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Journal of Experimental Marine Biology and Ecology 247 (2000) 99–112

www.elsevier.nl / locate / jembe

The impact of UV-B radiation and different PAR intensities

on growth, uptake of

C, excretion of DOC, cell volume,

and pigmentation in the marine prymnesiophyte, Emiliania

huxleyi

Kristine Garde , Caroline Cailliau

The International Agency for C Determination, VKI, Agern Alle 11, DK-2970 Hørsholm, Denmark Received 8 July 1999; accepted 20 December 1999

Abstract

The impact of UV-B radiation (280–320 nm) and different PAR (400–700 nm) intensities on 14

growth, uptake of C, excretion of DOC, cell volume, and pigmentation in the marine prymnesiophyte, Emiliania huxleyi, was studied in a laboratory experiment. In UV-B-exposed

algal cultures at UV-B doses .2.7 kJ m UV-B_BE, changes in incorporation and excretion rate 14

of C, and indications of DNA damage (in the form of termination of cell division and enlarged cell volume) were observed. Since the UV-B doses used are representative of UV-B doses in the top layer of clear ocean water and E. huxleyi is a common bloom-forming algae with a wide geographic distribution, it is suggested that current UV-B intensities have an impact on primary production and phytoplankton biomass.  2000 Elsevier Science B.V. All rights reserved.

Keywords: C-uptake; Cell volume; Phytoplankton; Pigmentation; UV-B radiation

1. Introduction

Phytoplankton cells live in a varying physical environment, being exposed to different light regimes depending on their position in the water column. The distribution of the plankton organisms within the water column is decisive for the UV-B (280–320 nm) dose to which the organisms are exposed. Vertical mixing will to a large extent control the distribution of the organisms, and be decisive for the time phytoplankton cells are at the water surface, where the UV-B intensity can be intensive, but also at depth where

*Corresponding author. Tel.:145-16-92-00; fax:145-16-92-92.

E-mail address: [email protected] (K. Garde)

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though some of the responses to either low-light or excessive light conditions appear to be similar to UV-B effects, the physiological changes in the algae cells may be completely different. For example can reduced growth in UV-B exposed algal cells originate from DNA damages (Mitchell and Karentz, 1993), damage to the photo-synthesis apparatus (Smith et al., 1992; Cullen and Neale, 1994), or changed nutrient metabolism (Behrenfeld et al., 1995; Garde and Gustavson, 1999). The aim of the present study was to assess the responses of algal cells to UV-B radiation and different PAR intensities. The impact of the different light regimes and light intensities on growth,

uptake of C, excretion of dissolved organic carbon (DOC), cell morphology, and pigmentation was conducted on a culture of the marine prymnesiophyte, Emiliania

huxleyi. We choose E. huxleyi as test organism because this algae is one of the most dominant algal species in oceanic waters (Brown and Yoder, 1994), also at higher latitudes where the impact of increased UV-B radiation due to ozone depletion is

believed to be most pronounced (Vernet and Smith, 1997; Bjorn et al., 1998).

2. Materials and methods

2.1. Culture conditions and irradiation

A non-calcifying flagellated strain of Emiliania huxleyi (Lohman) Hay and Mohler, isolated from Oslo Fjord in 1990, was grown in batch cultures at 158C in K30 medium (Keller et al., 1987). No coccolith production took place during the experiment. Prior to the experiment, four cultures of E. huxleyi were acclimated to a light intensity of 106

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mmol photons m s (light source: Pope 36 W m ) with a light / dark cycle of 16:8 h, in 3-l Erlenmeyer bottles. Three bottles were made of Pyrex and one of quartz. Several cell doublings were observed to allow for adequate light acclimation. The experiment started at the beginning of a light period, i.e. t50, when the algae were in an exponential growth phase. One bottle was kept at original light intensity, 106 mmol

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photons m s (ML), one exposed to lower light intensity, 53mmol photons m s

