Available online at www.ehs.or.

kr

Chlorophyll Fluorescence Based Copper Toxicity Assessment

of Two Algal Species
K. Suresh Kumar1, Young-Seok Han1,
Kyung-Sil Choo1, Jeong-Ae Kong1
& Taejun Han2
1

Institute of Green Environmental Research,

216 Business Incubator Center, Incheon National University,
177 Dohwa-dong, Nam-gu, Incheon 402-749, Korea
2
Department of Biology, Incheon National University,
Incheon 402-749, Korea
Correspondence and requests for materials should be addressed
to T. Han ([email protected])
Accepted 23 October 2008

Abstract
Pulse Amplitude Modulated (PAM) fluorometry is a
sensitive and rapid method used to assess toxic effect of pollutants in plants as well as algae. This study evaluates the difference in sensitivity of two marine macroalgae, Ulva pertusa and Ecklonia cava, to
copper. The photosynthetic efficiency of the algae
was measured as the ratio of variable to maximal
chlorophyll fluorescence (Fv /Fm) as well as maximum
electron transport rate (ETRmax). The algae were exposed to 0.125-1 mg/L of copper and their physiology was studied every 24 h for a period of 4 days. Increase in copper concentration caused proportional
decrease in the photosynthetic efficiency, particularly ETRmax, of both the species studied. Moreover, U.
pertusa proved to be more sensitive to copper than
E. cava, thus implying its use as a toxicity biotest
battery.
Keywords: Chlorophyll a fluorescence, Copper, Ecklonia
cava, Ulva pertusa, ETRmax, Fv /Fm

Introduction
Copper is considered one of the most toxic heavy
metal ions to algae and plants and is a potent inhibitor of photosynthesis1. Gledhill et al. realized the
significance of regulatory and biological perspectives
of bioavailable copper in seawater and copper speciation, and discussed its deleterious effects on marine
organisms, particularly macroalgae1. In marine algae,

copper is essential for metabolic processes like electron transport in photosynthesis and in various enzyme systems (e.g. amine oxidase, cytochrome c oxidase). However, excess copper results in toxic responses, including subtle changes in enzymatic activity to gross changes in cell structure and function
and inhibits photosynthesis2. The type and extent of
the responses of marine macroalgae to copper vary
according to the species under consideration.
Copper in seawater, mainly complexes wtih naturally occurring organic matter and this is known to
ameliorate toxicity to certain algae. The chemical form
(speciation) of copper in seawater is therefore an extremely important consideration when carrying out
tests to determine its toxicity, as different copper species can have different degrees of toxicity to marine
algae 1. Pulse-amplitude-modulated measurement
(PAM) of Photosystem II (PSII) chlorophyll fluorescence, a unique and universal technique to evaluate
eco-physiology, is applied in almost all studies that
address photosynthetic responses of plants and algae
to the environment3-5. The principle underlying chlorophyll fluorescence analysis is relatively straightforward. Light energy absorbed by chlorophyll molecules
in a leaf 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 fluorescence. These three processes
occur in competition, such that any increases in the
efficiency of one will result in a decrease in the yield
of the other two. Hence, by measuring the yield of
chlorophyll fluorescence, information about changes
in the efficiency of photosynthesis and heat dissipation can be gained5. Chlorophyll fluorescence is a
measure of the efficiency of photosynthesis and can
be used, therefore, as an indicator of health and vitality. In recent years, the screening of plant fluorescence
signatures is developing as a specific tool which could
be applied to detect the functioning and health status
of plants.
The present research aims to investigate the physiology of two marine alga (using PAM fluorometry)
exposed to various concentrations of copper. Furthermore, this work mainly employs temporal determination of the relative sensitivity of Ulva pertusa and
Ecklonia cava forming a native algal flora of South

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Toxicol. Environ. Health. Sci. Vol. 1(1), 17-23, 2009

Korea on daily basis to study the effect of time of exposure on the algal physiology.

