tmpF69A TMP

Ecotoxicology and Environmental Safety 104 (2014) 5171
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv
Review
Algal photosynthetic responses to toxic metals and herbicides assessed

by chlorophyll a uorescence
K. Suresh Kumar a, Hans-Uwe Dahms b, Jae-Seong Lee c, Hyung Chul Kim d, Won Chan Lee d,
Kyung-Hoon Shin a,n
a
Department of Environmental Marine Sciences, College of Science and Technology, Hanyang University, Ansan 426-791, Republic of Korea
Green Life Science Department, College of Convergence, Sangmyung University, 7 Hongij-dong, Jongno-gu, Seoul 110-743, Republic of Korea
c
Department of Biological Sciences, College of Natural Sciences, Sungkyunkwan University, Suwon 440-746, South Korea
d
Marine Environment Research Division, National Fisheries Research and Development Institute, Busan 619-705, Republic of Korea
b
art ic l e i nf o
a b s t r a c t
Article history:
Received 9 November 2013
Received in revised form
28 January 2014
Accepted 30 January 2014
Chlorophyll a uorescence is established as a rapid, non-intrusive technique to monitor photosynthetic

performance of plants and algae, as well as to analyze their protective responses. Apart from its utility in
determining the physiological status of photosynthesizers in the natural environment, chlorophyll a
uorescence-based methods are applied in ecophysiological and toxicological studies to examine the effect of
environmental changes and pollutants on plants and algae (microalgae and seaweeds). Pollutants or
environmental changes cause alteration of the photosynthetic capacity which could be evaluated by
uorescence kinetics. Hence, evaluating key uorescence parameters and assessing photosynthetic performances would provide an insight regarding the probable causes of changes in photosynthetic performances.
This technique quintessentially provides non-invasive determination of changes in the photosynthetic apparatus
prior to the appearance of visible damage. It is reliable, economically feasible, time-saving, highly sensitive,
versatile, accurate, non-invasive and portable; thereby comprising an excellent alternative for detecting
pollution. The present review demonstrates the applicability of chlorophyll a uorescence in determining
photochemical responses of algae exposed to environmental toxicants (such as toxic metals and herbicides).
& 2014 Elsevier Inc. All rights reserved.
Keywords:
Algae
Chlorophyll a uorescence
Photosynthesis
Toxic metal
Herbicide
Pulse amplitude modulated (PAM)
uorometry
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Parameters of chl a uorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Maximum quantum yield (F/Fm) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Effective quantum yield (PSII or F/Fm0 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Non-photochemical quenching (NPQ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.
Electron transport rate (ETR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Trace and toxic metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Mercury (Hg) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Copper (Cu) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Cadmium (Cd) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.
Chromium (Cr). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Herbicides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Diuron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Atrazine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
Simazine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.
Hexazinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5.
Glyphosate [N-(phosphonomethyl) glycine] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Antifouling biocides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
Irgarol 1051 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. Fax: 82 31 416 6173.

E-mail addresses: shinkh@hanyang.ac.kr, shinkh65@naver.com (K.-H. Shin).
http://dx.doi.org/10.1016/j.ecoenv.2014.01.042
0147-6513 & 2014 Elsevier Inc. All rights reserved.
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K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 104 (2014) 5171
5.2.
Tributyltin chloride (TBT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.
Sea-Nine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Web references . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Light constitutes the most abundant and constant type of energy
available on earth, that directly or indirectly provides majority of
energy used by biological systems for the past few billions of years.
The mechanism of conversion of light energy to chemical energy,
which is then used for metabolic processes, is called photosynthesis.
The photosynthetic complexes are exquisitely tuned to capture solar
light efciently; they then transmit the excitation energy to reaction
centers (RC), where long-term energy storage is initiated. The energy
transfer mechanism is often described by semi-classical models that
invoke hopping of excited-state populations along discrete energy
levels (Blankenship, 2002). Light-driven reactions of photosynthesis
then convert the physical energy of light into a stable electrochemical
potential, which is eventually stored as chemical energy through a
series of dark reactions (Sundstrm et al., 1999). These light reactions
occur in two closely coupled pigment systems: light energy is
absorbed by a network of so-called antenna pigments bound to
proteins and the excitation energy is very efciently transported via
chlorophyll a to the photochemical reaction center (RC) of photosystem II (PSII) and I (PSI), where the energy is converted into a stable
trans-membrane charge separation through a sequence of electrontransfer reactions (Amerongen et al., 2000). The part of absorbed
light energy not used in PSII photochemistry, is dissipated via nonradiative energy or chl a uorescence emission associated with the
PSII complex (Pullerits and Sundstrm, 1996; Popovic et al., 2003).
Concisely, light energy absorbed by chlorophyll molecules can
undergo one of three fates: it can be used to drive photosynthesis
(photochemistry), excess energy can be dissipated as heat or it can
be re-emitted as light-chlorophyll a uorescence. These three
processes occur in competition, such that any increase in the
efciency of one will result in a decrease in the yield of the other
two. Hence, by measuring the yield of chlorophyll (chl) a uorescence, information about changes in the efciency of photosynthesis and heat dissipation can be acquired (Maxwell and Johnson,
2000). Even though the amount of chl a uorescence is very small
(approximately 12 percent of the total light absorbed), its
measurement is straightforward and reliable. Chl uorescence is
a physical signal dened as the radiative energy evolved from deexciting chl a molecules ( 690 nm for PSII, 740 nm for PSI)
(Rohek et al., 2008). Govindjee (2004) explains how chl a
uorescence provides new and important information on the
composition of pigment systems, excitation energy transfer, physical changes in pigment-protein complexes, primary photochemistry, kinetics and rates of electron transfer reactions in PSII, the
sites of various inhibitors, and activators, as well as, lesions in
newly constructed mutants. Any modication in the photosynthesis or related biochemical or physiological processes would lead
to signicant changes in the yield and kinetics of dissipated
uorescence. Therefore, alteration of the photosynthetic capacity
evaluated by uorescence kinetics could be used to indicate
damage induced by pollutants or environmental changes. Chl a
uorescence measurement of PSII is a unique, rapid, non-intrusive
and universal technique that reveals information on plant performance and protective responses. This intriguing tool, evaluating
the ecophysiological status, could be applied in most studies that
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address photosynthetic responses of plants and algae in the

environment (Maxwell and Johnson, 2000; Adams and DemmigAdams, 2004; Murchie and Lawson, 2013). Several factors can lead
to a decrease (quenching) of chl a uorescence (for e.g., excessive
irradiance, low or high temperature, drought, toxic chemicals,
toxic metals, and herbicides). However, the interpretation of
uorescence signals and discrimination of the contributions of
each factor depends on an individual (Rohek et al., 2008). Its
rapid data generation, advanced software (Lazr and Nau, 1998),
and accurate statistical analysis provide a highly resolved relationship of the light and dark reactions of photosynthesis (Joshi and
Mohanty, 2004). Several reports demonstrate the advantages of
this technique (Juneau et al., 2007; Rohek et al., 2008); this
makes chl a uorescence a method of choice as compared to
routine biotests based on growth inhibition..
There has been a growing interest in the practical application of
chl a uorescence as a rapid and sensitive bioindicator of plant stress
in response to different chemicals in recent years (Kumar and Han,
2010). Chl a uorescence analysis equates to a bioanalytical tool
which can be used for evalualting exposure to mixtures of pollutants
acting by a common mode of action (Muller et al., 2008). Apart from
being helpful in determing temporal damage, it also provides an
effect-based real-time assessment of the impact of toxicants in
complex environmental conditions including mixtures. Endo and
Omasa (2004) suggested chl a uorescence measurements as an
effective tool for determing toxicity of herbicides to aquatic plants
and algae. Chl a uorescence is also used as a powerful tool to
investigate the ecophysiology of phytoplankton and to monitor its
biomass (Muller et al., 2008). Reports also suggest parameters of chl
a uorescence to be widely used to assess the abundance and activity
of phytoplankton in the natural habitat of algae, without affecting
their physiological state (Antal et al., 2001).
Among the various uorescence techniques, pulse amplitude
modulated uorometry (PAM) introduced by Schreiber et al. (1986),
and supplemented with the saturation pulse method can be
employed to obtain information on the functioning of the photosynthetic apparatus and photosynthetic activities of plants (Rohek
et al., 2008). These modulation uorometers in combination with
the application of saturating light pulses, generally provide
essential information beyond that obtained by conventional chlorophyll uorometers. This method (i.e. PAM) operates with three
kinds of light (modulated, actinic, and saturating), which facilitate
the analyses of uorescence-induction kinetics in photosynthesizers
and the evaluation of their primary productivity (Figueredo et al.,
2009). These uorometers provide information regarding several
acclimatization and adaptive mechanisms adapted to cope with
environmental stress, such as high light intensity, heat, chilling,
dehydration, salinity and malnutrition (Rohek et al., 2008). The
pulse amplitude modulation (PAM) uorescence permits in vivo
non-destructive determination of changes in the photosynthetic
apparatus much earlier than the appearance of visible injury
(Kumar and Han, 2010) Pulse amplitude modulated (PAM) uorometry and the saturation pulse technique can be useful in measuring toxicant-induced changes in the effective PSII quantum
efciency (Muller et al. 2008; Schreiber, 2004). Several types of
PAM including the Maxi Imaging-PAM, Diving PAM as well as the
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Fig. 1. Schematic representation of the light reactions of photosynthesis in algae. The four protein complexes (represented from left to right): photosystem II (PSII),
cytochromes B6/F (Cyt-b6/f), photosystem I (PSI), and ATP synthase. The continuous black arrows indicate linear electron transport. The following processes within the
photosystem II are schematically depicted: the light harvesting complex II (LHC II), phaeophytin (Phe), the primary electron acceptor quinone QA and a bound secondary
electron acceptor quinone QB. The plastocyanin (PC) is an electron transporter between Cyt-b6/f and P700 of PSI. The terminal electron acceptor downstream of PSI is a
ferredoxin (Fd) which with the ferredoxin-NADP reductase (FNR) reduces NADP to NADPH. ATP synthase that generates ATP from ADP Pi using chemiosmotic energy from
the Proton (H ) gradient created by splitting water and the translocation of protons. (b) Negative impact of heavy metals (HMs), herbicides (HCs) and antifouling compounds
(AFCs) on light reaction of algal photosynthesis (see the text for further information).
ToxY-PAM uorometer are known (http://www.walz.com/products/

chl_p700/pan/overview.html).
In recent years, aquatic ecosystems (lakes, ponds, rivers, sea and
oceans) have evidenced serious environmental degradation due to
various anthropogenic activities including indiscriminate disposal of
pollutants from both industrial and domestic sources. Chemical
contaminants including organochlorine compounds, herbicides,
domestic and municipal wastes, petroleum products and toxic metals
have adverse effects on most aquatic organisms. The substantial roles
played by algae in aquatic ecosystems are well-known. Microalgae are
important primary producers and suitable indicators of eutrophication and toxic stress (Vanselow, 1998), while, macroalgae (or seaweeds) are ecologically (Chennubhotla, 1996) and industrially
important (Klnc et al., 2013). Toxicants such as toxic heavy metals
and herbicides are known to interfere with the functioning of
photosynthesis and its associated metabolism in both micro and
macroalgae. Fig. 1 illustrates the inhibitory effects of several toxic
metals and herbicides on the photosynthetic apparatus. It is necessary
to realize that since photosynthesis forms the fundamental basis of
the food chain, therefore, even sublethal effects on primary producers
could impact the energy transfer throughout the food chain.
2. Parameters of chl a uorescence

