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B :B I O LO G Y

ELSEVIER Journal of Photochemistry and Photobiology B: Biology 34 (1996) 21-28

Effects of UV radiation on photosynthesis of phytoplankton exposed to

solar simulator light
H. Herrmann a, D.-P. H~ider a, M. K6fferlein b, H.K. Seidlitz b, F. Ghetti c,.
a institut3~r Botanik und Pharmazeutische Biologie, Erlangen, Germany
GSF-Forschungszentrum fiir Umwelt und Gesundheit GmbH, Expositionskammern, Neuherberg, Germany
c CNR Istituto di Biofisica, via San Lorenzo 26, 56127 Pisa, Italy

Received 2 May 1995; accepted 2 October 1995

~_bstract

A sunlight simulator was used to investigate the effects of short term irradiation on photosynthetic oxygen production and chlorophyll
fluorescence in the unicellular algae Dunaliella salina and Ochromonas danica. Oxygen evolution was measured with a Clark-type electrode
and initial (Fo) and maximal (Fro) fluorescence were determined by means of a pulse amplitude-modulated (PAM) fluorometer. Using
different long-pass filters, a UV exclusion study was performed comparing the effects of irradiation with full sun simulator light, with sun
fimulator light without UV-B and short wavelength UV-A radiation and without any UV component. In D. salina, exposure to full sun
simulator light caused a marked drop in photochemical efficiency indicated by a decline in the initial slope of the irradiance-response curve
tbr photosynthetic oxygen production and by a decrease in the ratio Fv/Fm, whereas only a slight inhibition of photosynthetic activity was
3bserved irradiating with PAR and UV-A radiation or with PAR alone. In the case of O. danica, a strong inhibition was observed under all
~rradiation conditions. The results are consistent with preliminary data obtained with outdoor experiments.

Keywords: Dunaliella salina; Oxygen evolution; Ochromonas danica; PAM fluorescence; Sunlight; Sunlight simulator; UV-B radiation; UV-A radiation

1. Introduction on the Earth' s surface, due to the depletion of the stratospheric

In recent years much effort has centred on acquiring irra-
diation systems with an emission spectrum simulating solar
radiation, in order to meet the requirement of reproducible
light conditions for ecophysiological plant research and, more
generally, in the field of environmental photobiology [ 1-3 ].
Such simulators allow experiments to be carried out in a
controlled environment, where irradiation conditions and cli-
matic factors can be reproduced at any time and disturbing
agents, often encountered in field experiments, can be
excluded.
The results of indoor experiments, extrapolated to the nat-
ural conditions after comparison with data from field exper-
iments, will allow prediction of the consequences of possible
global changes in the natural light environment. In this
respect, sunlight simulators could be a useful tool to deter-
mine whether terrestrial plants and aquatic photosynthetic
organisms are sensitive to present levels of solar UV radiation
and to forecast the effects of increasing ultraviolet irradiance
* Corresponding author. Tel.: + 39 50 513214, fax: + 39 50 553 501, e-
mail: GHE'I~rI @IB.PI. CNR.IT
ozone layer.
For phytoplankton organisms many studies reported the
deleterious effects of UV radiation on growth, metabolism,
motility, photo- and gravi-orientation, pigmentation and pho-
tosynthetic capability (Refs. [ 4,5 ] ). The contribution of UV-
B (280-315 nm) and UV-A (315--400 nm) radiation in
inhibiting photosynthesis was described for various phyto-
plankton species and/or communities under artificial [6-9]
and solar [ 10-16] irradiation conditions. UV-B radiation, in
marine phytoplankton exposed to different spectral distribu-
tion of solar light, was shown to be almost as effective in
inhibiting photosynthesis as UV-A radiation, although total
energy in the UV-B was about 10% of that in UV-A [ 11 ].
Moreover, the substantial role of short wavelength ( ~<350
nm) UV-A radiation in reducing carbon uptake was dem-
onstrated [ 15]. Larkum and Wood [9], using artificial irra-
diation, showed that the drop in photosynthetic oxygen
production caused by UV-B radiation in various species of
phytoplankton is stronger than inhibition due to PAR (pho-
tosynthetic active radiation) and that the observed decrease
in variable fluorescence is a fast and sensitive indicator of
UV-B effects.