0.5

(a)

LSD: 0.07

(b)

LSD: 0.10

(c)

LSD: 0.08

(d)

LSD: 5.26

0.4
0.3
Fo

Results and Discussion

0.2
0.1
0
0.8

Fm

0.6
0.4
0.2
0
0.8

Fv/Fm

0.6
0.4
0.2
0
60
50
40
ETRmax

Physiology of U. pertusa
There was a significant difference in the initial fluorescence yield between the control and the treated alga
(p0.001). On the first three days of exposure (Figure
1), an increase in copper concentrations from 0.1250.5 mg/L caused an increase in the initial fluorescence
yield (Fo) of U. pertusa, further increase in concentration (1 mg/L Cu) caused a decline in Fo. But after 4
days of exposure, all the concentrations caused inhibition of initial fluorescence yield.
Contrarily, maximum fluorescence yield (Fm) in case
of U. pertusa exposed to 0.125-0.25 mg/L Cu was
nearly similar to the control after one day, while 0.5
and 1 mg/L Cu caused a considerable decrease in the
maximum fluorescence yield. As the period of exposure increased, there was a decline in the maximal
=
fluorescence in presence of 0.5 and 1 mg/L Cu (p=
0.154).
Figure 1c demonstrates a decrease in the ratio of
variable to maximal chl fluorescence (Fv/Fm) of U.
pertusa exposed to different concentration of copper.
A decreasing Fv/F m was recorded with increasing
concentration as well as with increasing time of exposure (p0.05). Figure 1d vividly reveals the toxic
effect copper on photosynthesis of U. pertusa, particularly ETRmax (p0.001). Dramatic decrease in the
ETRmax was observed with increase in concentration
of copper (i.e. from 0.125-1 mg/L) after one day of
exposure. Though, on the following 2 days, there was
a rise in the ETRmax of the control as well as the alga
exposed to 0.125 and 0.25 mg/L Cu, which again
declined after the fourth day. Another noteworthy fact
is that, copper concentration 0.5 mg/L inhibited the
electron transport rate of U. pertusa.
In case of Fv/Fm, the NOEC recorded after the first
and second day of exposure was 0.125 mg/L, which
changed to 0.25 and 1 after the third and fourth day
respectively. When ETRmax was used as an endpoint,
NOEC values of 0.125 mg/L were obtained on the
first two days, while after 3 days of exposure to copper, an NOEC of 0.125 mg/L was obtained, on the following day the NOEC further increased to 0.25 mg/L.
Temporal daily fluorescence studies to evaluate the
physiological state of copper exposed U. pertusa with
respect to Fv/Fm yielded EC10s of 0.101 (0.046-0.172),
0.151 (0.000-0.3770), 0.086 (0.021-0.457) and 0.032
(0.004-1.120) mg/L after 1 to 4 days respectively.
When U. pertusa was studied for its ETRmax, lower

30
20
10
0
1

Days
Ctrl

0.125

0.25

0.5

Figure 1. Chlorophyll fluorescence parameters (a) Fo, (b)

Fm, (c) Fv/Fm, and (d) ETRmax measured on U. pertusa after
varying periods of exposure to a range of Cu concentrations.
=4) are shown.
Mean95% confidence interval (n=

EC10 values of 0.304 (0.024-0.049) and 0.027 (0.0190.041) mg/L were obtained.
The EC50 value for Fv/Fm of U. pertusa obtained
after a day of exposure was 0.481 (0.405-0.652) mg/
L, which gradually decreased to 0.367 (0.281-0.428)

Chlorophyll Fluorescence Based Copper Toxicity Assay

19

Table 1. NOEC, EC10 and EC50 values for the inhibition of Fv/Fm and ETRmax of U. pertusa exposed to copper.
Parameter

Day

NOEC

EC10 (95% CI range)

CV%

EC50 (95% CI range)

CV%

Fv /Fm

1
2
3
4

0.125
0.125
0.25
1

0.101 (0.046-0.172)
0.151 (0.000-0.377)
0.086 (0.021-0.457)
0.032 (0.004-1.120)