Chlorophyll uorescence which is successfully used as a noninvasive signal in plant biochemistry, physiology and ecology
(Cerovic et al., 1996), comprises a number of measurement
protocols that are used for different purposes. A detailed description of certain chl a uorescence parameters, their denitions,
photosynthetic signicance, and mutual relationships are provided
by Nedbal et al. (2000), Rohek (2002), Maxwell and Johnson
(2000) and Elahifard et al. (2013). Some of these parameters
include: PSII photosystem II; QA primary quinone acceptor of
Photosystem II; QB secondary quinone acceptor of Photosystem
II; NPQ non-photochemical quenching; qP photochemical
quenching; qE energy-dependent quenching; qI photoinhibtory quenching; qT quenching related to state transitions; pH
transthylakoid pH gradient PSII; F0 uorescence emission
measured when the primary quinone acceptor QA is oxidized
and non-photochemical quenching is inactive; F00 uorescence
emission measured when QA is oxidized and nonphotochemical
quenching is active; Fm, uorescence emission measured when QA
and the plastoquinone pool are reduced and non-photochemical
quenching is inactive; Fm0 uorescence emission measured
when QA and the plastoquinone pool are reduced and nonphotochemical quenching is active; F variable uorescence in
the absence of non-photochemical quenching (F0 Fm F0); F0
variable uorescence measured when nonphotochemical quenching is active (F0 Fm0 F00 ); F/Fm maximum quantum yield; PSII
(or F/Fm0 ) effective quantum yield; ETR Photosystem II-based
electron transport rate; and, ETRmax maximum electron
transport rate.
Nedbal et al. (2000) vividly describe the yield of chlorophyll
uorescence to depend fundamentally on the capacity of PSII to
carry out a stable charge separation between P680, the primary
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donor, and QA, the primary quinone acceptor of the reaction

center. When QA is oxidized, the reaction center is able to utilize
the light energy harvested by the antenna system for charge
separation, and the fraction of excitation lost to uorescence is
low, giving rise to lowest uorescence yields (F0 or F00 ). In contrast,
when QA is reduced, the reaction center is unable to undergo
stable charge separation, and the fraction of excitation lost to
uorescence is high, giving rise to maximum uorescence yields
(Fm or Fm0 ). Measurements of the steady state uorescence emission together with measurements of F0, F00 , Fm and Fm0 , provide
data for calculating photochemical yields and physiological conditions. Apart from being controlled by the photochemical processes in PSII, the yield of chlorophyll uorescence is strongly
modulated by mechanisms that limit the ow of the excitation
energy from the antenna system to the PSII reaction centers. For
e.g., when light intensities exceed the electron transport capacity
of the photosynthetic apparatus, non-photochemical quenching
comes into play, wherein a process that diverts excitation energy
into heat occurs within the antenna system (Nedbal et al., 2000).
However, certain researchers point out the use of (E) or the
efciency (quantum yield) of the conversion of absorbed energy in
photosynthetic reactions, where the value of would be proportional to the following: (i) to the relative number of functionally
active (f) and open (qP) PSII reaction centers in algal cells, (ii) to
the effciency of photochemical conversion of light energy in open
reaction centers (RC), [mM electron mE 1], and (iii) to the effciency of electron transfer from H2O to CO2 (e), [mMC
(mM electron) 1]; wherein some parameters like f, can be determined by measuring uorescence parameters F0 and F/Fm (Antal
et al., 2001).
Several articles provide the utility of chl a uorescence parameters in assessing the effects of toxicant or their combinations
(Rohek et al., 2008; Papageorgiou and Govindjee, 2004). This
review focuses on the utility of chl a uorescence parameters for
monitoring the effects of toxic metals and herbicides on microalgae and macroalgae (seaweeds), while referring to few most
important chl a uorescence parameters viz. maximum quantum
yield (F/Fm), effective quantum yield (PSII), non-photochemical
quenching (NPQ) and electron transport rate (ETR).
2.1. Maximum quantum yield (F/Fm)
The F/Fm is the maximum quantum efciency of PSII photochemistry, at which light absorbed by PSII is used for the reduction
of the primary acceptor (quinone QA). It can be calculated based on
the following equation:
F v =F m F m F 0 =F m PSII =qP
where, Fm is maximal and F0 is minimal uorescence from a darkadapted sample and qP represents photochemical quenching.
The dark adapted values of F/Fm reect the potential quantum
efciency of PSII, and are used as a sensitive indicator of plant
photosynthetic performance; the maximum theoretical values for
F/Fm are 0.65 for single turnover saturation pulses (Kolber and
Falkowski, 1993) and 0.83 for multiple turnover pulses
(Magnusson, 1997). Several researchers suggest optimal values of
F/Fm of most plants and macroalgae (seaweeds) to be around 0.83
(Bjrkman and Demmig, 1987; Chaloub et al., 2010). In practice,
maximum achievable F/Fm is known to vary between taxa as a
result of differences in pigment composition and cell structure
(Koblek et al., 2001; Suggett et al., 2009); for e.g., smaller taxa
appear to have lower F/Fm values (as low as 0.30.4 for the
smallest pico-eukaryotes) along with higher PSII values (Suggett
et al., 2009). It is evident that the environmental factors that
impact the PSII, directly or indirectly, will also impact measures of
F/Fm (Greene et al., 1992); a few dominant factors include light,
nutrient status and temperature (Wozniak et al., 2002). However,

Kroon et al. (1993) state that, several factors when combined
confound the interpretation of F/Fm and other uorescence
parameters. Brand (1982) also found that many marine phytoplankton species exhibit endogenous diel patterns in uorescence
parameters and explained that changes in cellular metabolism
could be due to varying environmental conditions. Since abiotic
and biotic stress, in the presence of light, commonly cause a
decreased F/Fm in plants, Baker (2008) suggested F/Fm measurements as a simple and rapid way of monitoring stress. Li et al.
(2010) also reported F/Fm as a robust indicator of photosynthetic
stress, stating that this parameter is particularly useful to determine thermal, salinity, dessication and other environmental stressors. Bioassays based on rapid F/Fm changes are particularly
helpful in laboratory testing of toxins, where multiple species
selection can be customized to the ecosystem or taxa of interest.
Choi et al. (2012) acknowledge the utility of portable uorometers
facilitate eld testing; nevertheless, they mention that distinct
species specic responses complicate interpretation of net F/Fm
signals from a community. Therefore, it is important that these
parameters are fully understood in order to obtain reliable interpretation; for e.g., the presence of phycobilisomes in the lightharvesting system of red algae generally results in a lower
maximal quantum yields (F/Fm) than that measured in green
and brown algae (Bchel and Wilhelm, 1993).
2.2. Effective quantum yield (PSII or F/Fm0 )
The PSII photochemical efciency in the light-adapted state,
also known as the effective, or steady state, quantum yield of
chlorophyll uorescence is typically denoted as F/Fm0 , F0 /Fm0 ,
Fq0 /Fm0 or PSII. Photochemical quenching parameters always relate
to the relative value of F0 m and Ft. It is calculated as F/Fm0 , F0 /Fm0 ,
Fq0 /Fm0 or using the equation
PSII F m0 F s =F m0 or 1 F s =F m0
A prime notation (0 ) used after a uorescence parameter indicates
that the sample is exposed to light that will drive photosynthesis
i.e., actinic light. The difference between Fm0 and F0 is designated as
Fq0 and results from quenching of Fm0 by PSII photochemistry. The
ratio Fq/Fm is theoretically proportional to the quantum yield of
PSII photochemistry prior to the application of the saturating light
pulse (Genty et al., 1989). PSII, measuring the efciency of PSII
photochemistry, is considered as one of the most useful parameters (Genty et al., 1989) that reveals the physiological state. In
addition, PSII provides a rapid method to determine the PSII
operating efciency under different light and other environmental
conditions (Baker, 2008). Deviations in effective quantum yield
(F/Fm0 ) reveal a reversible down regulation of PSII photochemistry rather than irreversible damage to the photosynthetic apparatus. Therefore, it is considered as a good eco-physiological
indicator to evaluate algae response to environmental stress.
Juneau et al. (2001) particularly explained the operational PSII
quantum yield at steady state of electron transport to be a
sensitive indicator of algal sensitivities to toxicants.
2.3. Non-photochemical quenching (NPQ)
NPQ depicts the relative increase in the sum of the rate
constants of the non-photochemical deactivation processes (uorescence emission, heat dissipation and spillover of excitation
energy from PSII to PSI) relative to the dark-adapted state (presuming no non-photochemical reduction of the PQ pool in the
dark) (Cosgrove and Borowitzka, 2010). Non-photochemical
quenching could be measured using the equation suggested by
Bliger and Bjrkman (1990):

NPQ F m =F m0 1 or F m F m0 =F m0
where Fm represents the maximum uorescence emission
recorded in dark-adapted condition while Fm0 represents the
maximum uorescence value recorded at discrete time intervals
during illumination. For plants, this value is expected to range
from 0.5 to 3.5 at saturating light intensities. However, the value of
NPQ markedly varies from species to species, for e.g., it is
estimated to be around 0.20.3 in marine picoeukaryote Ostreococcustauri, while in cyanobacteria and diatoms the NPQ values of
0.5 and 1.2 have been reported (Finazzi et al., 2006).
Owing to its high sensitivity and quickness of response, NPQ is
considered one of the most appropriate indicators to visualize
inhibitory effects (Kumar et al., 2010). An inhibition of the NPQ
could be linked to damage in the pigments; particularly, NPQ
inhibition have been observed when an organism is exposed to
toxic compounds (Ricart et al., 2010). However, Ivorra et al. (2000)
and Corcoll et al. (2012) reported that metal exposed biolms did
not show any response to PSII0 , PSII and NPQ.
2.4. Electron transport rate (ETR)
Calculating the photosynthetic electron transport rate is one of
the most common applications using uorescence signals; the
electron transport rate (ETR) can be obtained by means of the
expression:
ETR PFD AF F=F m0 MT F II
where PFD is the irradiance (M photons m 2 s 1) between 400
and 700 nm of the actinic light, AF is the absorption factor of the
algae at a given wavelength, which can be derived from absorbance (A) by using the equation: A log(1 AF), F/Fm0 (MT) is
the effective quantum yield at each irradiance and the subscript MT
indicates that the maximum uorescence was measured using a
multiple turnover ash, and FII is a factor that corresponds to the
absorption of 2 light quanta (one for each photosystem, i.e. PSI and
PSII) needed for the transport of one electron (Aguilera et al.,
2008). This value of FII might differ according to the species (for e.
g., in seaweed the thickness of the thallus inuences this factor)
and light acclimation (high versus low irradiances). FII for diatoms
and phaeocystis is 0.8 (80 percent of chl a associated with PSII),
and that for chlorophyta, phaeophyta and rhodophyta is 0.5, 0.80
and 0.15 respectively (Figueroa et al., 2003). ETR could also be
considered as an indicator of environmental stress in algae, for e.g.
copper toxicity studies carried out by Han et al. (2008) revealed
concentration dependent decrease in ETR of Ulva pertusa and
Ulva armoricana.
3. Trace and toxic metals

Heavy metals, generally dened as metals or metal elements
with a specic density 45 g/cm3, have a variety of applications,
for e.g., copper (Cu), cobalt (Co), chromium (Cr), cadmium (Cd),
ferrous (Fe), zinc (Zn), lead (Pb), mercury (Hg), manganese (Mn),
nickel (Ni), molybdenum (Mo) and vanadium (V) (Jrup, 2003;
Szyczewski et al., 2009). They pose a huge threat to ora and
fauna; therefore, their indiscriminate usage and disposal have
been comprehensively discussed globally. In photosynthetic
organisms, toxic heavy metals are known to cause a negative
impact on nearly all the components of the photosynthetic apparatus. Apart from decreasing the photosynthetic pigment content,
they harm the light and dark reactions of photosynthesis, chloroplast membrane structure, light-harvesting and oxygen-evolving
complexes, photosystems and constituents of the photosynthetic
55
electron transport chain and also inhibit the reductive pentose