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light intensity was regulated by adding or removing black tulle from the outside of the glass bottles. The black tulle reduced the light intensity equally at wavelengths from 200 to 700 nm. The algae in the quartz bottle were exposed (t50) to artificial UV-B

radiation (Phillips TL 20 W/ 12 RS) with an intensity of 0.52 W m (UV), in addition

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to the original light intensity, 106mmol photons m s . The UV-B tubes were covered by a film of cellulose acetate, which absorbs wavelength ,280 nm, i.e. UV-C radiation. In order to minimize the change of the filter properties of the film, the cellulose acetate was preburnt for 48 h at a distance of 1 m from four UV-B lamps. The UV-B spectra

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provided a biologically effective radiation (UV-BBE) of 0.185 W m (666 J m h ), when weighted with Setlow’s DNA spectrum normalised to 300 nm (Setlow, 1974).

Daily UV-BBE up to 1700 J m may be considered realistic UV-B intensities for temperate regions during summer months, according to Behrenfeld et al. (1993). The spectras and intensities of the PAR and UV-B lamps (Fig. 1) were measured with a spectroradiometer, OL-754 Optronics Laboratories, INC. The spectrophotometer was calibrated against a Deuterium lamp standard for the spectral range between 200 and 400 nm.

2.2. Experimental procedure

At the start of the experiment, each culture had a density of 0.75310 (60.0613

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10 ) cells per ml and was inoculated with 1200 mCi NaH CO3 solution (The

International Agency for C Determination, VKI, Hørsholm, Denmark), giving a final

concentration of 400 mCi l . Subsamples for cell number, cell morphology, uptake of

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C, excretion of DO C, and pigment concentration were taken regularly during the 28 h of the experiment.

2.2.1. Enumeration and cell volume

Samples for microscopic enumeration of algal cells were fixed in a Lugol solution and stored at 58C. At least 100 cells were counted, and the growth rates were calculated. The cell diameter for at least 50 cells was measured in the microscope, and cell volume was estimated by assigning the cells to the geometric shape of a sphere. The dimension was multiplied by a factor 1.1 to compensate for shrinkage due to fixation (Choi and Stoecker, 1989). For bacterial enumeration, samples were fixed in glutaraldehyde and stored at 58C. Acridine orange-stained samples were filtered onto 0.2-mm black polycarbonate filters and counted in an epifluorescence microscope (Hobbie et al., 1977).

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2.2.2. Determination of PO C and DO C

The activity of particular organic carbon (PO C) was determined from triplicate 5-ml subsamples filtered onto 0.45-mm cellulose nitrate filters (25 mm). To remove labeled, dissolved inorganic carbon, the filters were fumed in acid for 5 min and subsequently placed in a scintillation vial with 10 ml Ecoscint A scintillation cocktail. The filtrate

from the PO C filtrations was collected for determination of dissolved organic carbon

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Fig. 1. The spectras and light intensities to which cultures of Emiliania huxleyi were exposed. (A) Intensities of the PAR lamps, and (B) intensity of the artificial UV-B tubes. HL denotes high light (176mmol photons

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m s ), ML denotes Medium Light (106mmol photons m s ), LL denotes Low Light (53mmol photons

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m s ), and UV denotes exposure to UV-B (0.52 W m ). The UV was, in addition to the UV-B light, exposed to PAR with the same intensity as ML. The spectras and light intensities were measured outside the Erlenmeyer bottles. Note the difference in units of the Y-axes.

inorganic carbon and degassed for 24 h, after which Instagel scintillation cocktail was added. All radioactivity counting was carried out using a liquid scintillation counter (Beckman Model 1802). The release of extracellular organic material was expressed as

percentage of total assimilated C (Zlotnik and Dubinsky, 1989):

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% DOC excretion5DO C3100 /(PO C1DO C)

Dissolved inorganic carbon (DIC) was measured by acidification of 1 ml sample with a subsequent quantification of CO in an IR gas analyser.2

