in the pigment content (chl a and carotenoid) of the diuron exposed S. japonica can be visualized in Figure 2. As compared to chl a, reduction in
carotenoid content showed a uniform pattern of decline
in presence of increasing diuron concentrations. At
high concentrations of diuron (0.4 mg/L), 60.37%
reduction in carotenoid content was noted; hence it
could be stated that amongst the pigments studied,
carotenoid was more sensitive to diuron. Noticeable
changes in the chl a content was evident only at concentrations 0.025 mg/L. In presence of 0.4 mg/L
diuron, the chl a content was reduced to 0.132 mg/g
FW.
Overall, it could be said that, after a fortnight of
treatment, a significant decrease (p0.01) in the photosynthetic pigments (chl a and caroteniod) was observed in response to increasing diuron concentrations.
Deficiency in pigments could be due to photobleaching or by inhibition of their biosynthesis34.
Inhibition of the photosynthetic apparatus by diuron,
even at low concentrations, is likely to change pigment
content, reduce growth and alter biomass of critical
primary producers in estuarine habitats, which in turn
could induce changes in the composition of the community4. Although short-term acute responses to diuron, such as those exhibited by fluorescence indicators
are potentially reversible; diuron can cause long-term
damage by destroying chlorophylls and carotenoids
(e.g., xanthophyll) along with cell membranes35. Contrarily, few researchers36 did not encounter a clear correlation between the increasing herbicide concentration and the pigment content. The chlorophyll pigment
data were difficult to interpret, possibly because the
herbicide has not been shown to be an inhibitor of

191

pigment biosynthesis. However, in this study, it was

indeed difficult to interpret the diuron dependent
changes in the chlorophyll content, but rather its inhibitory effect on the carotenoid content was more evident and easy to decipher. Despite this, it could be
stated that lethal responses of diuron towards S. japonica was demonstrated as reduction in chlorophyll and
carotenoid content.

Effects of Diuron on Photosynthetic Activity

Chl a fluorescence measures and quantifies alteration in PSII activity caused directly or indirectly by
stress37. Based on the study of Magnusson et al.25,
Pulse amplitude modulation (PAM) fluorometry is
ideally suited to measure the sub-lethal impacts of
photosystem II (PSII)-inhibiting herbicides. Conrad et
al.38 clearly states the significance of measuring chlorophyll fluorescence yield to assess the toxic effect of
diuron. According to them triazines and derivatives
of phenylurea, which are often found in outdoor water
samples, induce specific changes in the yield of the
in-vivo chl a fluorescence of PSII. These changes are
correlated quantitatively with the concentration of the
herbicides and can therefore be used to set-up a lowprice monitor system.
According to Stanger and Appleby39, diuron induces
phytotoxicity by catalysing lethal photosensitized
oxidation in the cell. This may occur as a result of (a)
a greater concentration of oxidized chlorophyll caused
by an interruption of the electron flow and (b) due to
an inhibition of NADPH formation which is necessary
to maintain a functional carotenoid protective mechanism. Several investigators have shown that diuron is
a potent inhibitor of the Hill reaction in photosynthesis. Diuron owes its effectiveness to its ability to inhibit the flow of electrons from water to NADP. In the
presence of diuron both the c-type and the b-type cytochromes can be oxidized by PS I, but they cannot be
reduced by PS II. One therefore assumes that diuron
acts at a site between PS II and the cytochromes.
Diuron binds to the site of the exchangeable quinine
(QB) site on the D1 protein, which includes the P680
chlorophyll and the neighbouring phaeophytin (Ph)
that form the heart of the reaction centre. When bound,
this herbicide blocks forward electron transport beyond
the 1-electron reduction of the first stable electron acceptor, the bound quinone QA. The currently accepted
view is that damage to cells by PSII-binding herbicides occurs from pigment-mediated photogeneration
of active oxygen species. When bound, a back reaction in PSII occurs, resulting in the formation of the
P680+ Ph-radical pair40.
Most commercially available fluorometers commonly measure the minimal (Fo), maximal, (Fm) and vari-