1011-1344/96/$15.00 © 1996 Elsevier Science S.A. All rights reserved

SSDI 101 l- 1 3 4 4 ( 9 5 ) 0 7 2 4 5 - 4
22 H. Herrmann et al. / Journal of Photochemistry and Photobiology B: Biology 34 (1996) 21-28

10~ i ~ i i i 2. Materials and methods

10° Dunaliella salina was grown under white light (about 10

'E
,o. . . . . . . . . . . i .....................
~D
'~ 102 [
10-3 . . . . I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I
J light is emitted by an array of several lamp types. Metal halide
104 ] I
300 400
Wavelength [nm]
Fig. 1. Spectral distributions in the three experimental conditions of irradi-
ation: unfiltered sun simulator radiation, with quartz window covering the
samples (solid line) ; sun simulator radiation filtered with WG 360 (dashed
line) and GG 400 (dotted line) cut-off filters (-i.e. sun simulator radiation
deprived of UV-B and total UV components, respectively).
In this work, a sunlight simulator [3] was used in the
investigation of the effects of short term ( ~<30 min) irradi-
ation on the photosynthetic activity of two unicellular flag-
ellate algae: the halotolerant chlorophycean Dunaliella salina
and the freshwater chrysophycean Ochromonas danica. Pre-
liminary UV exclusion studies in the field (see below) had
shown in these two organisms quite different effects of expo-
sure to solar radiation and in this work the reproducibility of
these results in the controlled environment of the sun simu-
lator was tested.
In order to estimate the inhibition of photosynthesis caused
by UV-B and short wavelength UV-A radiation, the sunlight
simulator was used for a UV exclusion study, in which the
effects of irradiation in the following three experimental con-
ditions were compared: (a) full sun simulator light (A > 295
nm); (b) sun simulator light deprived of UV-B and short
UV-A wavelengths (A > 338 nm); (c) sun simulator light
deprived of almost all UV (A>385 nm). Photosynthetic
activity was probed by measuring oxygen production with a
Clark-type electrode and chlorophyll fluorescence by means
of a pulse amplitude-modulated fluorometer (PAM).
W m-2), in a light-dark cycle (15 h light-9 h dark), at 25
°C in a medium described by Johnson et al. [ 17]. Ochro-
monas danica (SAG 933-7, G6ttingen) was grown as
described by Tippit et al. [ 18] under continuous white light
(about 5 W m - z ) at 23 °C.
The experiments were performed in a climatized sun sim-
ulator especially designed for plant experiments [3]. The
( Osram HQI-T5 400W / D), q uartz-halogen ( Osram Halos-
i I I
500 600 700 tar 500W) and blue fluorescent (Philips TL36/18) lamps
provide the main lighting in the visible region. Excess IR
radiation is absorbed by a layer of circulating deionized water.
Extra quartz-halogen lamps located below the water filter
provide long-wave irradiation otherwise eliminated by the
water filter. UV-B fluorescence lamps (Philips TL40W/12)
were mounted below the water filter on the walls of the lamp
house. Short wavelength UV-B and UV-C radiation (i.e.
wavelengths shorter than 290 nm) are rejected by two layers
of borosilicate glass (Schott, TEMPAX) each 6.5 mm thick.
Thus an almost optimal spectral match of natural global radi-
ation is obtained. The spectral irradiance in the sun simulator
was determined by means of a radiometric calibrated double
monochromator system (Bentham M300, UK).
The organisms were exposed to simulator light in small
Petri dishes, kept in a water bath at a temperature of 23 _+2
°C. They were irradiated with unfiltered and filtered sun sim-
ulator radiation: WG 360 or GG 400 long-pass filters (3 mm,
Schott & Gen., Mainz, Germany) were used to cut off UV-
B and short wavelength UV-A or almost all UV, respectively.
A quartz window was used to cover the samples irradiated
with unfiltered sun simulator light in order to compensate for
reflection losses of the long-pass filters. In Fig. 1 the spectral
distributions obtained in the three different irradiation con-
ditions are shown. Table 1 shows the respective integrated
spectral irradiances.
Absolute photosynthetic oxygen production was deter-
mined at room temperature, before and after exposure, using
a Clark-type electrode inserted in a cuvette for liquid-phase
measurements (DW2/2, Hansatech Instruments, Norfolk,