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2.2.3. Pigments

For pigment analyses (i.e. chlorophylls and carotenoids), 50–200 ml samples were collected on glass fibre filters (Whatman GF / C, 47 mm) and immediately frozen in liquid nitrogen, after which they were stored until analysis. The filters were extracted in 100% acetone, sonicated on ice for 10 min and stored at 58C for 24 h (Bidigare, 1991). A mixture of 1.0 ml pigment extract and 0.3 ml H O were injected into a Shimadzu-2

LC10 HPLC system, and the pigments were analysed according to Wright et al. (1991), ¨

with modifications as described in Schluter and Havskum (1997). The HPLC system

was calibrated with pigment standards from The International Agency for C De-termination, VKI, Hørsholm, Denmark.

2.3. Statistical test of data

For all data collected an ANOVA: Two-factor analysis was used. P values ,0.05 were regarded as significant.

3. Results

The number of E. huxleyi cells in all light regimes was of the same order of magnitude during the first 15 h of exposure (Fig. 2A). Cell divisions took place during the dark period, and the cell number increased significantly in all but the UV-B-exposed light regimes. The cell volume in the cultures only exposed to PAR decreased parallel to

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the increase in cell number (from 58mm at 15 h to 24–31mm at 24 h in LL, ML and

HL), while the cell volume in the UV-B-exposed cells increased significantly to 80mm

during the first hours of the dark period but declined to 55 mm at the end, i.e. t524 (Fig. 2B). Microscopic observations revealed that the enlarged UV-B-exposed cells did not look normal, since the vacuole area within the cells increased very markedly. The change in cell volume and appearance was notable already after 4 h of exposure, i.e. 2.7

kJ m UV-BBE, in the UV-B-exposed culture. No difference in bacterial abundance was observed between the four light treatments (data not shown).

Uptake of C took place only during light periods in all light regimes (Fig. 3A).

PO C was significantly highest in the HL and ML cultures after 24 h of incubation. The

PO C in UV and LL cultures was only half the activity in HL and ML. Despite a lack

of cell division, the UV-B-exposed culture had the same incorporation rate of C as the

culture exposed to LL. The PO C was actually higher during the first 10 h of incubation in the UV-B-exposed culture compared to LL, but thereafter and for the rest of the

experimental period, PO C was slightly higher in LL than in UV. The activity per cell was highest in the UV-B-exposed cells after 24 h of incubation (data not shown), although growth based on cell division was not observed in the cells. The % DOC

excretion followed the levels of the PO C and increased during the experiment in HL and ML from 0.3 to 1.6%, while a smaller increase from 0.3 to 0.6% was found in LL and UV-B (Fig. 3C).

The algal cells from the UV and the ML cultures received the same intensity of PAR light, differing only in UV-B load. The variance in algal response between these two

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Fig. 2. (A) Cell number, and (B) cell volume for cultures of Emiliania huxleyi grown at four different light conditions. The horizontal bar indicates the dark period from 16 to 24 h. Abbreviations as in Fig. 1.

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cultures is thus regarded to be due to the added UV-B exposure. The PO C and DO C

were significantly inhibited at UV-BBE doses higher than 2.7 kJ m (Fig. 4). The inhibition increased as the UV-B dose increased. No recovery from the UV-B induced inhibition was observed after 8 h in the dark.

In general, the cultures exposed to only PAR light exhibited an identical increase in the concentration of chlorophyll a (chl a) during the light period, and the chl a concentration remained constant during the dark period (Fig. 5A). No changes in chl a concentration, however, were observed in the UV-B-exposed culture. The carotenoids, i.e. the sum of fucoxanthin (fuco), 199-hexanoyloxyfucoxanthin (199-hex), diadinox-anthin (DD), diatoxdiadinox-anthin (DT), and b-carotene, increased in the cultures exposed to PAR intensities alone, while the carotenoids in the UV-B-exposed cells were more or less constant during the experiment (Fig. 5B). The ratio between the carotenoids and chl

a was significantly lower in the LL, while no major difference was observed between

ML, HL and UV (Fig. 5C).