Toxicol. Environ. Health. Sci. Vol. 2(3), 188-199, 2010

0.6

0.6

0.15

LSD: 0.05

LSD: 0.05

LSD: 0.05

0.4
NPQ

0.1

Fo

Fm

0.4

0.2

0.2

0.6

10

0.05

0
ETRmax - LSD: 0.02

- LSD: 0.66

LSD: 0.06
8
Fv/Fm

ETRmax

0.4

0.2

0.4

0.15
0.1

4
0.05
0

0
Ctrl 0.00625 0.025
0.1
Diuron (mg/L)

0.4

2
0

Ctrl 0.00625 0.025

0.1
Diuron (mg/L)

0.2

192

Ctrl 0.00625 0.025

0.1
Diuron (mg/L)

0.4

Figure 3. Chlorophyll fluorescence parameters, Fo, Fm, Fv/Fm, NPQ, ETRmax and , measured for S. japonica after a fortnight of
=4) are shown.
exposure to a range of diuron concentrations. Mean95% confidence interval (n=

able chlorophyll fluorescence (Fv, where Fv=Fm-Fo)37;

and the ratio chl a variable fluorescence (Fv/Fm)41-44.
In this study too, the photosynthetic processes in S.
japonica were severely affected by diuron, as demonstrated by the decrease in chl a fluorescence. Significant changes in chl fluorescence parameters, observed
at low concentrations were also indicative of alternation in the structure of the photosynthetic apparatus.
This is in line with observations on the sensitivity of
PSII in aquatic plants exposed to pollutants reported
earlier4,45-48.

Effect on Minimal Fluorescence (Fo)

Minimal chl a fluorescence at a dark-adapted state
(Fo) is obtained when the photosynthetic cells are
exposed to light after the dark adaptation period. Thus,
because reaction centres will be maximally oxidized,
the absorbed energy will be used entirely to photochemistry and the fluorescence will be minimal. The
Fo of the exposed S. japonica discs differed significantly (p0.001) from the control (Figure 3).
Schreiber et al.49 reported that an increase in the Fo
signal occurs as the energy is dissipated non-photochemically, due to the incomplete oxidation of QA. On
the other hand, Muller et al.50 reported binding of PS
II herbicides to the plastoquinone (PQ) binding site on
the D1-protein (QB site) in PS II blocks reoxidation of
the primary electron acceptor QA, normally leading to
an increase in initial fluorescence yield Fo. Ralph11
reported minimum fluorescence increased with increase in exposure and concentration of diuron, where 1

mg/L treatment doubled the Fo signal within 1 h and

100 g/L doubled the Fo within 5 h (in case of Halophila ovalis). The results obtained herein for S. japonica match the aforesaid statement; a similar increase
in Fo was observed in presence of 0.00625-0.1 mg/L
diuron, however, further increase in the diuron concentration caused a slight decline in Fo of S. japonica.
Baker51 suggests that increase in Fo occurs as a result
of stress treatment. It is essential to note that Fo signifies the level of fluorescence when QA is maximally
oxidized, but in presence of high concentration of
stressors it might be possible that the PSII is damaged
to a greater extent, hence there is no significant pattern
of Fo, or, a decrease in Fo could be noted due to this.

Effect on Maximal Fluorescence (Fm)

Significant reduction (p0.001) in maximal fluorescence yield (Fm) could also be observed with increasing diuron concentrations i.e. at 0.1 and 0.4 mg/L.
From chl fluorescence perspective, when a PSII herbicides bind to the D1 protein and the PSII reaction
centres become closed upon illumination, the conversion of excitation energy into chemical energy is blocked and the maximal fluorescence yield is reached,
provided the herbicide is bound to all reaction centres
and illumination is sufficiently strong17. It is known
that DCMU significantly increased the Fo and Fm signals. This response could also be associated with
blocking of the electron transport from the primary to
secondary plastiquionone (QA to QB), resulting in an
increase in fluorescence emission. Since this herbi-

Effects of the Diuron on Physiology of S. japonica

cide blocks the reoxidation of QA, absorbed energy

cannot be used in photochemistry, and so it must be
non-photochemcially dissipated, hence the Fo signal
increases and approaches the Fm level11. In this study
too, along with the increase Fo, a decline in maximal
fluorescence (Fm) was observed at higher concentrations tested. Flagella et al.52 reported that increase in
Fo associated with a decrease in Fm could indicate that
the energy distribution within the light harvesting complex and PSII chlorophyll is affected and that the light
absorption capacity of all chlorophyll, including the
reaction centres, decreases.