19.86
36.24
83.47
-

0.481 (0.405-0.652)
0.367 (0.281-0.428)
0.350 (0.280-0.390)
0.711

6.04
5.25
5.41
-

ETRmax

1
2
3
4

0.125
0.125
0.125
0.25

0.034 (0.024-0.049)
0.027 (0.018-0.041)
0.059 (0.003-0.275)
0.219 (0.000-0.315)

9.17
11.62
63.81
18.26

0.210 (0.138-0.294)
0.155
0.285 (0.135-0.407)
0.356 (0.309-0.384)

8.98
11.80
2.60

Table 2. Maximum permissible limits set by various organizations for copper.

Organization
6

Global effluent guidelines

WHO guideline Expert Consultation
for the 4th Edition of the Guidelines
for Drinking-water Quality7
FAO Standards for Effluent Discharge
Regulations8
Water Environment Partnership
in Asia (WEPA)-Coastal Water Quality
Standards for Protecting Human Health9

Maximum limit

0.25 mg/L
2 mg/L
0.5 mg/L

0.02 mg/L

and 0.350 (0.280-0.390) mg/L after the second and

third day (Table 1). After the fourth day, the EC50
values obtained for Fv /Fm for the same was 0.711 mg/
L. As seen in Table 2, this fulfills the Global Effluent
Guidelines and is near the value of the WHO guidelines set for drinking water quality too6-7. Hence this
estimation of EC50 values for the ratio of variable to
maximal chl fluorescence (Fv/Fm) could be used as a
vital bioassay end point for indicating presence of
copper as a toxicant in water bodies. On the other
hand, for ETRmax, EC50 values of 0.210 (0.138-0.294),
0.155, 0.285 (0.135-0.407) and 0.356 (0.309-0.384)
mg/L were obtained after 1 to 4 days of exposure to
copper. Table 2 demonstrates the maximum permissible limits set by various organizations for copper6-9.
As viewed in Table 2, determination of ETRmax of U.
pertusa, fulfills most of the water quality criteria, and
hence could be used for determining alarming concentrations of copper in aquatic systems. Based on
the results obtained herein, determination of ETRmax
was more sensitive as compared to other parameters
tested i.e. the presence of low concentrations of copper could be easily detected by measuring its inhibitory effect on ETRmax of U. pertusa as an end point.

Physiology of E. cava
E. cava demonstrated almost similar Fo values irrespective of the concentration after the first and se-

cond day of exposure (Figure 2). Moreover, on the

first day of exposure the Fo recorded for the control
was higher than the exposed alga. After 3 days of
exposure to copper, a decreasing Fo could be recorded in presence of 0.125, 0.5 and 1 mg/L Cu. On the
fourth day, this alga demonstrated development of
resistance to copper, and in fact Fo values higher than
the first day could be recorded (p0.001). Another
noteworthy fact is that there was a steady increase in
the Fo values of the control on the third and fourth
day this may be credited to the laboratory environment adaption skill of E. cava. A similar trend was
observed in case of Fm recorded after 4 days of exposure.
After a day of exposure, nearly similar Fv/Fm values
were obtained irrespective of copper concentration.
On the other hand, a decline in the same was noted
with increase in exposure period (i.e. after the second
and third day of exposure). On the fourth day of exposure, there was a visible rise in the Fv/Fm value.
Studies of E. cava showed a rise in ETRmax in the
presence of 0.125 and 0.25 mg/L Cu after a day of
exposure as compared to the control; though, there
was a decrease in the ETRmax with rise in copper concentration. On the consecutive day, there was a significant increase in the ETRmax of the control. A decreasing trend of ETRmax was observed with increasing
concentration, after 3 days of exposure (p0.001).
Based on the above statements, it can be stated that,
the tested concentrations of copper had toxic effect
on the various photosynthetic parameters tested (Table
3). After exposure to copper, E. cava showed NOEC
values 0.125, 0.5, 0.25 and 0.5 mg/L on the first to
fourth day respectively when Fv/Fm was considered as
an endpoint. The EC10 values for Fv/Fm ranged from
0.094 on the first day to 0.694 (0.538-0.913) mg/L on
the fourth day. Maximum NOEC (1 mg/L) was recorded, using ETRmax as an endpoint, after one day of
exposure to copper. Considering the same endpoint,
EC10 and EC50 values of 0.318 (0.108-0.6280) and
0.962 mg/L were obtained in case of E. cava after a
day of exposure to copper. While on the subsequent