phosphate cycle (Appenroth, 2010). In addition, these toxic metals
cause inhibition of enzyme activities; they substitute the Mg2 site
in the ternary enzymeCO2metal2 complex, thereby impeding
the PSII reaction centers, for e.g., rubisco (bisphosphate carboxylase
oxygenase), or react with the enzyme-SH groups in proteins and are
responsible for the substitution of essential ions, enhancement of
photoinhibition and oxidative stress, the impediment of plastocyanin function, change in lipid metabolisms, and disturbances in the
uptake of essential microelements (Clijsters and Assche, 1985;
Kpper et al., 2002). Measuring photosynthetic activity has been
considered as a good screening method for detecting possible stress
conditions, especially involving heavy metals (Appenroth, 2010). For
e.g., the F/Fm of U. pertusa exposed for 7 days to Cu2 (0
2000 mg L 1), Hg2 (02000 mg L 1), Cr6 (016,000 mg L 1), and
Ni2 (016,000 mg L 1) declined from 0.76 to 0.31, 0.26, 0.08 and
0.17, respectivley, with corresponding EC50 (half maximal effective
concentration) values of 201.3, 195.2, 3429.3, and 5527.8 mg L 1,
respectively (unpublished data; Fig. 2a).
The impact of toxic metals, pesticides and other environmental
pollutants on photosynthesis has been more popularly discussed
in a plant perspective till date. In the present review we discuss
the use of chl a uorescence in determining the phytotoxicity of
toxicants from an algal perspective, including their mode of action.
3.1. Mercury (Hg)
Mercury, classied as a transition metal, is located in groups
312 of the periodic table. It is considered as the most toxic
element among those having no known physiological function
(non-essential elements) in algae. This metal inhibits photosynthesis and exerts severe adverse effects on different vital functions of
algae by acting on growth, cell permeability, chlorophyll content,
and nitrogen xation (Campanella et al., 2000). Mercury is known
to be a strong inhibitor of the photosynthetic electron transport
and may achieve that through inhibition at different sites such as
the water-splitting system, plastocyanin, the NADP-ferrodoxine
oxidoreductase enzyme, or at PSI and PSII (Singh et al., 1993;
Popovic et al., 2003). The toxic effect of mercury in some cases (for
e.g. Dunaliella tertiolecta) is shown to be associated with the
inhibition of the water-splitting system and consequently causes
a decrease of the PSII quantum yield (Samson and Popovic, 1990).
Even more, Hg2 decreases the capacity of the photosystem to
dissipate the excitation light energy via the photochemical pathway and is responsible for an increased uorescence life-time
(Juneau et al., 2001).
Several reports are available on the evaluation of different algal
species for their sensitivity to mercury using chl a uorescence or
PAM-uorometry. The transfer of excitation energy within phycobilisomes in Spirulina platensis cells is inhibited by the presence of
very low Hg2 concentrations (2.01 mg L 1) (Murthy et al., 1989).
Juneau et al. (2001) investigated Ankistrodesmus falcatus, Selenastrum capricornutum, Chlorella vulgaris, Microcystis aeruginosa and
Pediastrum biwae, and reported that uorescence parameters [viz.
the maximal PSII quantum yield (M), the operational PSII
quantum yield at steady state of electron transport (0 M), the
photochemical quenching value (qP) and the non-photochemical
quenching value (qN)], were appropriate indicators of the inhibitory effects of Hg2 . At the steady state of electron transport,
Juneau et al. (2001) observed 50 percent reduction in the operational quantum yield (0 M), when these algae were exposed to
9.34, 16.83, 61.78, 56.97 and 73.22 mg L 1 Hg2 , respectively, for
96 h. In another study, a 25 percent decrease of M and 61 percent
decrease in 0 M was reported for Hg2 exposed S. capricornutum
(5 h, 500 mg L 1) (Juneau and Papovic, 1999). Y. Wu et al. (2012)
demonstrated the utility of two-photon excited uorescence
56
Fig. 2. Effect of heavy metals and herbicides on the chlorophyll uorescence of macroalgae. (a) Ulva pertusa in a 96-well after being treated (7 days) with (i) Cu (0, 62.5, 125,
250, 500, 1000 and 2000 g L 1), (ii) Hg (0, 62.5, 125, 250, 500, 1000 and 2000 g L 1), (iii) Cr (0, 62.5, 125, 250, 500, 1000 and 2000, 4000, 8000 and 16,000 g L 1) and
(iv) Ni (0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 and 16,000 g L 1). (b) Sargassum hemiphyllum in a 96-well after being treated (1 h) with (i) diuron (0, 1.5625, 3.125,
6.25, 12.5, 25, 50, 100 and 200 g L 1), (ii) atrazine (0, 6.25, 12.5, 25, 50, 100, 200 and 400 g L 1), (iii) simazine (0, 31.25, 62.5, 125, 250, 500, 1000 and 2000 g L 1) and
(iv) hexazianone (0, 12.5, 25, 50, 100, 200 and 400 g L 1). (c) Undaria pinnatida in a 96-well after being treated (1 h) with (i) diuron (0, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100
and 200 g L 1), (ii) atrazine (0, 6.25, 12.5, 25, 50, 100, 200 and 400 g L 1), (iii) simazine (0, 31.25, 62.5, 125, 250, 500, 1000 and 2000 g L 1) and (iv) hexazianone (0, 12.5,
25, 50, 100, 200 and 400 g L 1).
lifetime imaging (TPE-FLIM) systems in uncovering adverse effects

of Hg2 on Thalassiosira weissogii wherein an injury to the
photosystem was caused due to algal exposure to Hg2 . Lu et al.
(2000) reported that an increase in non-photochemical quenching
(NPQ) of S. platensis often results in a decrease in F/Fm when
exposed to 20 mM of Hg2 . Methylmercury chloride (MeHg) at
concentrations above 0.20 mg L 1 is reported to decrease the F/Fm
ratio (which characterizes the maximal efciency of energy utilization in the PSII) of the green microalgae Chlamydomonas reinhardtii;
here, the degree of inhibition depended on the time of treatment and
was always higher under illumination conditions (50 mE m 2 s 1)
than under dark conditions (Kukarskikh et al., 2003). In addition,
MeHg treatment of algal cells indicated the damage on the donor
side of PSII and the damage of the electron transfer from QA to QB,
while the reduction of photochemical uorescence quenching (qN)
under MeHg treatment was due to an increase in the fraction of
closed reaction centers (QA-). Overall, methylmercury is known to
damage the photosynthetic electron transfer chain of C. reinhardtii at
several sites, showing a stronger inhibitory effect on photosynthetic

process than the effect of Hg2 (Kukarskikh et al., 2003). The
diatomeaceous algae Thalassiosira weissogii exposed to 0.20 and
0.02 mg L 1 methyl mercury (MeHg) has been reported to show a
slow decrease in the PSII activity, but the alga was not sensitive to the
same concentrations of Hg2 (Graevskaya et al., 2003).
Hg2 is also known to cause a reduction (48 percent) in the
F/Fm value of the seaweed Gracilaria edulis (Abu-Bakar and
Ahamad-Zakeri, 2012). Ahamad-Zakeri and Abu-Bakar (2013)
reported that 1 mg L 1 of Hg2 caused 82 percent reduction in
Fv/Fm of Caulerpa racemosa, 47 percent reduction in Caulerpa
lentillifera and 13 percent reduction in U. pertusa. Lu et al. (2000)
suggest that the decrease in the maximal efciency of PSII
photochemistry (F/Fm) observed by increasing the Hg content,
could be the result of an increased proportion of QB-non-reducing
PSII reaction centers. However, a decreased F/Fm could also be due
to an inhibition of the transfer of excitation energy from the
phycobilisome to PSII reaction centers.
3.2. Copper (Cu)

Copper serves as an essential component in metabolic processes of algae, playing vital functions in the electron transport
and various enzyme systems (for e.g., amine oxidase, cytochrome c
oxidase), and particularly works as a prosthetic group of the
chloroplastic antioxidant enzyme Cu/Zn superoxide dismutase. It
aids in the regulation of PSII-mediated electron transport either as
a part of a polypeptide involved in electron transport, or as a
stabilizer of the lipid environment close to electron carriers of the
PSII complex. Nevertheless, Cu is considered one of the most toxic
heavy metal ions to algae and plants and is a potent inhibitor of
photosynthesis (Gledhill et al., 1997; Janik et al., 2010). Cu is
considered as the second most toxic metal after Hg (Baumann
et al., 2009). Barn et al. (1995) summarized its direct impact on
photosynthesis stating that though it is a constituent of the
primary electron donor in PSI (the Cu-protein plastocyanin), high
Cu concentrations inhibit the photosynthetic electron transport
rate, especially the PSII, and both Cu deciency and Cu toxicity
interfere with pigment and lipid biosynthesis and, consequently,
with the chloroplast ultrastructure, thus negatively inuencing the
photosynthetic efciency. A slight increase in endogenous Cu2
concentrations (above optimum level) could interfere with various
metabolic pathways causing inhibition of photosynthesis, and a
number of deleterious effects at the physiological, biochemical,
and structural levels (Mishra and Dubey, 2005; Kumar et al., 2009;
Zhang et al., 2014; Hook et al., 2014). Kpper et al. (2002)
investigated the effect of Cu2 on the photosynthetic apparatus
of algae belonging to different phyla and proposed a Cu2 induced
decrease of variable uorescence (F) is a consequence of inhibition of electron donation to the primary photochemical reaction.
Cu2 probably induces a state analogous to photoinhibition in
algae, wherein trapping of an exciton in the PSII reaction center is
followed by its non-radiative dissipation. Furthermore, the inhibition of PSII activity by Cu2 has been reported to occur at the
Pheo-QA domain, which interferes with the stabilized charge
separation (P680 QA) (Kpper et al., 2002). Copper has been
shown to inhibit the photosynthetic electron transport through
damage to both, the donor and acceptor sides, of PSII (Ptsikk
et al., 1998). In fact, blockage of the electron transport by Cu2
could lead to a decrease in F/Fm (Karukstis, 1991).
Several researchers have projected the inhibitory effect of
copper on the photosynthesis of several species of microalgae,
and this fact makes microalgae the most suitable tool for monitoring the effect of Cu using the chlorophyll uorescence technique.
Though several reports suggest the detrimental impact of Cu on
the ultrastructure and functioning of macroalgae (Miao et al.,
2014), as well as microalgae (Lombardi and Maldonado, 2011), a
few of the studies revealing inhibitory effects of this toxicant on
algae have been outlined here. Cu2 is reported to cause important mitochondrial alterations, chloroplast disorganization and
changes in the protein and lipid composition in Euglena gracilis
(Einicker-Lamas et al., 2002). Kpper et al. (2002) reported
0.64 mg L 1 of Cu2 to cause a reduction in the Fm of the
microalga Scenedesmus quadricauda. Rocchetta and Kpper
(2009) observed a reduction in photosynthetic quantum efciency
of PSII of E. gracilis in the dark adapted state (measured by F/Fm)
after 48 h of exposure to Cu2 . These authors also found that
0.64 mg L 1 Cu2 treated E. gracilis had decreased oxygen evolution and photochemical quenching. Further, they report 0.64 and
3.17 mg L 1 Cu2 to cause 2030 and 6070 percent inhibition of
growth respectively after 96 h. Xia and Tian (2009) found Chlorella
pyrenoidosa exposed to 0.13, 0.32, 0.64, 1.27, and 2.54 mg L 1 Cu2
exhibited a signicant decrease of RC/CS (39.5 percent, 41.9
percent, 59.8 percent, 55.1 percent and 61.2 percent respectively);
here, RC/CS stands for the amount of active PSII reaction centers
57
per excited cross section. Moreover, 0.1 mg Cu2 L 1 is known to

cause a slight inhibition of photosynthesis in C. pyrenoidosa and
0.25 mg L 1 causes a total inhibition of the same (Wong and
Chang, 1991). Apart from affecting the photochemistry of PSII,
copper also slows down the synthesis of PSII D1 protein, thereby,
inhibiting the recovery from photoinhibition in C. pyrenoidosa
(Vavilin et al., 1995). Ou-Yang et al.,, (2012) reported a suppression
of photosynthesis, particularly the F/Fm of C. vulgaris in the
presence of 0.32 mg L 1 Cu2 . Sbihi et al. (2012) showed a
remarkable reduction in F/Fm in case of Cu2 (0.2 mg L 1)
exposed Planothidium lanceolatum and Isochrysis galbana, and
demonstrated that chl a uorescence parameters were more
sensitive than growth endpoints. Here, IC50 (half maximal inhibitory concentration) for growth of P. lanceolatum and I. galbana
were 0.62 mg L 1 after 72 h and 1.4 mg L 1 after 120 h, respectively. In another illustration, a 30 percent decrease in the maximum quantum yield (m) and quantum yield at steady state of
the electron transport (0 m) of Raphidocelis subcapitata have been
reported after 96 h exposure to 10 mg L 1 Cu2 (El Berdey et al.,
2000). Miao et al. (2005) elucidate that the PAM parameter 0 m is
more sensitive than the cell-specic growth rate; they report 5 h
EC50 values for the 0 m for the freshwater algae C. reinhardtii and
Pseudokirchneriella capricornutum were 13 and 30 mg L 1, respectively. These values were lower than the 24 h EC50 values (EC50s) of
growth inhibition (46227 mg L 1) mentioned in other studies.
According to Rijstenbil et al. (1994), long-term exposure to copper
resulted in a decrease in F0 as well as in Fm in the marine diatom
Ditylum brightwellii. On the other hand, Samson et al. (1988)
reported a decreased quantum yield of PSII in the Cu2 exposed
marine planktonic diatom D. tertiolecta. Nevertheless, it is necessary to note that even lower concentrations of Cu2 could inhibit
photosynthesis of diatoms (for e.g., 100 mg L 1 inhibits Nitzschia
closterium; Stauber and Florence, 1987), cyanobacteria (Anabaena
variabilis stressed with Cu2 at low light intensities; Kowalewska
and Hoffmann, 1989), as well as phytoplankton (Kowalewska et al.,
1992). Dewez et al. (2005) found that NPQ of Scenedesmus obliquus
exposed to 1 mg L 1 Cu2 was more sensitive than other uorescence parameters such as the proton induced uorescence
quenching (QEmax), the antenna size per photosystem II reaction
center (ABS/RC) and the value of the pH-dependent quenching
(QPQ). On the other hand, Perales-Vela et al. (2007) reported that
the sub-lethal Cu2 concentration in vivo caused a reduction of
the active PSII reaction centers and the primary charge separation,
decreasing the quantum yield of PSII, the electron transport rate
and the photosynthetic O2 evolution. Furthermore, they suggested
that the NPQ increases as Cu2 increases in the culture medium;
the effect of Cu2 on NPQ is inversely correlated with the effect on
ETR of Scenedesmus incrassatulus when the concentration ranged
from 0 to 3.145 mM. Interestingly, Bielmyer et al. (2010) observed
signicant concentration dependent decrease in PSII signicantly
in the algal symbionts of A. cervicornis; however the NPQ signicantly increased for the same range of Cu2 concentration (i.e.
020 mg L 1). Interestingly, Rocchetta and Kpper (2009) exposed
E. gracilis to Cu2 concentrations ranging from 0.5 to 50 mM and
obtained negative NPQ values that usually correlated with higher
PSII in actinic light compared with the dark-adapted Fv/Fm.
An apparent decline in the maximum quantum yield of PSII
measured by F/Fm has also been visualized in U. armoricana
(100 mg L 1 Cu2 ) and U. pertusa (250 mg L 1 Cu2 ) (Han et al.,
2008). Mamboya et al. (1999) studied the effect of Cu2 on the
photosynthetic efciency (F/Fm) of Palmaria boergesenii, reporting
that both concentration and exposure time had a signicant effect in
inhibiting the photosynthetic efciency. Cu2 is also known to cause
a 30 percent and 23 percent reduction in the F/Fm of G. edulis and
Gracilaria manilaensis (Abu-Bakar and Ahamad-Zakeri, 2012). Interestingly, Ahamad-Zakeri and Abu-Bakar (2013) reported 2 mg L 1 Cu
58
to reduce the F/Fm of C. lentillifera; they also state that no F/Fm could
be detected in case of C. racemosa exposed to 2 mg L 1 Cu. Even
more, 0.64 mg L 1 of Cu2 is known to reduce the yields of the
macroalgae Chondrus crispus, Palmaria palmata and Ulva intestinalis
(Baumann et al., 2009). Exposure to 250 mg L 1 Cu2 for 7 d caused a
signicant reduction in F/Fm, Fm, F0 and Fv of Gracilariopsis longissima
(Brown and Newman, 2003). Kumar et al. (2009) reported that the
IC50s for F/Fm and ETRmax of Cu2 -exposed (macroalgae) U. pertusa
were 0.35 and 0.28 mg L 1, respectively; this difference in response
of F/Fm and ETRmax reects an inhibition downstream of PSII,
suggesting a down-regulation of the PSII electron transport as a
result of inhibition of the Calvin cycle enzymes and Rubisco. Besides,
the IC50 for ETRmax of Cu2 -exposed Ecklonia cava is reported to be
0.5 mg L 1 after 72 h (Kumar et al., 2009). Han et al. (2008) studied
U. pertusa and reported NPQ to be a sensitive parameter which
declined at 25 mg L 1; in this case, the copper-induced decrease in
NPQ was explained by a decrease in the rate of electron transport in
the pathway P680-pheophytin-QA-QB or to an increase in the rate
of the back reaction, QA -P680