Effect of Diuron on Non-Photochemical

Quenching (NPQ)
Two types of quenching occur in chloroplast: the
photochemical quenching (qP) directly dependent to
the electron transport, and the non-photochemical
quenching (NPQ) representing all non-radiative mechanisms involved in dissipation of excess absorbed
light energy. NPQ is composed of three different
components according to their relaxation kinetics in
darkness following a period of illumination, as well
as their response to specific inhibitors. Many works
have shown that whatever the pollutant mode of action,
NPQ was one of the most appropriate indicator for
inhibitory effect because of its high sensitivity and
quickness of response53.
Significant reduction in the NPQ (p0.001) was
recorded in the presence of increasing diuron concentrations (0.025-0.4 mg/L) for S. japonica. Krause and
Weis41 suggested that change or decrease in NPQ,
may be caused in vivo under physiological condition
by three major mechanism: a) energy dependent
quenching (qE) caused by the intra-thylakoid acidification during light driven proton translocation across
the membrane, b) quenching related to state 1-state
2 transition (qT) regulated by phosphorylation of
LHC, II and c) photoinhibitory quenching (qI) related to photoinhibition of photosynthesis. Because these
components are related to different events of the photosynthetic process, it is expected that their susceptibility
may vary with herbicides action sites. Suresh Kumar
and Han54 reported NPQ of Lemna sp. to decrease
significantly at all external concentrations of herbicides, especially notable decline in NPQ was reported
in presence of 0.0125 mg/L diuron.
Han et al.55 suggested that the presence of xenobiotic (copper) may cause decrease in ETR in the pathway
P680 Pheophytin QA QB, or an increase in the
rate of the back reaction, QA- P680+; this may be
responsible for the decline in NPQ. Similarly in the
present study, the decline in NPQ observed in the diuron treated S. japonica might be justified to be due to

193

the probable decrease in ETR. Noteworthy decline in

NPQ was observed at 0.025-0.4 mg/L diuron treatments here.
According to Brack and Frank56, triazines and urea
herbicides cause a parallel decrease in photochemical
and non-photochemical quenching as a consequence
of a block in the electron-transport chain. Without
electron transport, the proton-motive force cannot be
established. Therefore, non-photochemical quenching
also is reduced.

Effect on Maximum Quantum Yield (Fv/Fm)

The determination of Fv/Fm is based on PSII bioenergetics and chl a fluorescence induction kinetics.
The light energy absorbed by the chlorophyll molecules may undergo three pathways: either be used to
drive photosynthesis (photochemistry), excess energy
can be dissipated as heat, or it can be re-emitted as
light-chlorophyll fluorescence, which is known as nonphotochemical quenching57. These three processes
occur in competition, such that an increase in the efficiency of one will result in a decrease in the yield of
the other two. Hence, by measuring the yield of chl
fluorescence, information about changes in the efficiency of photochemistry and heat dissipation may be
obtained. The Fv/Fm has been used as a tool to rapidly
obtain information regarding the PSII physiological
activity of cells. In this way, the parameter is used to
investigate the effects of environmental conditions
such as nutrient concentration, temperature variation,
and different light intensities in algal cells57.
Inhibitory effect of diuron on the photosynthetic
apparatus of S. japonica was best demonstrated by
optimal quantum yield of PSII (measured as Fv/Fm).
Severe damage to PSII caused by increasing concentrations of diuron was evident by the steep decline in
Fv/Fm values. An EC10 of 0.0014 (0.0010-0.0017) mg/L
and EC50 of 0.0109 (0.0026-0.0218) mg/L was recorded for optimal quantum yield.
In aquatic pollution, Fv/Fm has been used to investigate the toxicological behaviour of pesticides, toxic
metals, polycyclic aromatic hydrocarbons, and contaminated water on the photosynthetic apparatus of plant
cells57. Percival et al.37 compared three kinds of oak
trees and reported that diuron caused maximal damage
to leaf photosynthetic system between 3 and 5 weeks
as manifest by the greatest reduction in Fv/Fm and increased in Fo values. In a study with marine diatoms
Macedo et al.57 confirmed Fv/Fm is a more sensitive
parameter than growth for monitoring effect of a herbicide (bentazon). Macedo et al.57 obtained EC50 of
13.00 mg/L for the herbicide DCMU based on Fv/Fm,
which was higher than the EPA (2000) EC50 value i.e.
0.16 mg/L (48 h). Juneau et al.58 stated 100 mg/L of