Table 1
Integrated spectral irradiances for UV-B, UV-A and PAR ranges of the sun simulator and of the sun (in Tramariglio, about 40o36 ' N, 8° 11' E, Alghero, Sardinia,
Italy, May 1993

Irradiation conditions UV-B UV-A PAR

( W m -2) ( W m 2) ( W m -2)

Sun simulator Unfiltered 1.6 47.6 336

Filtered with WG360 0.0 22.0 ( h > 338 nm) 336
Filtered with WG400 0.0 1.3 318
Sun Unfiltered 1.1 45.2 390
Filtered with WG360 0.0 18.5 (A > 338 rim) 367
Filtered with WG400 0.0 2.0 364

Data are calculated from spectral irradiance measurements performed by means of a double monochromator spectroradiometer.
 H. Herrmann et al. / Journal of Photochemistry and Photobiology B: Biology 34 (1996) 21-28
UK). In order to stimulate photosynthetic oxygen production,
samples were irradiated with actinic white light provided by
a quartz-halogen lamp filtered by an infrared absorbing filter.
Irradiance-response curves were determined varying the irra-
diance by means of neutral density filters. The photosynthetic
activity of the algal samples was calculated as femtomoles of
oxygen per cell per minute. Oxygen consumption due to
respiration was measured for each sample, kept in the dark.
As respiration was not significantly affected by the different
irradiation conditions, the absolute value of oxygen con-
sumption determined in the dark was subtracted from the
absolute values of oxygen production under light irradiation,
m order to evaluate the net photosynthetic oxygen production
rate.
In vivo chlorophyll fluorescence was measured at room
temperature with a pulse amplitude modulation fluorometer
(PAM 101, Waltz, Effeltrich, Germany). Before and after
exposure to simulated solar radiation, F o (initial fluorescence,
all reaction centers are in an open state), Fm (maximal fluo-
rescence, all reaction centers are closed), Fv (variable fluo-
rescence, F v = F m - F 0 ) and the ratio F J F m were
:letermined. Before each measurement, the cells were dark
adapted for 5 rain. Fo was induced by low fluence rate red
light pulses (0.031 W m 2. emission peak at 650 nm) emitted
~y an LED (type USBR, Stanley) for 1 /~s at a frequency of
1.6 kHz. Fm was induced by the application of a saturating
!~ulse of white light (770 W m -2; 800 ms) using a fiber optic
-on nected to a halogen cold light lamp (KL 1500 electronic:
Schott, Mainz, Germany).
Analogous field experiments were performed at the Center
tbr Advanced Research in Photobiology (Tramariglio, about
-~0~36' N, 8°11' E, Alghero, Sardinia, Italy, May 1993). In
Fig. 2 the spectral irradiance of natural sunlight (measured
:luring experiments by means of a double monochromator
spectroradiometer, Optronic 752, USA) is plotted together
wilh the unfiltered spectral irradiance of the sun simulator.
101
I0o
'E
I0'
10 2
10 3
10-~ { I
300 400 500 600
Wavelength [nm]
Fig. 2. Spectral irradiance of the sun simulator (dotted line) compared to
'.he spectral irradiance of natural sunlight (solid line) measured during field
,_~xperiments,performedat the Center for AdvancedResearchin Photobiol-
ogy (Alghero, Sardinia) in Summer1993.
23
Table 1 shows the integrated spectral solar irradiances meas-