.
3.3. Cadmium (Cd)
Cadmium occurs naturally in ores along with zinc, lead and
copper. Furthermore, this oxophilic and sulfophilic element forms
complexes with various organic particles and thereby triggers a
wide range of reactions that collectively put the aquatic ecosystems to risk (Jiang et al., 2013). It is present as a pollutant in
phosphate fertilizers. Cadmium compounds are used as stabilizers
in PVC products, color pigment, several alloys, and in rechargeable nickelcadmium batteries (Jrup, 2003). Metallic cadmium has mostly been used as an anticorrosion agent (cadmiation). Cadmium is considered as an important contaminant of
natural waters because of its high toxicity at low concentration
(Deckert, 2005). Cadmium causes displacement of the metal cofactors (Zn2 and Ca2 ) from undened protein targets or directly
binds amino acid residues, including cysteine, glutamate, aspartate
and histidine (Faller et al., 2005). The utility of chl a uorescence
techniques in determining toxicity of this toxic metal has been put
forward by several researchers, for e.g., the utlity of PAM to
quantify the toxic effect of Cd2 on the electron transport chain
in the PSII (Miao et al., 2005). Cd2 reduces both chlorophyll
content and Chl a/b ratio. It also inhibits chlorophyll formation,
decreases Rubisco activity, inhibits both PSI and PSII and enhances
lipoxygenase activity (Mishra and Dubey, 2005).
In the algal scenario, Cd2 accumulation in chloroplasts could
lead to an inhibition of the photophosphorylation as in the case of
Euglena sp. (Mendoza-Cozatl et al., 2002). Cd2 alters the phycobilisome structure of the bluegreen alga Anacystis nidulans, which
results in a decline of absorbed light energy, thereby inhibiting
photosynthesis (Gupta and Singhal, 1996). Sbihi et al. (2012)
reported a signicant reduction in F/Fm of 0.1 mg L1 Cd2
exposed P. lanceolatum and I. galbana. This indicated that the chl
a uorescence parameter was more sensitive than the growth
parameter (wherein IC50 for growth were 0.25 and 0.91 mg L 1
after 72 h and 120 h, respectively) (Sbihi et al., 2012). Though Cd2
inhibits both PSI and PSII activity, Husaini and Rai (1991) state that
the PSII is more sensitive to Cd2 than the PSI. In contrast, Zhou
et al. (2006) mentioned that the inhibitory site of Cd2 in
M. aeruginosa is not located at the PSII or PSI level, but is probably
situated on the ferredoxin/NADP -oxidoreductase enzyme at the
terminal of the electron transport chain. According to Zeng et al.
(2012), Cd-induced increase of average chlorophyll uorescence
lifetime of T. weissogii was mainly contributed by the inhibition of
the electron transport chain. More recently, Ou-Yang et al. (2013)
studied C. vulgaris over a long-term 96-h exposure to different Cd
concentrations using ow cytometry and pulse amplitude modulated
uorometry, and revealed the sequence of sensitivity of these toxicity

endpoints were: cell yieldPSII Eesterase activity 4 F/Fm 4 chl a
uorescenceEcell viability. Wang et al. (2013) reported 0.55 times
reduction in the F/Fm of C. pyrenoidosa (0.28) on exposure to 200 mM
of Cd for 96 h, whereas the NPQ value was reduced from 0.039 to
0.007 which was more sensitive than F/Fm; here, under high
concentration of Cd (200 mM), the deactivation of NPQ, indicated
failure to protect the photosynthetic apparatus.
In the macroalgal context, a decreased photosynthetic performance and relative electron transport rate (rETR) have been
observed in Cd2 -exposed Hypnea musciformis (Bouzon et al.,
2012). The photosynthetic parameter rETR provides a conjecture
of the rate of electrons pumped via the PSII into the photosynthetic chain (Schreiber, 2004). Therefore, malfunctioning of the
rETR undeniably implies that there is a damage caused to the PSII.
It is also suggested that the temporal characteristics of chl
uorescence can be utilized as a non-invasive biomarker for
indicating Cd2 toxicity in algal cells (Zeng et al., 2012). Apart
from the electron transfer rate, Cd2 also inuences the optimum
quantum yield of photosynthesis. High Cd2 concentrations (0.3,
0.9, and 2.7 mg L1) produce distinct signs of disturbance in the
PSII visualized by low optimum quantum yields (F/Fm) in Ulva
rigida (Barraza and Carballeira, 1999). Jiang et al. (2013) demonstrated a reduction in relative growth rate (RGR), F/Fm, and actual
photochemical efciency of PSII (yield) of two Ulva spp. treated
with Cd2 , stating that these reductions were greater in Ulva
prolifera than in Ulva linza. They also reported that U. linza had
a better adaptation to Cd2 than U. prolifera; this was because
U. linza had a higher nutrient content, as well as, a stronger
osmotic adjustment and photosynthesis ability.
3.4. Chromium (Cr)
Chromium is a transition element comprising the seventh most
abundant metal in the earth's crust. Trivalent [Cr (III) or Cr3 ] and
hexavalent [Cr (VI) or Cr6 ] species comprise the stable forms of
Cr; however, there are other valence states which are unstable and
short lived in biological systems. Cr (VI), the most toxic form of Cr,
is usually associated with oxygen to form chromate (CrO24 ) or
dichromate (Cr2O27 ) oxyanions. These molecules can easily go
through cell membranes as an alternative substrate for the sulfate
transport system (Shanker et al., 2005). The toxicity of Cr (VI)
compounds has been traced to the reactive intermediates (formation of dOH radicals from H2O2 via a Fenton reaction) generated
during the reduction of Cr by living cells (Shi and Dalal, 1990). As
observed in the case of Chlamydomonas (Rodrguez et al., 2007),
this toxic metal tends to generate reactive oxygen species (ROS),
which can attack thylakoid lipids (mainly unsaturated fatty acids).
This initiates peroxyl-radical chain reactions, destroying membranes and damaging indirectly structural pigment-protein complexes located in chloroplast membranes. Particularly, 10 mM Cr6
is reported to inhibit the growth (6070 percent) of E. gracilis and
decrease the F/Fm, but, its effect was less severe than Cu2
(Rocchetta and Kpper, 2009). Hrcsik et al. (2007) reported that
Cr6 caused an enhanced destruction of the reaction centers and a
reduction in the quantum yield of the PSII reaction center of the
alga C. pyrenoidosa, further stating that this inhibitory effect
increased with increasing concentration of Cr6 . In case of S.
obliquus, 1 mg L 1 Cr6 is known to decrease F/Fm and degrade
the electron transfer system, thereby impairing electron transfer
between PSII and PSI (Khalida et al., 2012). Cr6 (1 g cm 3) has a
marked toxicity on photosynthetic pigments, photosynthesis,
nitrate reductase activity and protein content of Glaucocystis
nostochinearum (Rai et al., 1992). In an assessment of Cr6 toxicity
on the freshwater green alga Monoraphidium convolutum, Takami
et al. (2012) found concentrations higher than 1 mg L 1 caused
Table 1
Properties of few commonly used herbicides.
S. No.
Hebicide
Diuron
Atrazine
Simazine
Hexazinone
Molecular Formula C9H10Cl2N2O
C8H14ClN5
C7H12ClN5
C12H20N4O2
IUPAC name
3-(3,4-dichlorophenyl)-1,1-dimethylurea
2-chloro-4-ethylamino-6isopropylamino-s-triazine
6-chloro-N,N-diethyl-1,3,5-triazine-2,4diamine
3-Cyclohexyl-6-dimethylamino-1-methyl-1,3,
5-triazine-2,4-dione
233.1
215.68
201.657
252.31
Water 46.62
Methanol 43,356.6
Ethanol 36,829.8
Hexane 23.31
1-octanol 19,114.2
Water 33
Methanol 15,000
Ethanol 17,000
1-octanol 10,352.6
Ethanol 1000
DMSO 20,000
DMF 20,000
Water 5
Methanol 400
Chloroform 900
Light petroleum 2
Water - 29,800
Methanol 617,500
n-hexane 530
n-octanol 134,700
acetone 79,000
Structure
Soubility (mg L
1
pKa
1.7
There are no dissociable hydrogens within
the normal environmental pH range (pH 4
9)
1.62
1.8
Kow
2.85
2.75
2.18
1.2
Literature
Green et al (2000); http://www.apvma.gov.

au/products/review/docs/
diuron_environment.pdf
https://www.caymanchem.com/pdfs/
13375.pdf; Raevsky et al (2007); http://
monographs.iarc.fr/ENG/Monographs/
vol73/mono73-8.pdf
http://www.epa.gov/safewater/pdfs/
factsheets/soc/tech/simazine.pdf; http://
pmep.cce.cornell.edu/proles/extoxnet/
pyrethrins-ziram/simazine-ext.html
http://www.fao.org/leadmin/templates/agphome/documents/
Pests_Pesticides/Specs/Hexazinone_2012.pdf;
http://www.cdpr.ca.gov/docs/emon/pubs/fatememo/hxzinone.pdf;
http://www.epa.gov/oppsrrd1/REDs/0266.pdf; Magnusson et al (2010)
S.No
Herbicide
Glyphosate
Irgarol-1051
TBT
Molecular formula
C3H8NO5P
C11H19N5S
C12H27ClSn
IUPAC name
N-(phosphonomethyl) glycine
2-methylthio-4-tert-butylamino-6cyclopropylamino-s-triazine
Tributyltin chloride
169.07
253.4
325.505
Molecular weight
Structure
Molecular weight
59
5.5
Arnold et al. 1997; see Laughlin et al. (1986)

http://datasheets.scbt.com/sc-218604.pdf;
Jongbloed and Luttik (1996);
SRC (1997); http://www.bjmytimes.com/ciba/ciba/1051.pdf
4.1
3.2
IUCLID (2000); http://ec.europa.eu/food/

plant/protection/evaluation/existactive/
list1_glyphosate_en.pdf
Kow
Literature
6.25
pKa1 2.3 at 20 1C (phosphate acid)
1.14.3
pKa2 5.7 at 20 1C (secondary amine)
pKa3 10.2 at 25 1C (carboxylic acid (HSDB 2006))
Water 1
Water 6
butylene glycol 150,000
propylene glycol 10,000
octanol 50,000
butylacetate 150,000
ethylglycolmonomethylether 100,000
methylisobutylketone (MIBK) 90,000
) Water 11,600 at 25 1C, pH 2.5
Methanol 231
n-hexane 26
n-octanol 20
Ethyl acetate 12
pKa
Solubility (mg L
1
S.No
Table 1 (continued )
60
a signicant increase in the electron transfer rates (ETR) and