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Toxicol. Environ. Health. Sci. Vol. 2(3), 188-199, 2010

diuron (5 h exposure period) to cause a 55% decline

in the operational PSII quantum yield of seagrass Halophila ovalis; additionally it also decreased the maximal PSII quantum yield (but to a lesser extent). They
also stated that recovery of seagrasses exposed to diuron was very slow. Lootens and Vandecasteele59 proposed a new and cheap chl a fluorescence imaging
system, and reported the average Fv/Fm of Zea mays
L. exposed for 30 minute to 34.3 mM diuron was 0.464
while that of the control was 0.768; moreover, they
also obtained Fv/Fm values of 0.594 and 0.791 for the
same treated and control plant respectively using PAM
2000. Lambert et al.12 reported EC50 for Fv/Fm of diuron exposed Chara vulgaris, Myriophyllum spicatum
and Apium nodiflorum as 4,033, 5,000 and 5,000
ng/L (after 14 days). Lambert et al.12 also mentioned
that diuron poses a potential risk to its relative growth
rate of M. spicatum and did not cause any detectable
risk to the Fv/Fm.
Diuron inhibits the photoreduction side of PSII; it
strongly blocks the re-oxidation of the primary electron
acceptor (QA), and the magnitude of the minimum
fluorescence emission increases considerably, causing
a decrease in the variable fluorescence (Fv)11. In the
present study too, a long-term reduction in the maximum potential quantum yield of photosynthesis (indicated by a suppression of the chl fluorescence parameter Fv/Fm) synonymous with damage to PSII reaction centres was observed in the presence of increasing diuron concentrations. Inhibition of effective quantum yield has been used to examine the sub-lethal toxicity of herbicides towards a variety of microalgae,
with some being sensitive to diuron at environmentally
relevant concentrations; furthermore, this parameter
has been successfully applied to obtain EC50 values
for a number of herbicides, using marine microorganisms in both dual channel ToxY PAM and I-PAM
instruments25.
In the present study pertaining to S. japonica, a
lowering of Fv/Fm is indicative of a loss in photochemical energy conversion efficiency and/or damage at
the level of PSII reaction centres. Likewise, Jones40
noticed an appreciable increase in photoinactivation
of PSII reaction centres and a reduction in Fv/Fm, as a
result of the combination of DCMU dose and irradiance intensity.

Effect on electron transport rate (ETRmax)

Eguchi et al.60 stated that chl a fluorescence imaging
enables us to measure directly the spatial and temporal
changes in photosynthetic electron transport; further,
diagnosis of the developmental stage of photosynthetic
organs and of functional injuries caused by biotic and
abiotic stresses could also be conducted using the tech-

nique. In the presence of diuron (or DCMU), QA becomes fully reduced upon illumination and cannot be
oxidized by QB, thus photosynthetic electron flow is
blocked after the one-electron reduction of the bound
QA61. This inhibition of linear electron transport to
the cytochrome b6/f-complex results in a shortage of
reduced nicotinamide-adenine dinucleotide phosphate
(NADPH), which is essential for the reduction of carbon dioxide17. Further, Fedtke and Duke62 also reported diuron to cause inhibition of electron transfers in
PSII by substituting at one of the two plastiquinone
binding sites responsible for electron transfer to the
cytochrome complex. This mode of action presumably
is responsible for the decreased CO2 assimilation and
drastically reduces rETR63.
On the other hand, ETRmax and the initial slope ()
of light response curve also depicted a similar inhibition pattern like that of Fv/Fm. Eguchi et al.60 further
report that diuron inhibits the photosynthetic electron
transport, which consequently caused disappearance
of the chl a fluorescence quenching. Diuron acts to
inhibit electron transfers in PSII by substituting at one
of the two plastiquinone binding sites responsible for
electron transfer to the cytochrome complex. This
mode of action presumably is responsible for the drastically reduced rETR. Further, although short-term
acute responses to diuron, such as exhibited by fluorescence indicators in the greenhouse, are potentially
reversible, diuron can cause long-term damage by
destroying chlorophylls and carotenoids (e.g., xanthophyll) along with cell membranes. Such damage can
be observed in the resulting reflectance spectra as
overall increases in leaf spectral reflectance and higher
ratios of accessory pigments to chlorophyll35. Thus
this explanation accounts for the decline in the ETRmax
observed in case of S. japonica studied herein.