ured under the three experimental conditions.
3. Results and discussion
The data points of the irradiance-response curves of pho-
tosynthetic oxygen production were fitted with the hyperbolic
saturation function y = a . x~ ( b + x) and the calculated para-
meters a and b were used to estimate the light-saturated rate
of photosynthetic oxygen production and the optimal pho-
tosynthetic irradiance value, respectively [ 19]. An inhibitory
effect due to irradiation is indicated not only by a decrease in
the light-saturated rate of photosynthetic oxygen production,
but also by a decline in the maximum light utilization effÉ-
ciency, i.e. the initial slope of the curve [ 19]. This is given
by the ratio a/b, derivative of the hyperbolic function for
x = 0. Standard errors for parameters a and b were obtained
from the fitting procedure and by means of them the uncer-
tainty in the ratio a / b was calculated.
Chlorophyll fluorescence parameters were used for esti-
mating the degree of inhibition of photosynthetic activity
observed after exposure to the different irradiation condi-
tions. Inhibition of photosynthesis, observed within minutes
to hours in response to excessive irradiation, is associated
with a decrease in Fv, whereas changes in Fo may vary [20].
The consequent decrease in the ratio (Fv/Fm) of variable to
maximal chlorophyll fluorescence (which in phytoplankton
organisms has a maximum value of approximately 0.65, inde-
pendent of growth irradiation conditions) is correlated with
a loss of effective absorption cross-section of PSII and, hence,
with a decline in the maximum light utilization efficiency,
i.e. the initial slope of the irradiance-response curves of pho-
tosynthetic oxygen production [ 19,20]. Repetition of meas-
urements on different samples exposed to the same irradiation
conditions allowed an estimate of errors in Fo and Fm of about
5%, from which the uncertainty in the ratio Fv/Fm was cal-
culated to be about 7%.
In Dunaliella salina, exposed to unfiltered sun simulator
radiation, a marked decline in the initial slope of the irradi-
ance-response curve of photosynthetic oxygen production
was observed, whereas in the samples exposed to radiation
without UV-B and short wavelength UV-A or without UV
the initial slope only slightly changed in comparison with the
unexposed control sample (Fig. 3 ( A ) ) . After 30 rain of
irradiation, in fact, the initial slope decreased to 30% of the
control value in the case of unfiltered samples, whereas it
decreased only to approximately 80% in filtered samples
(Fig. 3). This observed decline in the initial slope of the
curve seems due to an increase in the optimal photosynthetic
h --
700 irradiance value. In fact, whereas the parameter b was signif-
icantly higher in the case of the unfiltered sample (control:
2 6 + 5 W m 2; unfiltered: 8 0 + 2 0 W m-2; WG360:30_+5
W m-2; GG400:37 _+5 W m - 2 ) , the saturation parameters
a did not significantly differ (control: 5.2 +_0.5; unfiltered:
5.0+_0.5; WG360: 5.7_+0.5; GG400:6.0+_0.5 W m 2).
24 H. Herrmann et al. / Journal of Photochemistry and Photobiology B: Biology 34 (1996) 21-28