quantum yields of photosystem II (PSII) after a short-term (2 h)
of exposure; however, there was a subsequent decline in both
parameters after 48 and 72 h. In macroalgae, nal et al. (2010)
observed high Cr6 (52 and 260 mg L 1) concentrations to cause a
signicant decrease in the F/Fm of Ulva lactuca.
In aquatic ecosystems, mixtures of metals are more realistic
rather than single metals; in addition, these metals could also
occur along with other contaminants. Therefore, it is essential to
evaluate the combined or cumulative effect of metals or metal
mixtures on photosynthesis. Wong and Chang (1991) demonstrated that various bimetallic combinations of Cu2 , Cr6 and
Ni2 interacted synergistically on growth, photosynthesis and
chlorophyll a synthesis of C. pyrenoidosa. Ou-yang et al. (2012)
directed studies on the effect of ve heavy metals on the growth
and photosynthesis of C. vulgaris. Qian et al. (2009) elaborated
combined effects of Cu2 and Cd2 on the growth and
photosynthesis-related gene transcription of C. vulgaris. However,
more vivid studies need to be carried out on the utility of chl a
uorescence in case of algae exposed to mixtures of metals or
multiple stressors in combination. In this regard, Juneau et al.
(2001) evidenced heterogeneous sensitivity of different algal
species to the same pollutant, and suggested the need to use a
battery of species to evaluate the effects of mixtures of pollutants
in aquatic systems.
Reports suggest that the environmentally relevant concentration of mercury, copper, cadmium and chromium (as per the WHO
guidelines for surface water) were 0.01, 2.0, 0.003 and 0.05 mg.
L 1 respectively (Armah et al., 2010; Q. Wu et al., 2012). As
witnessed above, the chl uorescence technique is well-versed
in detecting environmentally relevant concentrations, and could
be used to detect the impact of environmental pollutants on
aquatic photosynthetic organisms.
4. Herbicides
Contemporary agricultural practices often necessitate extensive
use of herbicides (for the inhibition of growth of weeds and
mosses, and to protect agricultural crops). After dispersion and
subsequent soil leaching or runoffs, these herbicides nd their way
into water bodies and threaten aquatic biota (Kumar et al., 2010).
Solubility and partitioning behavior of herbicides, generally determine their impact on non-target aquatic organisms in the water
column or sediments; they also determine the persistence of the
herbicide, which in turn would inuence the potential exposure
period (Wilson, 2006). Table 1 summarizes the characteristics of
few of the commonly used herbicides, especially comparing their
solubility.
Muller et al. (2008) state that pesticides are ubiquitous in
human impacted aquatic environments, and environmentally
realistic concentrations of a range of phytotoxicants have been
shown to impact aquatic ecosystems by exerting selective pressure
and altering phototrophic species assemblages. Especially the PSII
herbicides compete with plastoquinone for binding to the D1
protein (QB) binding site in PSII. Binding disrupts electron ow and
excitation energy is re-emitted as uorescence rather than driving
photochemical processes (Muller et al., 2008). Chl a uorescence
has been used for many years to study the effect of environmental
factors such as herbicides. Juneau et al. (2007) explained that with
the development of very sensitive uorometers, these methods
have become very useful in ecotoxicological studies. These authors
disregarded the use of maximal PSII quantum yield to evaluate
toxic effects of herbicides due to its relatively low sensitivity.
Furthermore, they recommended the use of operational PSII
quantum yield and non-photochemical quenching since these
latter parameters integrate the entire physiological state of the

plant and are more sensitive (Juneau et al., 2007). On the other
hand, triazine herbicides (atrazine and simazine), as well as diuron
are reported to signicantly increase the F0 and Fm signals, and this
response is associated with blocking of electron transport from
primary to secondary plastiquinone (QA to QB), resulting in an
increase in chl a uorescence. Since these herbicides block the
reoxidation of QA, absorbed energy cannot be used in photochemistry (Conrad et al., 1993).
As for the mode of action of herbicides and their effects on
algae, Figueredo et al. (2009) evaluated the effects of diuron on
phytoplankton using PAM uorometry. These authors stated that
the operational quantum yield (0 m) and the ETR were both
sensitive parameters for measuring the toxic effect of DCMU on
algae. However, Figueredo et al. (2009) also mention that the
potential quantum yield (m) could also be used but with lower
sensitivity. Eullaffroy and Vernet (2003) studied herbicide phytotoxicity in algae, and noted drastic increases in the magnitude of
F684/F735 chl a uorescence ratios in the presence of herbicides and
also reported phytotoxic effects on the electron transport wherein
the magnitude of the effect was as follows: diuron3,4dichloromethylphenylurea (DCPMU) 4metribuzin 4atrazine 4terbuthylazine 4paraquat 4 3,4-dichlorophenylurea (DCPU). In another
report, Magnusson et al. (2008) compared effects of PSIIinhibiting herbicides viz. diuron, hexazinone and atrazine, on
two tropical benthic microalgae Navicula sp. (Heterokontophyta)
and Nephroselmis pyriformis (Chlorophyta). These authors concluded that PAM uorometry is an ideally suited, rapid and reliable
technique to measure the sub-lethal impacts of PSII-inhibiting
herbicides on microalgae. In fact, Magnusson et al. (2010) carried
out investigations on the acute effects of the PSII-inhibiting
herbicides diuron, tebuthiuron, atrazine, simazine, and hexazinone, herbicide breakdown products [desethyl-atrazine (DEA) and
3,4-dichloroaniline (3,4-DCA)] and binary mixtures, using three
tropical benthic microalgae; Navicula sp. and Cylindrotheca closterium
(Ochrophyta) and N. pyriformis (Chlorophyta), and one standard
test species, Phaeodactylum tricornutum (Ochrophyta), in a highthroughput Maxi-Imaging PAM (Maxi-IPAM) bioassay; they report
all binary mixtures to exhibit additive toxicity. A real-time image
analysis of the chl a uorescence emission from a photosynthetic
organism provides substantial information regarding the dynamics of
photosynthetic changes indicating a progressive decline in the rate of
photosynthesis when the organism is exposed to xenobiotics. A realtime image exemplifying the extent of damage caused by herbicides
such as diuron, atrazine, simazine and hexazinone on macroalgae
Sargassum hemiphyllum and Undaria pinnatida (1 h exposure visualized with Maxi-Imaging PAM using a 96-well plate) along with its
respective declining F/Fm have been projected in Fig. 2 (b and c);
here, the EC50s for S. hemiphyllum exposed to diruon, atrazine,
simazine and hexazinone were 10.2, 94.6, 226.7 and 65 g L 1
respectivley, while the same for U. pinnatida were 37.2, 119, 194.8
and 57.7 g L 1 respectivley (unpublished data).
For PSII herbicides, studies have demonstrated that lower EC50s
are usually obtained when F/Fm is considered as the response
parameter, in contrast to the EC50s obtained through algal growth
assays (Schreiber et al., 2002; Macedo et al., 2008); this shows the
superiority of the chl uorescence technique. Juneau et al. (2007)
recommend the systematic determination of EC50 value to be
useful in this context. In addition, Seery et al. (2006) particularly
compared the germination of gametes of Hormosira banksii with
its chl a uorescence measurements, and concluded that the
uorescence bioassay was precise, time-saving, and highly sensitive i.e. the EC50s ( percent PSII Inhibition) for Diuron, Irgarol and
Bromacil were three, four and three orders of magnitude (respectively) lower than EC50s generated from the germination bioassays. A few commonly used herbicides (along with few biocides)
61
and their mode of action, as well as their algal toxicity visualized

using various uorescence parameters are detailed below.
4.1. Diuron
Diuron or DCMU {3-(3,4-dichlorophenyl)-1,1-dimethylurea}, a
phenylurea herbicide and a potent inhibitor of photosynthesis, has
been used in agriculture for more than 50 years to inhibit the
growth of a wide variety of annual and perennial weeds and
mosses. It is also used as an antifouling agent in paints (Kumar
et al., 2010). It induces phytotoxicity by catalyzing lethal photosensitized oxidation in cells. This may occur as a result of (i) a
greater concentration of oxidized chlorophyll caused by an interruption of the electron ow, and (ii) due to an inhibition of NADPH
formation which is necessary to maintain a functional carotenoid
protective mechanism (Kumar et al., 2010). 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 ow of electrons from water to NADP. In the presence
of diuron both the c-type and the b-type cytochromes can be
oxidized by PSI, but they cannot be reduced by PSII; this indicated
that diuron might be acting at a site between PSII and the
cytochromes (Kumar et al., 2010). Diuron binds to the site of the
exchangeable quinine (QB) site on the D1 protein, which includes
the P680 chlorophyll and the neighboring phaeophytin (Ph) that
forms the core of the reaction center. When bound, this herbicide
blocks forward electron transport beyond the 1-electron reduction
of the rst stable electron acceptor, the bound quinone QA (Kumar
et al., 2010). 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

dPh-radical pair
(Jones, 2004; Kumar et al., 2010). Conrad et al. (1993) have clearly
stated the signicance of measuring chl a uorescence yield to
assess the toxic effect of diuron.
Diuron is reported to affect F/Fm of phytoplankton at very low
(10 g L1) environmentally relevant concentrations (Devilla et al.,
2005). Diuron and 3-(3,4-dichlorophenyl)-1-methylurea (i.e. DCPMU)
are also reported to cause a signicant inhibition of algal photosynthesis (Eullaffroy and Vernet, 2003). Magnusson et al. (2008)
reported sub-lethal impacts of PSII-inhibiting herbicides on two
tropical benthic microalgae Navicula sp. (Heterokontophyta) and N.
pyriformis (Chlorophyta), stating the order of toxicity (EC50 range)
as: diuron (3.737.69 mg L 1)4hexazinone (6.3127.75 mg L 1)4
atrazine (28.04133.72 mg L 1) for both algal species. In another
approach, Endo and Omasa (2004) evaluated images generated by
a microscopic imaging system to diagnose the photosynthetic activity
of Spirogyra distenta at different growth stages after treatment with
diuron. Monitoring the increase in the chl a uorescence ratio
F684/F735, Schreiber et al. (2007) proposed a new phytotoxicity
bioassay based on chl uorescence imaging of algal suspensions in
multiwell plates and quantied inhibition of the PSII quantum yield,
Y(II), assessed with the saturation pulse method. They particularly
illustrated the response of P. tricornutum to diuron reporting
44 ng L 1 of diuron to cause phytotoxicity. Similarly, Muller et al.
(2008) developed a novel rapid exposure assay involving the marine
diatom P. tricornutum and the freshwater chlorophyte C. vulgaris PSII
herbicides; the technique was found to be sensitive, with a detection
limit of 2.3 ng L 1 for the herbicide diuron. Kristoffersen et al. (2012)
in their study on Haematococcus pluvialis created nutrient-stressed
conditions, by using two-photon excitation provided by laser generated pulses in the femtosecond range and a Leica microscope setup.
They demonstrated that DCMU-inhibited green-stage cells displayed a signicant increase in chl a lifetime for the short component, while the DCMU inhibition did not signicantly affect the
lifetime of red-stage cells. Legrand et al. (2006) stated that the
62
F/Fm of microphytobenthic communities decreased by 64 percent
in the presence of 10 mg L 1 diuron. Nonetheless, Choi et al. (2012)