Effects of Diuron on Non-enzymatic

Antioxidant Activity
Oxidative stress occurs when a plant is subjected to
biotic and abiotic stresses due to production of ROS34.
Aspects of herbicide-induced, chlorophyll-mediated
oxidative stress are reviewed by Bowyer et al.61 and
Jones40. According to S tajner et al.64, herbicides could
be completely degraded due to microbial decomposition and subjected to photodecomposition under the
influence of UV light and ozone under field conditions; these photo-decomposed forms include DCPMU
(1-(3,4-dichlorophenyl)-3-methyl urea), DCPU (1(3,4-dichlorophenyl urea) and DCA (3,4-dichloroaniline)65,66. Further, it is important to note that herbicides
lost through photodecomposition produce toxic reactive oxygen species (ROS) such as O2-, OH, H2O2
and activated oxygen 1O2 as degradation products67.

Effects of the Diuron on Physiology of S. japonica

80
DPPHscavenging activity (%)

LSD: 6.21
60

40

20

0
Ctrl

0.00625

0.025

0.1

0.4

Diuron (mg/L)

Figure 4. Non-enzymatic antioxidant activity of S. japonica

measured after a fortnight of exposure to a range of diuron
=4) are
concentrations. Mean98% confidence interval (n=
shown.

These ROS inhibit chloroplast development, decrease

seed viability and root growth, stimulate leaf abscission and desiccation, and cause peroxidation of essential membrane lipids in the plasmalemma and intracellular organelles (i.e. lipid peroxidation). Cellular
components susceptible to damage by free radicals
are polyunsaturated fatty acids in membranes, proteins,
carbohydrates, nucleic acids and pigments 68. Apart
from this, Teisseire and Vernet69 reported that diuron
caused a depletion of ascorbate (one of the major nonenzymatic antioxidant) and weakly stimulated antioxidative enzyme in Lemna minor. Consequently, they
concluded that the diuron effect was probably not
attributable to herbicide strengthening of antioxidative defences of L. minor.
Studies on non-enzymatic antioxidant activity of S.
japonica assessed using DPPH scavenging assay
showed decreasing antioxidant activity with increasing concentrations of diuron. A significant (p0.001)
reduction (35.56%) in the scavenging activity at elevated concentrations of diuron i.e. 0.4 mg/L (Figure
4) was recorded. Thus it might be hypothesized that
this decreasing antioxidant capacity of S. japonica
was due to overloading of high levels of ROS produced in the diuron treated thallus, although further
studies need to be conducted to confirm this hypothesis.

Conclusions
Seaweeds play a pivotal role in the marine ecosystems, providing food, shelter and habitat to a range of
other marine organisms. Toxic impacts of pollutants

195

on seaweeds therefore have far-reaching consequences

including bioaccumulation of toxicants, decreases in
species biomass, abundance and diversity, or significant shifts in the dominance of a particular species.
Despite this reasoning, there remains a paucity of toxicological data for seaweed species and as such, limited data to implement protective measures to sustain
the same70. Based on the present study, it could be
stated that diuron is extremely inhibitory to the photosynthetic apparatus and also causes significant reduction in growth of S. japonica. It is obvious to envisage the devastating effect that this phenyl urea herbicide would cause to other seaweeds as well as aquatic
dwellers. Amongst the various parameters tested, a
notable decline in chlorophyll fluorescence (particularly Fv/Fm and ETR) was recorded as compared to the
growth parameters (RGRFW and RGRarea) and pigment
content. This study also hold immense importance in
the field of toxicological studies, i.e. based on this
investigation it could be concluded that chlorophyll
fluorescence parameters are highly sensitive and could
be used as potential tools to investigate the effect of
herbicide on macro and microalgal species.