20

Toxicol. Environ. Health. Sci. Vol. 1(1), 17-23, 2009

200
LSD: 12.89

(a)

LSD: 36.63

(b)

LSD: 0.07

(c)

LSD: 34.57

(d)

Fo

150
100
50
0
500
400

Fm

300
200
100
0
0.8

Fv/Fm

0.6
0.4
0.2
0
250

ETRmax

200
150
100
50
0
1

Days
Ctrl

0.125

0.25

0.5

Figure 2. Chlorophyll fluorescence parameters (a) Fo, (b)

Fm, (c) Fv/Fm, and (d) ETRmax measured on E. cava after
varying periods of exposure to a range of Cu concentrations.
=4) are shown.
Mean95% confidence interval (n=

two days, a decreasing trend of EC50s was observed.

PAM fluorometry offers new vistas and advantages
for aquatic toxicological studies owing to their speed
and sensitivity10. In addition to considering sensitivity, it is necessary to employ the most sensitive algal
species in any reliable bioassay to detect the presence
of pollutants. For the development of a new bioassay,
the determination of algal sensitivity to specific pol-

lutants is essential because algal species may have

variable sensitivity to different pollutants10.
In the present study, differences in the response of
Ulva pertusa and E. cava to copper have been evaluated. The use of copper in antifouling paints, in the
treatment of diseases of fishes, in agricultural chemicals, and as algicides has increased the need to study
the effects of this metal on aquatic organisms11. Values
of EC50 for the fluorescence parameters (Fv/Fm and
ETRmax) indicate that U. pertusa is a more sensitive
species, as compared to E. cava within the concentration range used in the current study. Of the various
endpoints measured, ETR max was found to be the
most sensitive. The threshold concentration that resulted in a significant reduction in ETRmax was 0.5
mg/L for U. pertusa compared with 1 mg/L for E.
cava. Copper toxicity mechanisms vary according to
species and environmental conditions12. It has been
suggested that elevated concentrations of copper disrupts the cell membrane permeability of U. lactuca,
leading to potassium ion loss. This creates a severe
ionic imbalance, causing gradual cell death, as well
as has direct toxic impact on photosynthetic metabolism13.
It was observed that U. pertusa subjected to low
concentrations of copper (0.125 mg/L Cu) exhibited
higher Fo than those of the control group, especially
during the first 3 days of exposure (Figure 1a). As Fo
is an index of photosynthetic pigment complex and
shows an increase with increasing damage the present
results indicate that the low concentration of copper
may have caused chlorophyll destruction in the algae5.
Differing copper concentrations have been shown
to affect photosynthesis process in several algae. Han
et al. reported no change in Fv/Fm and Fo in U. pertusa exposed to Cu concentrations less than 100 g/L
although chl a declined significantly14. Barraza and
Carballeira reported greater toxicity of copper on
chlorophyll fluorescence of U. rigida as compared to
cadmium in their study of Fv/Fm12. Wilson and Freeberg demonstrated toxic concentration ratios (0.07-5
mg/L copper, 24-48 h) for four microalgae using fluorescence techniques, while Barraza and Carballeira
demonstrated U. rigida to be more resistant to copper
(0.3 mg/L)12,15. A similar pattern was observed in the
present work, wherein U. pertusa was resistant to
0.25 mg/L copper while it was inhibited by 0.5 mg/L
copper. Mamboya et al. studied effect of copper on
the photosynthetic efficiency (Fv/Fm) of P. boergesenii
and reported that both concentration and exposure
time had a significant effect in inhibiting the photosynthetic efficiency16. They also reported a significant interaction between copper concentration and
exposure time in inhibiting the photosynthetic effici-