particularly assessed 5 commonly found freshwater algal species
and the USEPA model species Pseudokirchneriella subcapitata, and
found that their maximum quantum yield of PSII (F/Fm) declined
rapidly over o20 min in response to low concentrations of the
photosynthesis-specic herbicide diuron. Furthermore, the maximum inhibition (78.4 percent) of F/Fm was observed at a Km value
of 32.63 mg L 1 for P. subcapitata (Korshikov) Hindak (CPCC 37, syn.
UTEX 1648) after 80 min exposure (Choi et al., 2012). On the other
hand, diuron (0.21 mg L 1) was reported to cause 50 percent
inhibition of the uorescence yield of PSII in Chlorella fusca (Conrad
et al., 1993). Knauert et al. (2008) reported that the 0.98 mg L 1 of
diuron inhibited 47.7 percent of PSII quantum yield of freshwater
phytoplankton. Besides diuron (5 mg L 1) signicantly reduced F/Fm
of Elodea canadensis to 57 percent and 80 percent on day 2 and 5,
while it reduced F/Fm of Potamogeton lucens by 55 percent on day 5
(Knauert et al., 2010). Fai et al. (2007) reported EC50 value of growth
inhibition in S. capricornutum to be 10 mg L 1 for diuron, whereas
EC50 values of F/Fm, F/Fm0 and NPQ were 7, 8.6 and 12.3 mg L 1
respectively. Kottuparambil et al. (2013) summarized marine
microalgae data set for diuron wherein they mention inhibiton of
F/Fm0 i.e. Quantum efciency under ambient light conditions.
Rodriguez-Mozaz et al., (2012) evaluated combined effects of diuron
and temperature on changes in photosynthetic efciency (Yeff),
photosynthetic capacity (Ymax), photochemical quenching (qP) and
non-photochemical quenching (NPQ) using a Diving PAM uorometer and reported that most of these parameters of Scenedesmus
vacuolatus were affected, except the non-photochemical
quenching (NPQ) mechanisms, which was inhibited only after 12 h
of exposure, suggesting damage in the pigments where the NPQ
took place (Rodriguez-Mozaz et al., 2012). Cao et al. (2013) reported
2 mM DCMU to cause complete restrainment of the NPQ of
Nannochloropsis sp.
Numerous reports elaborate the inhibitory values of chl a
uorescence parameters of various herbicide exposed algae, however, a few of them have been discussed herein. According to the
APVMA (2005) the EC10s for the effective quantum yield [Y(II)] of
Navicula sp. and N. pyriformis were 1.0 or 1.1 mg L 1 respectivley,
while the EC50s for the same were 5.5 and 5.9 mg L 1, respectively.
Nevertheless, the 24 h EC10 of F/Fm for diuron exposed Desmodesmus subspicatus and P. tricornutum has been reported to be
1.81 mg L 1 and 0.74 mg L 1, respectively (see Escher et al., 2006).
Magnusson et al. (2010) reported the EC50 values of Y(II) of diuron
exposed Navicula sp., N. pyriformis (Chlorophyta), P. tricornutum
(Ochrophyta), and C. closterium (Ochrophyta) were 2.6, 2.06, 2.71
and 4.4 mg L 1, respectively. However, the same authors earlier
reported the EC50 of effective quantum yield F/Fm of Navicula sp.
(Heterokontophyta) and N. pyriformis (Chlorophyta) were 5.5 and
5.9 mg L 1, respectively (Magnusson et al., 2008).
There are several other reports that project EC50 values for the
F/Fm of DCMU exposed algae, for e.g., in Pseudokirchnirella subcaptita, P. tricornutum, C. vulgaris, and Seriatopora hystrix (0.0024,
0.018, 0.020 and 0.0023 mg L 1 respectively; see Kumar and Han,
2011), and Chara vulgaris (16.8 ng L 1; Lambert et al., 2006).
According to EPA (2000) the 24 h EC50 for ecotoxicity of the DCMU
herbicide based on the growth of P. tricornutum is 10 mg L 1, while
a 24 h EC50 of F/Fm was 3.0 mg L 1 (Schreiber et al., 2002). Fai et al.
(2007) stated that the EC50 of growth rate of S. capricornutum was
10.5 mg L 1 after 72 h; they also stated that the EC50 of maximal
PSII quantum yield (m) and operational quantum yield (m) of
S. capricornutum measured after 1 h was 7 mg L 1 and 8.6 mg L 1,
respectively; this emphasized on the precision and time-saving
virtues of the chlorophyll uorescence technique.
Amongst the reports on macroalgae, severe damages to PSII
caused by increasing concentrations of diuron evident by the steep
decline in F/Fm values of Saccharina japonica, are reported by

Kumar et al. (2010). They obtained an EC50 of 0.0109 mg L 1 for
optimal quantum yield of S. japonica, but no electron transport was
evidenced at 0.4 mg L 1. Reis et al. (2011) observed a reduction in
the YPSII values as well as the ETRmax of Kappaphycus alvarezii
chilling stress strongly limits the electron ow through thylakoids
of K. alvarezii. This possibly occurred due to a rigidifying effect on
thylakoid membrane which caused a slower mobility of PQ
molecules. DCMU (1.65 mg L 1) also caused 50 percent inhibition
of the effective quantum yield (F/Fm) of H. banksii, while the
germination was less sensitive to DCMU (inhibited by
4650 mg L 1), thus proving that this uorescence parameter was
more sensitive than the germination end point (Seery et al., 2006).
Interestingly, several reports demarcate the adverse impacts of
diuron on the algae isolated from corals; two such reports
demonstrating the effect of diuron on these photosynthetic
symbionts of corals are presented here. Jones et al. (2003)
reported 450 percent reduction in photochemical efciency of
algae isolated from a coral Stylophora pistillata within 30 s of
exposure to a 12 mg L 1 diuron solution. On the other hand,
Owen et al. (2003) reported that algal symbionts isolated from
Madracis mirabilis, Diploria strigosa and Favia fragum exposed to
diuron (6 h; 10 mg L 1) showed a 2050 percent reduction in the
net 14C (H14CO3 ) incorporation.
In addition, diuron is also reported to inhibit corals as well as
seagrasses; for e.g., 1 mg L 1 diuron is reported to cause 1525
percent reduction in the effective quantum yield of photosynthesis
of Acropora millepora and Pocillopora damicornis, while 10 mg L 1
caused a 7585 percent reduction (Negri et al., 2005). The F/Fm
of the corals Acropora formosa, Porites cylindrical and Montipora
digitata were inhibited by 5.1, 4.3 and 5.9 mg L 1 diuron, respectively (10 h, 23.31 mg L 1) (APVMA, 2005). Diuron (100 mg L 1,
5 h) caused 55% decline in the operational PSII quantum yield of
the seagrass Halophila ovalis and it also decreased the maximal
PSII quantum yield to a lesser extent (Juneau et al., 2007).
Chesworth et al. (2004) found that the F/Fm values of 10 day
diuron exposed Zostera marina EC50 was 3.2 mg L 1.
4.2. Atrazine
Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) is
a selective systemic s-triazine herbicide used primarily to control
broadleaf plants and grassy weeds. It inhibits the rate of photosynthesis (especially PSII), chlorophyll content, and cell numbers,
therby causing damage to the micro- and macroora of rivers, lakes,
estuaries and marine ecosystems (Brard and Pelte, 1999; Plumley
and Davis, 1980). Prolonged exposure to atrazine not only inhibits
photosynthesis but also leads to slow starvation in plants or a more
rapid response that is believed to be due to the production of
secondary toxic substances (for e.g., reactive oxygen species)
(Shimabukuro et al., 1976). Atrazine being algistatic inhibits photosynthesis by blocking the electron transport during the Hill reaction
of PSII. However, algal responses to atrazine vary widely depending
on the concentrations used, the duration of exposure, and the algal
species tested. Numerous researchers have either examined or
summarized the effects of atrazine on algae and phytoplankton
(Huber, 1993; Solomon et al., 1996; DeLorenzo et al., 2001).
Encapsulating a few reports, it could be stated that atrazine
also inhibits the photosynthesis of: S. vacuolatus, Nitzschia palea,
Fragilaria var. ulna, green algae, and diatoms (whose effective
quantum yield or PSII showed 50 percent inhibition when
treated with 0.03, 0.1, 0.065, 0.043 and 0.095 mg L 1 atrazine
respectively for 1 h; Schmitt-Jansen and Altenburger, 2007), E.
canadensis and P. lucens (where F/Fm were inhibited 62 percent
and 33 percent, respectively, in the presence of 70 mg L 1 atrazine
after ve days; Knauert et al., 2010), E. canadensis (50 percent
inhibition F/Fm was reached at 75 mg L 1; Vervliet-Scheebaum

et al., 2008), and Chara canescens (50 percent inhibition of F/Fm
occurred in the presence of 0.27 mg L 1 after 60 min; Kster and
Altenburger, 2007). Knauert et al. (2008) demonstrated that
1.09 mg L 1 atrazine inhibited 45.6 percent of the PSII quantum
yield of freshwater phytoplankton. Furthermore, Legrand et al.
(2006) observed 60 percent reduction in the F/Fm of microphytobenthic communities in the presence of 100 mg L 1 atrazine; while, Vallotton et al. (2008) observed 45 percent and 70
percent inhibition of S. vacuolatus F/Fm in the presence of
125 mg L 1 and 520 mg L 1 atrazine, respectively, after 30 min of
exposure. However, after 24 h, the inhibition values were only 51
percent at 125 mg L 1 and 63 percent at 520 mg L 1. High atrazine
concentrations (2.16 mg L 1; 7 day exposure) reduced the rate of
photosynthesis and cell number of Nitzschia sigma and Thalassiosira uviatilis signicantly (Plumley and Davis, 1980). Atrazine
(2.16 mg L 1; 5 days) is also reported to reduce the primary
productivity (from 191.4 to 29 mg C m 2 h 1) of microecosystems (Plumley and Davis, 1980). On the other hand, Choi et al.
(2012) reported that in the sensitive species Synura petersenii low
concentrations of atrazine ( o 2.16 mg L 1) affected F/Fm. They
further reported that the threshold concentration of atrazine
( o 0.02 mg L 1) required for detection of changes in F/Fm in the
three most sensitive species for C. reinhardtii Dang, Navicula
pelliculosa (Breb.) Hilse and P. subcapitata (Korshikov) Hindak
was similar to (EC50) concentrations for effects on chl a uorescence parameters in P. subcapitata (0.070.08 mg mL 1). Atrazine
also inhibits the F/Fm of freshwater periphytic algae (Dorigo and
Leboulanger, 2001).
Reports elucidating the EC50s of various photosynthetic parameters of atrazine-exposed algae are available, these include:
P. subcapitata, Anabaena os-aquae, and N. pelliculosa (EC50s for
effective quantum yield F/Fm were 59.5, 87 and 123 mg L 1,
respectively, after 48 h; Brain et al., 2012); Navicula sp. (Heterokontophyta) and N. pyriformis (Chlorophyta) (EC50s for effective
quantum yield PSII were 99 mg L 1 and 28 mg L 1, respectively;
Magnusson et al., 2008); A. falcatus, Pandorina morum, Pediastrum
boryanum, Chlamydomonas snowii, Aulacoseira granulata var. angustissima, Fragilaria crotonensis, Phormidium muscicola, Microcystis
osaquae, Aphanizomenon os-aquae and Anabaena spirodes (EC50
values of the F/Fm were 0.02, 0.04, 0.01, 0.03, 0.09, 0.43, 0.003,
0.007, 0.01 and 0.006 mg L 1, respectively; Deblois et al., 2013);
Pseudokirchneriella subcaptita, P. tricornutum, C. vulgaris, and
S. hystrix (EC50s of F/Fm were 0.009, 0.045, 0.103 and 0.045
mg L 1, respectively (see Kumar and Han, 2011)); Skeletonema
costatum (EC50 of F/Fm was 0.050 mg L 1; Walsh et al., 1988);
Chlorella (EC50 was 0.71 mg L 1 for atrazine inhibited uorescence
yield of PSII; Conrad et al., 1993); S. capricornutum [EC50 of
maximal PSII quantum yield (m) and operational quantum
yield (0 m); 1 h 77 mg L 1, and 71.7 mg L 1; Fai et al., 2007]; as
well as, Navicula sp., N. pyriformis (Chlorophyta) P. tricornutum
(Ochrophyta) and C. closterium (Ochrophyta) (EC50s of Y(II) were
36, 14.2, 33.6 and 76.7 mg L 1 respectivley, Magnusson et al., 2010).
In addition, Kumar and Han (2011) cited reports showing 50
percent inhibition of photosynthesis in C. pyrenoidosa, Chlorella
sp., Scenedesmus sp., and Anabaena inaequalis exposed to 0.139, 0.5,
1, 1, 0.3 mg L 1 of atrazine, respectively. Fai et al. (2007) reported
EC50 of growth inhibition in atrazine exposed S. capricornutum to be
81.4 mg L 1, whereas EC50s of F/Fm, F/Fm0 and NPQ were 77, 71.7
and 73.9 mg L 1 respectively.
4.3. Simazine
Triazine compounds are inhibitors of non-cyclic ETR in
photosynthesis and cause chlorosis and necrosis in susceptible
plant tissues (Oneal and Lembi, 1983). Simazine (6-chloro-N,
63
N-diethyl-1,3,5-triazine-2,4-diamine), rst introduced in 1956 by