Materials and Methods

Sample Collection and Culture Maintenance
S. japonica was collected from sites near Ahnin on
the well-conserved eastern coast of Korea (37.4
N,
129.1
E). Unialgal stock cultures were maintained in
artificial seawater medium, prepared by dissolving
commercial sea salts (Coralife, Energy Savers, California, USA) in deionized water (salinity 35) appended with 1 mM KNO3, 0.1 mM K2HPO4, and three
vitamins (1 mM vitamin B1, 0.1 M vitamin B12 and
D-biotin) as nutrients, at 10
C and 60 mol photons/
m 2/s of white fluorescent light (FL400, Kum-Ho,
Seoul, South Korea) under a 12 : 12 h L : D photoperiod. The alga was allowed to adapt in the laboratory
environment for 24 h before conducting the tests.
Discs (Cork borer no. 4) were carefully excised from
the actively growing basal region of healthy thalli of
S. japonica and used for the studies.
Test Conditions
Different environmentally relevant target concentrations of diuron (CAS 330541, Sigma, 0.00625 to 0.4
mg/L) were prepared by adding the required stock to
aeration flasks (capacity 500 mL) containing 400 mL
of artificial seawater medium (pH 8.4 adjusted using
0.5 N NaOH). To avoid any contamination, the glassware was soaked in 10% HNO3 for 24 h, rinsed with
deionized water and oven dried prior to use. Analyti-

196

Toxicol. Environ. Health. Sci. Vol. 2(3), 188-199, 2010

cal grade reagents were used throughout the test.

The test constituted a fortnight (15 days) of exposure
to diuron under the aforesaid environmental conditions
(i.e. culture maintenance). The following parameters
were evaluated after the test period.

Relative growth rate

Change in area of the seaweed disks followed by 15
days of exposure to diuron under experimental conditions was measured using a computer-assisted image
analyzer (MV200, Samsung, Seoul, Korea). Relative
growth rates (RGR) were calculated using the following equation:
ln An-ln A0
=mmmmmmmmmm100
RGRarea (% day-1)=
t
where, A0 and At are the area of disk at time zero and
after t days, respectively.

Pigment Content
Pigment concentrations of each sample disc was
determined using a Specord spectrophotometer (S10,
Zeiss) after extraction in 100% methanol for 24 h in
the dark at 4
C. Chlorophyll a (chl a) and carotenoid
content of the methanolic extracts were measured
according to the method outlined by Hipkins and
Baker71.
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 a fluorescence readings
were measured. Fm, the maximum fluorescence yield
of dark-adapted samples, and Fo, the initial fluorescence yield, and NPQ were recorded. The maximum
quantum yield of photosystem II (PSII) in the darkadapted 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 photons m2/s72. Maximum electron transport rate (ETRmax) was derived from the
=[1-exp (-*I/
hyperbolic tangent formulation, ETR=
Pt)]*exp (-*I/pt) where indicates electron transport rate under light-limited conditions, adapted from
Platt et al.73.
Antioxidant Activity
Non-enzymatic antioxidant activity of each the algal
discs were measured by evaluating DPPH(1,1-diphenyl-2-picryhydrazyl) scavenging activity. Each thallus disc was homogenized in 2 mL of absolute ethanol
with a mortar and pestle and the extract centrifuged at

10,000 rpm for 15 min at 4

C. An aliquot of (0.5 mL)
of the supernatant was mixed with a 0.5-mM DPPH
ethanol solution (0.25 mL) and 100 mM of acetate buffer (pH 5.5, 0.5 mL). After incubating in the dark for
30 min, the absorbance of the mixture was measured
at 517 nm to determine DPPH scavenging activity74.
Activity is expressed as (Acontrol-Atreated)/Acontrol
100%, where Acontrol and Atreated are the absorbance at
517 nm of control and diuron treated samples.

Statistical Analysis
Analysis of variance (ANOVA) was performed to
confirm significant differences in response. Multiple
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 EC10s (effective concentration at which
10% inhibition occurs) and 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 research was financially supported by Korea
Ministry for Food, Agricultures, Forestry & Fisheries,
and supported by Mid-career Research Program through NRF grant funded by the MEST (2007-0055521).

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