Chlorophyll Fluorescence Based Copper Toxicity Assay

21

Table 3. NOEC, EC10 and EC50 values for the inhibition of Fv /Fm and ETRmax of E. cava exposed to copper.
Parameter

Day

NOEC

EC10 (95% CI range)

CV%

EC50 (95% CI range)

CV%

Fv/Fm

1
2
3
4

0.125
0.5
0.25
0.5

0.094
0.103 (0.047-0.957)
0.256 (0.086-0.352)
0.694 (0.538-0.913)

1
1
1

ETRmax

1
2
3
4

1
0.125
0.25
0.05

0.318 (0.108-0.624)
0.038 (0.025-0.078)
0.268 (0.000-0.356)
0.496 (0.252-0.636)

21.15
23.59
34.52
17.34

0.962
0.741
0.522 (0.346-0.795)
0.861

ency of P. boergesenii. Nevertheless, in the present

study, both algae (U. pertusa and E. cava) showed a
decline in the mean photosynthetic efficiencies of
every treatment (exposure concentrations) which was
significantly different from each other (p0.05).
Copper has been shown to inhibit photosynthetic
electron transport through damage to both the donor
and acceptor sides of PS II17. In principle, blockage
of electron transport by Cu could lead to a decrease
in Fv/Fm18. In U. pertusa, changes in ETRmax paralleled those of Fv/Fm, whereas in E. cava, ETRmax showed a significant decrease at lower Cu concentrations
than did Fv/Fm. The difference in response of Fv/Fm
and ETRmax reflects inhibition downstream of PS II,
suggesting the down-regulation of PS II electron transport is a result of inhibition of Calvin cycle enzymes
and Rubisco14. Nielsen et al. reported that the ETRmax
of both high and low light (50 mol/m2/s) fronds had
increased after five days exposure to highest Cu treatment and remained elevated after 13 days3. Wu et al.
studied that the effect of different concentration of Cu
on colonial and unicellular form of Microcystis and
stated that the ETRmax of both forms decreased with
exposure time19. This study also suggests the time and
concentration dependent changes in ETRmax of both
species tested.
Comparison of the various photosynthetic parameters studied using these two algal species, revealed
U. pertusa was more sensitive to copper than E. cava.
Moreover, a significant reduction of ETRmax was observed in case of U. pertusa as well as E. cava, in
presence of 0.25-1 mg/L Cu. Thus ETRmax could be
considered a more sensitive parameter, which could
be used as an endpoint to detect the presence of copper.
Copper at low concentration acts as a micro-nutrient, favouring some physiological activities of the
algae. It is concluded that the degree of toxicity depends on both the concentration and exposure time.
Ulva species are generally considered to be tolerant
to heavy metals compared with many other macroalgae. But this study reveals that E. cava species ap-

pears to be more tolerant to Cu exposure than the U.

pertusa in terms of photosynthesis. PAM technique is
frequently used to research the mechanisms of toxicity and resistance in the physiology of marine algae as
well as land terrestrial plants. In addition, the use of
PAM fluoremetry to detect the presence of Cu is a
non-destructive eco-friendly method with high precision. The measurement of ETRmax as an endpoint is
recommended to test aquatic toxicant by using U.
pertusa.