i i ~ i f i i i t
0.18

100
0.16
'-'7
"U 75 &
~ 0.14

>
50
t~ 0.12

25 A .............. ~ o.oo; I
i
I
i
I
i
I I I I I I I
0.60
0 5 10 15 20 25 30
Time [mini ,..-y 0.50

0.40

•-~ 0.30
~D 4.0
¢)
"7 3.5 0.20
"~ 3.0 0.00 ~
~ 2.5 0.70
,-- 2.0 /11 i~.1 ~ 1 i 0.60
o / / ~ 1 1 I t l l i l
.-.1
i..s 0.50
d

m tLE 0.40
0.5 > 0.30
rr.
0.0 0.20
©
L i i i i _~ 000;
0 10 20 30 40 50 60 70 I I t I I I

lrradiance [W.m-2]
Fig. 3. (A) Relative maximum light utilization efficiency for photosynthetic
oxygen production at non-saturating irradiance in Dunaliella salina exposed
for different time intervals to unfiltered sun simulator radiation (circles) and
to sun simulator radiation filtered with WG 360 (squares) and GG 400
(triangles) cut-off filters; data are shown as percentage of the initial slope
of the control. (B) Irradiance-response curves of photosynthetic oxygen
production of DunalieUa salina before (diamonds, continuous line) and
after 30 min of exposure to unfiltered sun simulator radiation (circles, long
dashed) and to sun simulator radiation filtered with WG 360 (squares,
dashed) and GG 400 (triangles, short dashed) cut-off filters. The curves are
the result of fitting the data points with the hyperbolic saturation function
y=a.x/(b+x).
Under the same experimental conditions, F o for the irra-
diated samples decreased from 0.17 to about 0.15 within the
first 7-8 min of exposure, without further significant changes
after a prolonged exposure (Fig. 4). Fm showed a fast reduc-
tion from 0.58 to about 0.35-0.40 in the first 7 rain, but,
whereas in the filtered samples it slowly decreased to about
0.30-0.35 after 30 min exposure, in the unfiltered one it
diminished further to 0.19 (Fig. 4). Consequently, the ratio
Fv/Fm decreased from the control value (0.71) more in the
unfiltered samples (0.26, after 30 min) than in those irradi-
ated with filtered light (0.52-0.57, after 30 rain) (Fig. 4).
In Ochromonas danica the initial slope of the irradiance-
response curves of photosynthetic oxygen production
decreased to 30-40% of the control value after 10 min of
irradiation under all experimental conditions (Fig. 5). In
contrast to what was observed in the case of D. salina, the
saturation parameter a decreased to about 65% in all irradi-
0 5 I0 15 20 25 30
Time [min]
Fig. 4. Values of Fo, Fm and of the ratio Fv/Fm in Dunaliella salina exposed
for different time intervals to unfiltered sun simulator radiation (circles) and
to sun simulator radiation filtered with WG 360 (squares) and GG 400
( triangles ) cut-off filters.
ated samples (control: 5.3+0.5; unfiltered: 3.8+0.5;
WG360: 3.2+0.5; GG400: 3.4+0.5), thus indicating a
decline in the light-saturated rate of photosynthetic oxygen
production. The parameter b increased under all irradiation
conditions and it was higher in the case of the unfiltered
sample (control: 30_+5 W m 2 ; unfiltered: 80+_5 W m - 2 ;
WG360:46_+6 W m - 2 ; GG400:59_+7 W m - 2 ) . After 5
min of irradiation, all samples showed an increase in Fu from
0.17 to about 0.21-0,23, followed by a saturation, whereas
Fm decreased slightly faster in the unfiltered samples than in
the filtered ones (Fig. 6). Consequently, after 10 min irra-
diation, the ratio Fv/Fm dropped from the control value
(0.57) to 0.20 in the unfiltered sample and to about 0.30 in
the other ones (Fig. 6).
According to the literature, exposure to an excess of white
light causes the formation of singlet oxygen mediated by the
triplet state of P680, with the consequent inhibition of elec-
tron transfer through PSII, the proteolytic degradation of the
Dl-polypeptide and, eventually, the disassembly of the PSII
complex [ 21,22 ]. The predominant effect of photoinhibition
is a decrease in photosynthetic efficiency at limiting light
levels and in the ratio of variable to maximal chlorophyll
fluorescence (F,,/Fm). Reductions in the light-saturated rate
 H. Herrmann et al. / Journal of Photochemistry and Photobiology B: Biology 34 (1996) 21-28 25

i i i i i i I I I I I f

0.24

0.22
'-'7.
" 75 = 0.20

0.18
~ 50
0,16
•~ 25
A 0.00; I I i I [ I
I I I I I I
0 t I I I I I
0.40
0 2 4 6 8 10

Time [minl "7.

0.35

4.5 E
f'~ 0.30
"-~ 4.0 B . *
"7,= 3.5
0.00; I t I I I I
"6 3.0 r t T i i i
0.60
2.5
o 2o ~zJ~~q ~- "-7. 0.50
¢9
= 1.5
~i 0.40