the Swiss company J. R. Geigy (Cremlyn, 1990), is a part of the
triazine family (six-member ring containing three carbon and
nitrogen atoms) and is also used to control germinating annual
grasses and broad-leaved weeds. Simazine binds to the plastiquinone B (QB) protein binding site on the D1 protein of PSII complex
in chloroplast thylakoid membranes; this prevents the transfer of
electrons from plastiquinone A (QA) to QB (Ahrens 1994). In this
manner, it interrupts photosynthetic electron transport, CO2
xation and the production of ATP and NADPH2 (Ahrens, 1994).
Simazine has also been popularly used in aquarium algicides for
effectively controlling unicellular and attached lamentous algae at
0.51.0 mg L 1 concentration. Studies on lakes and ponds suggest
that simazine is most effective against blue-green algae, moderately effective against green algae, while diatoms and agellates
are least sensitive (Jellyman et al., 2006). Nevertheless, though this
herbicide nds its way to the marine ecosystems, its impact on the
photosynthesis of marine ora has been sporadically discussed.
Simazine is particularly of aquatic-use having devastating impact
on algae, for e.g., 1.01 mg L 1 inhibits chlorophyll production in the
cyanobacterium Oscillatoria lutea (74 percent) and the green alga C.
vulgaris (42 percent) (see Oneal and Lembi, 1983). Moreover,
photosynthesis of Ankistrodesmus braunii a non-lamentous species is also reported to be inhibited by 0.95 mg L 1 simazine
(Oneal and Lembi, 1983). Turbak et al. (1986) observed 50 percent
inhibition of S. capricornutum photosynthesis due to exposure to
simazine (0.02 mg L 1). Besides, simazine (0.8 mg L 1) is reported
to signicantly reduce the F/Fm of Protosiphon botryoides from 0.77
to 0.57 (Kobbia et al., 2001).
Studies on various algae substantiate the appropriateness of
investigating chl a uorescence as a tool for examining the
impact of simazine on algae. The EC50 values of several simazine
exposed algae have been reported as follows: P. subcapitata, P.
tricornutum, C. vulgaris, and S. hystrix (EC50 of F/Fm were 0.15
1.24, 0.4, 0.076 and 0.15 mg L 1, respectively, see Kumar and Han,
2011); S. capricornutum (EC50 values were 1.41 mg L 1 for chl a
uorescence; Bozeman et al., 1989); Chlorella (EC50 for uorescence yield of PSII was 0.44 mg L 1; Conrad et al., 1993); S.
costatum (EC50 of F/Fm was 0.01 mg L 1, Walsh et al., 1988); as
well as, Navicula sp., N. pyriformis (Chlorophyta), P. tricornutum
(Ochrophyta) and C. closterium (Ochrophyta) [EC50 of Y(II) were
157, 24.4, 101 and 242 mg L 1, respectively, Magnusson et al.,
2010]. Particularly, Bonilla et al. (1998) observed EC50s for shortterm inhibition of photosynthesis in phytoplankton (0.07
0.20 mg L 1), periphyton (0.090.24 mg L 1) and epipsammon
(0.220.57 mg L 1) communities exposed to simazine, while
Goldsborough and Robinson (1988) reported EC50 of simazine
exposed periphyton photosynthesis as 0.8 mg L 1. Additionally,
simazine also inuences the photosystems of coral symbionts
(EC50 value of F/Fm of coral 0.150 mg L 1, see Kumar and Han,
2011). It also inuences O2 evolution in marine unicellular algae,
i.e. Chlorococcum sp., D. tertiolecta, I. galbana and P. tricornutum
(EC50 after 90 min were 2.5, 4.0, 0.60 and 0.60 mg L 1 respectively) (Walsh, 1972).
4.4. Hexazinone
Hexazinone (rst developed in 1968 and registered as a noncropland herbicide in 1975), controls a broad range of annual,
biennial and perennial herbaceous weeds, vines and woody plants.
It is used during periods of active growth for herbaceous weed
control, site preparation and hard-wood control (Sung et al., 1985).
Hexazinone is a systemic herbicide that inhibits photosynthesis in
susceptible plants, diverting highly reactive molecules into a chain
reaction that destroys chloroplasts and cell membranes and
other vital compounds (Tu et al., 2001). Apart from inhibiting
64
photosynthesis, hexazinone inhibits the synthesis of RNA, proteins, and lipids in plants at higher levels of exposure. It is usually
applied as a pre-emergent herbicide. However, in order to activate
hexazinone, it is essential for the soil to be moist (by rain or
irrigation). It works by binding to a protein component of the PSII
complex, which blocks the electron transport, thereby resulting in
a chain reaction wherein the triplet-state chlorophyll reacts with
oxygen (O2) to form singlet oxygen (O), and both the chlorophyll
and singlet oxygen strips hydrogen ions (H ) from unsaturated
lipids in cell and organelle membranes, producing lipid radicals
(Tu et al., 2001). These lipid radicals in turn attack and oxidize the
other lipids and proteins, resulting in the loss of cell and organelle
membrane integrity, loss of chlorophyll and carotenoids, leakage
of cellular contents, cell death, and ultimately the death of the
plant (WSSA, 1994).
There could be plausible and substantial damage caused by the
runoff of hexazinone after its application in terrestrial areas. This
potential toxicant could, thereafter, cause inhibition of photosynthesis to various aquatic plants as well as algae (Durkin et al.,
2005). High concentrations of hexazinone, could lead to signicant
losses of algae and macrophytic biomass, which in turn enters the
food chain that ultimately could impact sh and wildlife species
(GIST, 2013). Though several reports suggest a substantial inhibition of net photosynthetic activity by hexazinone, these reports
are either based on ow-through tests, chl a-specic productivity,
or involve measurements of chlorophyll content, algal biomass or
physiological activity measured using 14C uptake (Kreutzweiser
et al., 1995; Durkin et al., 2005).
Nevertheless, Jones and Kerswell (2003) obtained an EC50 of
8.8 mg L 1 for the F/Fm0 of S. hystrix during a short-term (10 h)
exposure to hexazinone. Magnusson et al. (2008) reported EC50 of
effective quantum yield (PSII) of Navicula sp. (Heterokontophyta)
to be 16 mg L 1 and N. pyriformis (Chlorophyta) was 6.2 mg L 1. In
yet another study, these authors reported EC50-values of Y(II) of
Navicula sp., N. pyriformis (Chlorophyta), P. tricornutum (Ochrophyta) and C. closterium (Ochrophyta) exposed to hexazinone as
5.7, 2.4, 6.6, and 6.9 mg L 1, respectively (Magnusson et al., 2010).
Magnusson (2009) validated the utility of PAM uorometry as a
rapid and reliable technique to measure sub-lethal toxicity of PSIIinhibitors in microalgae (N. pyriformis and P. tricornutum), and
elucidated the order of toxicity of herbicides based on their (EC50s)
as: diuron (3.77.69 mg L 1) 4hexazinone (6.3127.75 mg L 1)
4atrazine (28.04133.72 mg L 1) for both algal species.
4.5. Glyphosate [N-(phosphonomethyl) glycine]
Glyphosate found its application as an herbicide in 1970. It is
anionic at physiological pH levels. It is active as a salt with various
cations, for e.g. sodium or isopropylamine salts (Duke and Powles,
2008). Amrhein et al. (1980) suggested that the phosphanoglycine
herbicide glyphosate inhibited the aromatic amino acid biosynthesis at the site of chorismate mutase or prephenate dehydratase;
this clearly indicated that glyphosate does not interfere with the Zscheme of photosynthesis. Though photosynthesis is not regarded
to be a primary inhibitory target of glyphosate, reports state that
this toxicant denitely inuences the process of photosynthesis
(Yanniccari et al., 2012). For e.g., Olesen and Cedergreen (2010)
suggest that glyphosate affects the photosynthetic electron transport indirectly by inhibiting sink processes. According to Choi et al.
(2012), the rapid and dramatic inhibitory effect of glyphosate on F/
Fm may involve the disruption of integrated carbon and nitrogen
assimilation which serves as a sink for photochemically derived
energy. The enzyme, 5-enolpyruvylshikimate-3-phosphate synthase
(EPSPS), a key enzyme belonging to the shikimate pathway, is
responsible for the biosynthesis of the essential aromatic amino
acids, phenylalanine, tryptophan and tyrosine. Glyphosate, the only
commercial inhibitor of EPSPS, is currently the most used herbicide

for this particular application (Duke and Powles, 2008).
Glyphosate has been reported to inhibit the photosynthetic
electron transport (Hill reaction) and O2 evolution in both wild
type and mutant cells of Anabaena doliolum at high concentrations
(200400 mg mL 1); here, chl a uorescence studies of both
strains unveiled quenching of uorescence emission at lower
doses (20100 mg mL 1) and an increase in uorescence emission
at higher concentrations of glyphosate (100200 mg mL 1)
(Shikha and Singh, 2004). Choi et al. (2012) assessed the utility
of F/Fm with 5 commonly found freshwater algal species and
P. subcapitata (the USEPA model species), and revealed that the
F/Fm responded rapidly and in a dose-dependent way to the toxin
glyphosate (o15.22 mg L 1) which has a mode of action not
specic to photosynthesis (and particularly does not directly target
the PSII). Moreover, the percent suppression of F/Fm by glyphosate
increased exponentially with concentration. In that study, involving the effect of diverse toxic water pollutants (including glyphosate) on chl a uorescence of freshwater microalgae, Choi et al.
(2012) mentioned that the rapid suppression of F/Fm offers
considerable time-saving advantages (over traditional 4 day
growth bioassays). It allowed a larger number of algal species,
toxins and toxin concentrations to be tested in the laboratory with
fewer resources. These authors evidenced a rapid suppression of
F/Fm occurring in response to glyphosate, thereby suggesting that
these changes could reect the entire cell physiological stress
(Choi et al., 2012). In Neosiphonia savatieri, an increase in minimal
uorescence intensity (F0) and decrease in the relative variable
uorescence intensity at the J step, VJ (FJ F0)/(Fm F0) have been
reported with increasing concentrations of glyphosate and incubation time. This might be due to the fact that glyphosate probably
affects the membrane organization (phycobilisome attachment or
internal antenna of PSII) (Pang et al., 2012). Here, the maximum
PSII photochemical efciency F/Fm (Fm F0)/Fm for dark-adapted
N. savatieri decreased signicantly from 0.65 to 0.24 after 5 min
exposure of 4 mg L 1 glyphosate, this signicant reduction in
F/Fm illustrates that the energy absorbed by N. savatieri was not
efciently used for the electron transport but dissipated to heat
(Pang et al., 2012). Sheehan et al. (1998) conducted comparative
studies on nine algae from three different classes: the chlorophyceae (Micromelum minutum MONOR1 and MONOR2), the eustigmatophyceae (Nannochloropsis (NANNP1 and NANNP2), and the
bacilliarophyceae (Cyclotella cryptica T13L, Callitrismulleri CHAET9,
Amphora AMPHO17, Nitzschia pusilla NITSC12, and Navicula saprophila
NAVIC1). They revealed that all the strains were sensitive to the
herbicide glyphosate (RoundUp), which affects the shikimic acid
pathway. Studies on effect of glyphosate on alginate immobilized and
free Selanastrum species reveal that the percentage inhibition of the
relative uorescence increased in a curvilinear fashion with increasing
glyphosate concentration with the median inhibitory concentration
(IC50) being 26.3 mg L 1 for immobilized cells while that of the free
cells was 7.8 mg L 1 (Bozeman et al., 1989). In another context, Van
Rensen (1974) observed a 50 percent reduction in oxygen evolution
by a culture of the green alga Scenedesmus sp. after 6090 min
exposure to 118 mg L 1) glyphosate.
Nonetheless, certain investigators reveal the utility of PAM uorometry in a different manner. Dam and Wijtze (2012) revealed how
dissolved herbicides and elevated sea surface temperatures combinedly disrupt the fragile symbiotic relationship of coral or foraminiferan species with their autotrophic micro-algal endosymbionts.
5. Antifouling biocides
Apart from the toxic effects of herbicides, there are a number of
biocides that are incorporated into a variety of substances; for e.g.
certain booster biocides are introduced to antifouling (AF) paints

to improve their efcacy against these photosynthetic organisms
(Voulvoulis et al., 1999). However, they have an adverse impact on
algae (especially macroalgae) and few of them are either algistatic
or algicidal. Many different booster biocides that have been added
to AF paints include: chlorothalonil, dichlouanid, Irgarol 1051s,
TCMS pyridine, thiocyanatomethylthio-benzothiazole (TCMTB),
diuron, dichloro-octyl-isothiazolin (DCOIT, Sea Nine 211s), zinc
and copper pyrithione (Zinc and Copper Omadines), and zineb
(Dafforn, 2009; Dafforn et al., 2011). A few of the most widely used
and studied booster biocides are discussed here.
5.1. Irgarol 1051
Irgarol 1051 (2-methylthio-4-tert-butylamino-6-cyclopropylamino-s-triazine) is a relatively new triazine herbicide (inhibitor of
photosynthesis) that has found its application as an algicide in
antifouling paints for boats and vessels. It is typically effective
against freshwater and seawater algae, and less against animals
(Albanis et al., 2002). This compound was introduced as a
replacement for organotonins in antifouling paints (Hall et al.,
2005) with detrimental impacts on the whole aquatic ecosystem.
Irgarol 1051 contamination has been detected in many estuaries,
marinas, harbors, and coastal areas (Hall et al., 1999). Irgarol has
been described as a most potent PSII inhibitor of algal photosynthesis. It displaces the QB quinone and prevents the electron
transfer, and this mode of action results in oxidative stress,
including photooxidation of chlorophyll and cell necrosis (Hall
and Gardinali, 2004). Additionally, its biochemical mode of action
against algal growth is reported to occur via the inhibition of the
photosynthetic electron transport in chloroplasts (Hall et al., 1999).
Irgarol 1051 affects the F/Fm of phytoplankton even at very low
(0.03 g L1) environmentally relevant concentrations (Devilla
et al., 2005). According to Dahl and Blanck (1996), Irgarol 1051
causes inhibition of periphyton photosynthesis (EC50 0.82 nM); in
addition it was approximately 70 times more toxic to algal
communities as compared to atrazine. Brard et al. (2003) too
conrm higher toxicity of Irgarol (as compared to atrazine) by
performing microplate and short-term uorescence tests on periphyton and phytoplankton. Nystrm et al. (2002) studied chl a
specic uorescence in phytoplankton and algae, thereby, demonstrating short-term toxicity of Irgarol 1051 to Potamogeton pectinatus. These authors particularly indicated low F/Fm values in the
presence of high concentrations of this biocide.
According to the US EPA (2000), the freshwater species bacillariophyte N. pelliculosa has been identied as one of the most
sensitive alga with a 5 d EC50 of 0.1 mg L 1. Buma et al. (2009)
stated that the 72 h EC50 of F/Fm0 of T. weissogii, Emiliania
huxleyi, Tetraselmis sp. and Fibrocapsa japonica was 0.327, 0.604,
0.230 and 0.110 mg L 1, respectively, but the 72 h EC50 of the
growth rate was 0.303, 0.406, 0.116 and 0.618, respectively. Here,
it is necessary to note that both the photosynthetic measurements
and the growth rate showed a similar sensitivity. Guardiola et al.
(2012) summarized the inhibition of the algal photosynthetic
activity (D. tertiolecta, Synechococcus sp., E. huxleyi, Fucus vesiculosus, Enteromorpha intestinalis, and U. intestinalis) and seagrass
(Z. marina) by Irgarol-1051 (which inhibits the PSII electron
transport). Moreover, Owen et al. (2003) reported a 4050 percent
reduction of net 14C (H14CO3 ) incorporation in algal symbionts
isolated from M. mirabilis, D. strigosa and F. fragum exposed to
10 mg L 1 Irgarol 1051 for 6 h. Irgarol 1051 concentration up to
0.23 mg L 1 negatively impacted the photosynthetic activity of the
green alga U. intestinalis (Tolhurst et al., 2007). In addition,
0.17 mg L 1 Irgarol 1051 is reported to cause 50 percent inhibition
of the F/Fm of H. banksii (Seery et al., 2006), while the 72 h EC50
of the F/Fm of E. intestinalis was 2500 ng L 1 (Scarlett et al., 1997).
65
Kottuparambil et al. (2013) summarize the effect of irgarol on the