Methods
Collection and Maintenance of Samples
Ulva pertusa and Ecklonia cava were collected
from sites near Ahnin on the well-conserved eastern
coast of Korea (37.4
N, 129.1
E). Unialgal stock cultures were maintained in artificial seawater medium,
prepared by dissolving commercial sea salts (Coralife, Energy Savers, California, USA) in deionized
water (salinity 35) appended with 1 mM KNO3, 0.1
mM K2HPO4, and three vitamins (1 mM vitamin B1,
0.1 M vitamin B12 and D-biotin) as nutrients, at 15

C and 10-15 mol photons/m2/s of white fluorescent

light (FL400, Kum-Ho, Seoul, South Korea) under a
12 : 12 h L : D photoperiod.
Toxicity Assay
Disks ( 4 mm) were prepared from the middle
region of healthy thalli of each alga with the help of
cork borer. Toxicity of copper was tested using a concentrated copper standard solution (CAS No. 744050-8, Junsei Chem., Japan) prepared in de-ionized
water acidified with 1 N hydrochloric acid (HCl) or 1
M HNO3. The target concentrations (0.125-1 mg/L)
were achieved by adding the required stock to aeration flasks (capacity 250 mL) containing 100 mL of
artificial seawater medium (pH 8.4 adjusted using 0.5
N NaOH). To avoid any contamination, the glassware
was soaked in 10% HNO3 for 24 h, rinsed with deionized water and oven dried prior to use.

22

Toxicol. Environ. Health. Sci. Vol. 1(1), 17-23, 2009

Table 4. Protocol for bioassay.

Test type
Test organism
Salinity
Temperature
Light quality
Photon irradiance
Photoperiod
Test vessel
Test specimen/flask
Test duration
Test endpoint

Non-static, non-renewal
Ulva pertusa and Ecklonioa cava
26-35
10 and 15
C0.5
C
Cool-white fluorescence
80-100 mol photons/m2/s
12 : 12 h LD
250 mL Conical flask
5 disk
1-4 days
Inhibition of photosynthesis

comparison test by the least significance difference

(LSD) was then carried out to find out significant differences in response from controls. Results are reported
as NOECs (highest concentration with no significant
difference from controls), EC10s (effective concentration at which 10% inhibition occurs) and EC50s (effective concentration at which 50% inhibition occurs)
with 95% confidence intervals estimated by the linear
interpolation method (ToxCalc 5.0, Tidepool Science,
California, USA).

Acknowledgements
The short-term (1-4 days) exposure experiments
were conducted to evaluate temporal variability in
both algae. Algal disks were exposed to copper by
dispensing them into above mentioned flat bottom
aeration flasks containing different concentrations of
copper. Unenriched artificial seawater media (0 mg/L
copper) was used to maintain control disks. The protocol for the bioassay using Ulva pertusa and Ecklonia cava is summarized in Table 4.
Chlorophyll a Fluorescence

Chlorophyll a fluorescence was measured using

Imaging Pulse Amplitude Modulated (I-PAM, Walz,
Effeltrich, Germany) fluorometer and (Diving-PAM).
Samples were initially dark-adapted for 15 min before
measuring chlorophyll (chl) fluorescence. Fm, the maximum fluorescence yield of dark-adapted samples and
Fo, the initial fluorescence yield, were recorded. The
maximum quantum yield of PSII in the dark-adapted
state is expressed as the ratio of variable to maximal
chl fluorescence (Fv/Fm), derived from (Fm-Fo)/Fm.
Rapid light curves were measured using 10 s pulses
of actinic light increased stepwise from 0 to 1,517
mol photons/m2/s 20. Maximum electron transport rate
(ETRmax) was derived from the hyperbolic tangent
=[1-exp(-*I/Pt)]*exp(-*I/pt)
formulation, ETR=
where indicates electron transport rate under lightlimited conditions, adapted from Platt et al.21.

Endpoints
A temporal comparison of the relative sensitivity of
both the alga to copper was determined using several
endpoints. The endpoints chosen for the experiment
included the initial fluorescence (Fo), maximum fluorescence yield (Fm), the ratio of variable to maximal
chl fluorescence (Fv/Fm), and maximum electron transport rate (ETRmax).
Statistical Analysis
Analysis of variance (ANOVA) was performed to
confirm significant differences in response. Multiple

This work was financially supported by Korea Ministry of Environment (091-061-046) and Korea Ministry for Food, Agricultures, Forestry and Fisheries.
The authors thank the anonymous reviewers for helping in publication of this manuscript.

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