~. 1.0 0.30
e-
0.5 0.20 -Q

© o.o I i i ~ i i
/

0 25 50 75 ]00 125
lrradiance [W.m2]
Fig. 5. ( A ) Relative maximum light utilization efficiency for photosynthetic
oxygen production at non-saturating irradiance in Ochromonas danica
exposed for different time intervals to unfiltered sun simulator radiation
(circles) and to sun simulator radiation filtered with W G 360 (squares) and
GG 400 (triangles) cut-off filters; data are shown as percentage of the initial
slope of the control. ( B ) h'radiance-response curves of photosynthetic oxy-
gen production of Ochromonas danica before (diamonds, continuous line)
and after 10 min of exposure to unfiltered sun simulator radiation (circles,
long dashed) and to sun simulator radiation filtered with W G 360 ( squares,
dashed) and GG 400 (triangles, short dashed) cut-offfilters. The curves are
the result of fitting the data points with the hyperbolic saturation function
y=a.x/(b+x).
of photosynthetic oxygen production are also observed when
severe damage to PSII occurs [22].
In D. salina exposed to radiation of wavelength longer
than 385 nm or 338 nm (i.e., with the GG400 and the WG360
filters, respectively) the absence of decrease in photosyn-
thetic efficiency and the slight reduction in FJFm indicated
a lack of pronounced photoinhibition (or, at least, a fast
recovery after irradiation treatment). This may partly be
explained by the capability of this organism to undergo State
1-State 2 transitions with a time course of a few minutes
[23]: at high irradiance levels the plastoquinone pool is
reduced and the light harvesting complex LHC-2 becomes
phosphorylated, with a consequent decrease in PSII cross-
section [ 19,23 ]. This rapid and reversible mechanism (lead-
ing to a decrease or to an increase in PSII cross-section at
high or low irradiance levels, respectively) regulates adap-
tation to changes in irradiance in the natural environment
0.00 ~ I I l I I I
0 2 4 6 8 10
Time [mini
Fig. 6. Values of Fo, Fm and of the ratio Fv/Fm in Ochromonas danica
exposed for different time intervals to unfiltered sun simulator radiation
(circles) and to sun simulator radiation filtered with W G 360 (squares) and
GG 400 (triangles) cut-off filters.
[ 19] and could account for the slight reduction in maximum
light utilization efficiency, observed in the samples irradiated
with filtered light, when transferred from the growth low
irradiance conditions to the sun simulator chamber and then
back to low irradiance conditions for oxygen and fluorescence
measurements.
Moreover, the ability to rapidly adjust the Chl a/Chl b
ratio, altering the relative levels of PSII core complex and
light harvesting complex LHC-2, has been observed in D.
salina [ 23 ]. Even if it was shown that this reversible adap-
tation mechanism to changes in light intensity has a half time
of about 3 h [23], it may partly contribute to reduce the
effects of high irradiance levels on D. salina exposed to
radiation of wavelength longer than 385 nm or 338 nm.
D. salina is also well known to accumulate t-carotene
more than any other known alga [ 24,25 ] and, even though
the levels of salinity and irradiance of our growth conditions
are lower than those at which the maximum of B-carotene
content was observed [ 24 ], also a slightly higher concentra-
tion of carotenoids in the chloroplasts could increase the rate
of quenching of triplet states and singlet oxygen, thus pre-
venting, at least for the relatively short times of these exper-
iments, the occurrence of photodamage requiring long
26 H. Herrmann et al. /Journal of Photochemistry and Photobiology B: Biology 34 (1996) 21-28

recovery times. Actually, it was observed that even in carot- i i i i i

1.25
enoid-poor cultures ofD. salina photon flux densities greater 2..
than 1500/~Einstein m 2 s - 1 (value comparable to the irra-
1.00
diance in the sun simulator) were necessary for inhibiting "7
photosynthesis [ 24].
On the other hand, the data obtained for D. salina show -~
O 0.75

that short exposure to solar levels of UV-B and short wave-

length UV-A radiation cause a marked drop in photochemical "O 0.50
-~,
efficiency, indicated by the decline in the initial slope of the
"0
2¢i"///
irradiance-response curve for photosynthetic oxygen produc- p 0.25
tion and by the pronounced decrease in the ratio Fv/Fm. The
absence of reduction in the light-saturated rate of photosyn-
e-L

0.00
0
I I I 1 I I I I
thetic oxygen production, however, does not indicate a severe
0 25 50 75 100 125 150 175
damage to PSII population [22]. According to our data no
lrradiance [W.m2]
inhibition seems to be caused by long wavelength UV-A
radiation.
These results are consistent with the outcome of previous
investigations showing that UV-B photons, at fluence rates 100 ' B

higher than those contained in sunlight at the Earth's surface,

are more effective than photons of the visible range in inhib- ,- 75

iting PSII activity and in inducing D1 protein degradation

[26,27 ], through photosensitization pathways [ 28]. In par- 50

ticular, Greenberg et al. [28] showed, under artificial irra-

diation conditions, that the degradation rate of the D 1 protein -~ 25
tz
has a maximum in the UV-B range and it is significantly
enhanced under natural sunlight in comparison to sunlight 0
filtered with a GG400 long-pass filter, removing all UV light. Control GG400 W G 3 6 0 Unfiltered