F0 /Fm0 of T. weissogii, E. huxleyi, Tetraselmis sp. and F. japonica
thereby reporting EC50s of 0.33, 0.60, 0.23, 0.11 mg L 1.
Nonetheless, there are other reports that emphasize the use of
chl uorescence in determining the impact of Irgarol on corals and
their photosynthetic symbionts, as well as seagrasses; for e.g.
Jones and Kerswell (2003) reported EC50 of F/Fm0 of the corals S.
hystrix and A. formosa 0.7 g L1 and 1.3 g L1, respectively.
Irgarol concentrations as low as 63 ng L 1 are reported to effect
the carbon uptake of inshore corals (M. mirabilis), while concentrations as low as 100 ng L 1 reduced the net photosynthesis of
intact corals after an 8 h exposure (Owen et al., 2002). As for
seagrasses, Chesworth et al. (2004) obtained an EC50 value of
1.1 mg L 1 for F/Fm of 10 d exposed Z. marina. Irgarol toxicity to
Zostera capricorni in both the laboratory and the eld was
evidenced by Macinnis-Ng and Ralph (2003), who observed
incomplete recovery of PSII quantum yields; however, after 1 h
exposure to 100 mg L 1 Irgarol, the F/Fm0 values in the laboratory
and eld dropped to 0 units.
5.2. Tributyltin chloride (TBT)
TBT was rst used in freshwater systems to eradicate molluscs
harboring the parasitic worm Schistosoma. However, since the
early 1960s TBT was introduced as a biocide in marine antifouling
paints (Lewis, 1998). Commercial antifouling formulations containing TBT are the major source of organotin contamination in
coastal waters. Most reports suggest the harmful impact of this
compound on several other parameters rather than photosynthesis; for e.g., it inhibits germination of spores of a fucoid alga
Phyllospora cosmosa (Burridge et al., 1995), spore attachment of
Ulva conglobata, chlorophyll contents of marine microalga Tetraselmis tetrathele, Nannochloropsis oculata and Dunaliella sp.
(Rumampuk et al., 2004), photosynthetic pigment content of the
marine eustigmatophyte N. oculata (Sidharthan et al., 2002), as
well as, growth rate, chlorophyll pigments, carbohydrate and
protein content of the marine microalga Tetraselmis (Yoo et al.,
2007). However, Sargian et al. (2005) observed changes in the
photochemical yield (F/Fm) of planktonic assemblages indicating a
signicant damage to the photosynthetic apparatus in both TBTadded treatments, with or without ultraviolet light (UV-B) exposure. They hypothesized that, the TBT added to the microcosms,
induced a negative feedback on the photosynthetic electron
transport and ultimately damaged PSII reaction centers of the
cells, and this was evident by reduction in the F/Fm values.
Furthermore, they explained that the lipophilic character of TBT
allowed this compound to penetrate the thylakoid membrane
where it can act as a proton carrier, inhibiting photophosphorylation (ATP synthesis). Moreover, a major ATPase site of TBT action is
the CF0 segment of the CF0CF1-ATPase complex. Hence, TBT may
affect the ATP synthesis by modifying the CF0 structure by the
subsequent generation of free radicals. Arrhenius et al. (2006)
observed that TBT-inhibited periphyton photosynthesis EC50
(0.02 mg L 1). Jensen et al. (2004) reported a 60 percent reduction
in the net photosynthetic activity of the seagrass Ruppia maritima
from tributyltin (TBT) spiked and impacted sediments.
5.3. Sea-Nine
Guardiola et al. (2012) mention the impact of Sea-Nine 211 (4,
5-dichloro-2-n-octyl-4-isothiazolin-3-one) or DCOIT as an inhibitor of PSII electron transport. On the other hand, based on F/Fm
measurements of natural phytoplankton communities, Devilla
et al. (2005) ranked the toxicity of few biocides as: Irgarol
1051s 4zinc pyrithione Sea-Nine 211s 4diuron. Thereby, these
authors suggest Sea-Nine to be more toxic than diruon.
66
Despite the fact that chl a uorescence is ubiquitous in ecophysiological studies, it is essential to note that the interpretation of data
remains a bit complex and at times controversial (Maxwell and
Johnson, 2000). However, Maxwell and Johnson (2000), realizing the
simplicity of measurements associated with chl a uorescence, quote
that analyzing the kinetics of uorescence rise remains controversial;
therefore, it would probably be best to avoid this in case of lack of
theoretical footing. Enrquez and Borowitzka (2010) mention about the
methodological problems and misconceptions in the interpretation chl
a uorescence signal. Figueroa et al. (2003) also state that it is essential
to carry out, an optimization of the PAM instrumentation to meet
accurately the low chorophyll uorescence emission of macroalgae;
for e.g. the presence of phycobilisomes in the light-harvesting system
of red algae results in generally lower maximal quantum yields (F/Fm)
than that measured in green and brown algae (Bchel and Wilhem,
1993). It is also essential to consider the right parameter; for instance,
Mallick and Mohn (2003) registered a signicant rise the nonphotochemical quenching (qN) of the metal-treated samples as compared to
its F/Fm, but they mention that this rise was not so distinct.
Exceptionally certain reports suggest some classical parameters to be
more sensitive than the uorescence parameters, for e.g., Ahmed and
Hder (2010) reported that the motility parameters show higher
sensitivity to Cd than chlorophyll uorescence parameters such as
F/Fm and ETRmax. Nevertheless, recent reports emphasize on the
utility of chl a uorescence as a tool for detecting environmental
pollutants such as metal and herbicides (Heimann, 2013).
In summary, it could be stated that chl a uorescence could be
considered as a convincing indicator of toxicity as it provides reliable
information regarding the effect of pollutants on the physiological
state of algae. Due to the ease of measurement and the rapid response,
chl a uorescence is particularly used to study toxicity of contaminants
dissolved in water. Although different chl a uorescence parameters
have been examined, most studies have focused on single species and/
or a narrow range of toxins. These parameters could be considered as
sensitive indicators of toxic metals, herbicides as well as antifouling
biocides. In-depth knowledge regarding the correlation between
growth and photosynthetic uorescence parameters facilitates the
validation of photosynthetic uorescence parameters as convenient
toxicity biomarkers. In other words, as certain parameters are good
indicators of environmental pollution, they could be easily be utilized
as endpoints in various ecotoxicity studies. However, the selection and
analysis of an appropriate uorescence parameter and its careful
calibration could provide a rapid, high-throughput evaluation of the
condition of any natural ecosystem. Nevertheless, it is very essential to
understand the principles underlying this technique for accurately
interpreting the measurements. As this technique is highly sensitive
and involves sophisticated instrumentation with high precision, it may
demand initial monitoring, followed by thorough handling skills and
proper protocols. It is also essential to compare the sensitivity of
various traditional end-points (such as growth measurements) with
various chl a uorescence parameters and, thereafter, consider its
applicability as a biomarker. Moreover, one should realize that algal
species vary in their sensitivity to toxins. Moreover, it is essential to
carry out further research on the applicability of chl a uorescence
measurement in the eld where more complex natural conditions
prevail.
6. Conclusions
Recording chl a uorescence has been well-established as a
versatile non-invasive tool for investigating differences in physiological processes occurring during the life cycle of a plant or alga
recently. It reveals a wide range of internal characteristics of the
plants or algae under natural conditions. Chl a uorescence
provides information on the efciency of energy conversion at
PSII reaction centers and hence it could be used as a sensitive

indicator for detecting damage to the photosynthetic apparatus.
Several studies have particularly emphasized the multifold applicability of chl uorescence techniques in evaluating the stress
physiology of algae (micro- as well as macroalgae). This technique
is useful in analyzing impacts of various environmental pollutants
such as toxic metals, herbicides, or their combinations. It allows
exceptionally rapid and accurate measurements of phytotoxicity as
compared to other established monitoring tools. It facilitates
indirect assessment of pollutant concentrations, and enables the
screening of large number of samples for the monitoring of abiotic
stress as well as toxicant damage in algae at early stages before the
damage symptoms appear visually. In terms of man-power,
materials, equipment and laboratory amenities, chl a uorescence
is rather inexpensive and less time consuming as compared to
chemical analytical techniques and algal growth assays. Various
advancements (such as imaging instrumentation and softwares)
and improvizations (such as portability) have facilitated the use of
chl a uorescence as a monitoring tool for environmental stress at
a global scale. Using this technique, large number of algal species,
toxins and toxin concentrations could be simultaneously and
rapidly tested in the laboratory with ease and few resources.
However, it is essential to understand the principles underlying
this technique for accurately interpreting the measurements. This
review discusses algal chl a uorescence as a powerful tool in
evaluating the photosynthetic responses of algae exposed to toxic
pollutants, and puts forward its aptness as a bioindicator in
monitoring environmental toxicants.
Acknowledgments
We thank Prof. Martin Podhurst (Sangmyung University, Republic of Korea) for language correction. The current research was
nancially supported by the National Research Foundation (NRF2012R1A2A2A02012617) and by a grant from Marine Biotechnology
Program funded by Ministry of Land, Transport and Maritime Affairs
of Korean Government. This study was approved by National
Fisheries Research and Development Institute (RP-2013-ME-080).
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Ecotoxicology and Environmental Safety 104 (2014) 5171

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

Algal photosynthetic responses to toxic metals and herbicides assessed

Chlorophyll a uorescence is established as a rapid, non-intrusive technique to monitor photosynthetic

Corresponding author. Fax: 82 31 416 6173.

address photosynthetic responses of plants and algae in the

ToxY-PAM uorometer are known (http://www.walz.com/products/

2. Parameters of chl a uorescence

donor, and QA, the primary quinone acceptor of the reaction

nutrient status and temperature (Wozniak et al., 2002). However,

Bliger and Bjrkman (1990):

3. Trace and toxic metals

electron transport chain and also inhibit the reductive pentose

lifetime imaging (TPE-FLIM) systems in uncovering adverse effects

several sites, showing a stronger inhibitory effect on photosynthetic

3.2. Copper (Cu)

per excited cross section. Moreover, 0.1 mg Cu2 L 1 is known to

of the back reaction, QA -P680

uorometry, and revealed the sequence of sensitivity of these toxicity

Molecular Formula C9H10Cl2N2O

Green et al (2000); http://www.apvma.gov.

Arnold et al. 1997; see Laughlin et al. (1986)

IUCLID (2000); http://ec.europa.eu/food/

a signicant increase in the electron transfer rates (ETR) and

latter parameters integrate the entire physiological state of the

and their mode of action, as well as their algal toxicity visualized

PSII occurs, resulting in the formation of the P680

F/Fm of microphytobenthic communities decreased by 64 percent

in the presence of 10 mg L 1 diuron. Nonetheless, Choi et al. (2012)

decline in F/Fm values of Saccharina japonica, are reported by

inhibition F/Fm was reached at 75 mg L 1; Vervliet-Scheebaum

N-diethyl-1,3,5-triazine-2,4-diamine), rst introduced in 1956 by

commercial inhibitor of EPSPS, is currently the most used herbicide

certain booster biocides are introduced to antifouling (AF) paints

Kottuparambil et al. (2013) summarize the effect of irgarol on the

PSII reaction centers and hence it could be used as a sensitive

throughput phytotoxicity assay (Maxi-Imaging-PAM). J. Environ. Monitor. 8,

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