The chl a concentration per cell was more or less constant in all light regimes during

the first 7 h of incubation, around 190 fg chl a cell , whereupon it increased at different rates in all light regimes until the dark period (Fig. 6A). The highest increase in chl a per

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Fig. 3. (A) PO C, (B) DO C, and (C) estimated % DOC excretion for cultures of Emiliania huxleyi grown at four different light conditions. The horizontal bar indicates the dark period from 16 to 24 h. Abbreviations as in Fig. 1. Error bars in A and B represent the standard deviation (n53).

cell was found in ML, with a concentration up to 357 fg chl a cell . The cell

concentration of fuco was significantly higher in LL, 266 fg fuco cell , than in any of the other light regimes while there was no change in cell concentration of fuco in UV (Fig. 6B). In contrast, the cell concentration of 199-hex was highest in ML, 318 fg

199-hex cell , and lowest in LL (Fig. 6C). HL and UV also displayed an increase in the 199-hex content per cell during the light periods (Fig. 6C). The cellular concentration of

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Fig. 4. The effect of the biologically effective UV-B dose, UV-BBE(weighted with Setlows DNA spectrum

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(Setlow, 1974)) on the PO C and DO C in Emiliania huxleyi cultures exposed to UV-B (UV) as percentage of the control(ML). The vertical bar indicates 8 h in the dark.

during the entire incubation period. The highest DT concentrations per cell were found

in ML and HL during the light period, 11 and 12 fg DT cell , respectively, but the DT concentration declined in the dark period, also in UV (Fig. 6E).

4. Discussion

In nature, plankton organisms are exposed to large variations in radiation and exposure time, owing to the absorption of light (e.g. by phytoplankton, debris, and DOC) and the vertically mixing of the water column. Exposure to severe UV-B doses have repeatedly been found to cause serious damage to plankton organisms, which was also observed in the present study where cessation of cell division (Fig. 2A) and enlarged

algal cells (Fig. 2B) were found at UV-B doses .2.7 kJ m UV-BBE. These damages could result from damage to DNA, which is one of the major lethal and mutagenic effects of UV-B (see Mitchell and Karentz (1993) for review). Gieskes and Buma (1997) also measured thymidine dimers as well as changes in growth rates when E. huxleyi was exposed to UV-B irradiation in the same order of magnitude as the one used here. As mentioned earlier, the UV-B doses used in the present study are within the range of ambient intensities during summer months for temperate regions (Behrenfeld et al., 1993), which implies that UV-B radiation may induce DNA damage in naturally occurring plankton cells living near the ocean surface. In support of this theory, DNA damage has been observed in naturally occurring bacterioplankton (Jeffrey et al., 1996), and increase in cell volume and inhibited cell division, potential signs of DNA damage, were found in a marine diatom exposed to ambient light intensities in Oregon, USA (Behrenfeld et al., 1992).

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Fig. 5. (A) The concentration of chl a and (B) the sum of carotenoids, and (C) the carotenoid / chl a ratio in cultures of Emiliania huxleyi grown at four different light conditions. The horizontal bar indicates the dark period from 16 to 24 h. Abbreviations as in Fig. 1.

repaired by a time-dependent process stimulated by UV-A and PAR or by dark repair (Quesada et al., 1995; Ekelund, 1996; Gieskes and Buma, 1997). Work by Behrenfeld et al. (1992) has suggested that the changes in cell volume upon UV-B exposure are a reversible process. In the present experiment, the cell volume in the UV-B-exposed algal cells decreased by the end of the dark period (Fig. 2B), but increased immediately when the light was switched on again, which supports the theory raised by Behrenfeld et al. (1992). The UV-B exposed algal cells were, however, not exposed to UV-A so the