These authors suggested that this degradation process is pho-
tosensitized by plastoquinone, instead of chlorophyll [ 28 ].
Preliminary field experiments on D. salina showed results
analogous to those obtained in the sun simulator. After 30
min of exposure to natural sunlight, the initial slope of irra-
diance-response curves of photosynthetic oxygen production
was reduced to about 25% of the control (Fig. 7), a value
very close to that of the sun simulator experiments. In this
case, however, filtered sunlight also caused inhibition of pho-
tosynthetic activity, gradually reduced by cutting UV radia-
tion (Fig. 7). This could be explained by the fact that PAR
was higher and UV-B lower in the field experiments in com-
parison with the simulated ones and the inhibitory effect of
UV-B could have been partly concealed by a general
photoinhibitory effect. In the field experiments too, the cal-
culated saturation parameters (control: 1.6 + 0.1 ; unfiltered:
1.6 + 0.1 ; WG360:1.5 _ 0.1 ; GG400:1.6 ___0.1 ) do not indi-
cate any significant reduction in the light-saturated rate of
photosynthetic oxygen production.
On the other hand, exposing D. salina to the light of a
Philips TL20W/12 UV lamp (with UV-B irradiance com-
parable to that of the previously described experiments and
with UV-A and PAR irradiances 10- and 100-times lower,
respectively) a similar decrease in the initial slope of irradi-
ance-response curves of photosynthetic oxygen production
was observed (Fig. 8). In this case, however, the light-satu-
rated rate of photosynthetic oxygen production decreased
from 1.5 + 0.1 in the control to about 0.9 + 0.1 and 0.6 + 0.1
after irradiation for 30 and 60 min, respectively, thus indi-
Fig. 7. (A) Irradiance-response curves of photosynthetic oxygen production
of Dunaliella salina before (diamonds, continuous line) and after 30 min
of exposure to unfiltered sun radiation (circles, long dashed) and to sun
radiation filtered with WG 360 (squares, dashed) and GG 400 (triangles,
short dashed) cut-off filters. The curves are the result of fitting the data
points with the hyperbolic saturation function y = a. x~ (b + x). (B) Relative
maximum light utilization efficiency for photosynthetic oxygen production
at non-saturating irradiance in Dunaliella salina exposed for 30 min to
unfiltered sun radiation and to sun radiation filtered with WG 360 and GG
400 cut-off filters; data are shown as percentage of the initial slope in the
control.
cating the occurrence of severe damage to PSII [22]. This
effect was probably due to the presence of UV-B wavelengths
shorter than 295 nm, which in the sun simulator are rejected
by the TEMPAX glass.
O. danica, even after short exposure times, underwent pho-
toinhibition under all experimental conditions. As irradiation
without UV caused a strong decrease in photosynthetic activ-
ity, a possible UV-B or UV effect may hardly have been
detected taking into account the fact that UV-B radiation was
about 0.4% and UV about 12% of the irradiance in the range
280-700 nm. The decrease in the ratio Fv/Fm is due both to
an increase in Fo and to a decrease in Fro. It is assumed that
this increase in Fo reflects a photoinhibition of PSII [29] : in
fact, in red algae [30] as well as in Ulva rotundata [31] an
increase in Fo after photoinhibitory treatment was observed
and, in the case of U. rotundata, it was suggested that the
rapid decrease in Fv/Fm caused by an increase in Fo may
reflect damage to PSII [ 31 ]. In the case of O. danica, prelim-
inary analogous experiments in the field had shown a com-
 H. Herrmann et al. /Journal t~'Photochemistry and Photobiology B." Biology 34 (1996) 21-28 27

Acknowledgements
1.25
The authors gratefully a c k n o w l e d g e financial support by a
7i 1.00 grant from the E C Research Project E n v i r o n m e n t ( C o n t r a c t
E V 5 V - C T 9 1 - 0 0 2 6 ) and a grant from the State o f Bavaria
0.75 ( B a y F O R K L I M , Project D I I I / l ). T h e y also are indebted to
S. Puntoni, L. Barsanti and V. Passarelli for kindly providing
0.50 Dunaliella s a l i n a and O c h r o m o n a s danica for the
experiments.
o 0.25 ~ / , ~ Q t1~ _.11_.". . . . _.ll--ll ~ -
e'~

O~ 0.00
I I I I i I

0 25 50 75 100 125 150 175

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Irradiance [W.m "2]
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