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Fig. 6. The pigment concentrations per algal cell in Emiliania huxleyi culture grown at four different light conditions. The figures from A–E each represent a pigment, chl a chlorophyll a; fuco, fucoxanthin; 199-hex, 199-hexanolyloxyfucoxanthin; DD, diadinoxanthin; DT, diatoxanthin. The horizontal bar indicates the dark period from 16 to 24 h. Abbreviations as in Fig. 1.

potential repair mechanisms would either be due to PAR light or dark repair. The balance between UV-B exposure and the concurrent intensities of UV-A and PAR, mainly determined by the vertical mixing of the water column, is therefore of importance for the UV-B-damage caused to plankton assemblages. Jeffrey et al. (1996) revealed that photoproducts (i.e. cyclobutane pyrimidine dimers, CPD) induced in natural bacterial plankton by UV-B exposure, accumulated in the surface water during

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calm weather, while in windy periods with strong vertical mixing, CPD was evenly distributed in the water column. An accumulation of enlarged algal cells in the surface water during calm weather, in addition to reduced food supply because of lack of cell division, would have serious consequences for phytoplankton grazers and may alter the interactions between organisms in the entire food web. Although cell enlargement upon UV-B exposure has been confirmed in several laboratory studies (Karentz et al., 1991; Van Donk and Hessen, 1995; Buma et al., 1996), this kind of abnormal-looking cell has not been reported as currently observed in the surface of the ocean layers.

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The uptake of C was also affected by UV-B doses .2.7 kJ m UV-BBE(Fig. 3A), but the effect was not as pronounced as the potential signs of DNA damage. The

UV-B-exposed algal cells continued to take up C even though cell division had

stopped, but the incorporation rate of C was reduced (Fig. 4) and corresponded to the incorporation rate measured in the algal cells exposed to low PAR (Fig. 3A). The artificial sun-lamps used as the source of UV-B in this study, however, produced irradiance more damaging to DNA than to photosynthesis, in contrast to UV-B in nature,

which can explain the different response in uptake of C and cell morphology. The exudation of organic matter by algal cells has been suggested to be an active release of excess photosynthates that accumulate when carbon fixation exceeds incorpo-ration into new cell material (Fogg, 1983). In accordance with this assumption, the % DOC excretion has been found to be positively correlated to light stress, i.e. both high and low light intensities (Zlotnik and Dubinsky, 1989). In the present study, however, the lowest % DOC excretion was observed in the algal cells exposed to light stress situations, i.e. UV-B radiation and low light intensity (Fig. 3C). This result remains unexplainable, but the % DOC excretion levels were in the order 0–2% for all light treatments, which is negligible compared to levels previously reported, e.g. 5–50%

(Zlotnik and Dubinsky, 1989), and up to 90% (Fogg et al., 1965). The DO C was

correlated to the PO C (Fig. 3) and was not biomass dependent (compare Fig. 2A and Fig. 3C), which implies that the exudation could not solely be explained by passive diffusion, as suggested by Bjørnsen, (1988).

The carotenoid / chl a ratio has been implied to increase upon UV-B exposure (Goes et al., 1994; Quesada et al., 1995; Roos and Vincent, 1998). In the present study, there was no difference in the carotenoid / chl a ratio of UV-B-exposed algae and cells exposed to HL and ML, while the algae exposed to low light exhibited a lower ratio (Fig. 5C). Assuming that HL and ML are light saturating conditions for E. huxleyi in this study, the observed variations in the carotenoid / chl a ratio indicate that the ratio reflect the response to light in general rather than UV-B alone. There was no decrease in any pigment concentration in the UV-B-exposed algal cells during the light period (Fig. 6), implying that there was no photobleaching of the pigments (Nielsen and Ekelund, 1993;

Donkor and Hader, 1996).

The chl a content per cell surprisingly peaked at the highest light intensities (Fig. 6A), probably because of an endogenous circadian rhythm. Since the algae had been adapted to the light cycle for four days, the chl a content seemed to be related to an endogenous circadian rhythm rather than to the current light regime (Post et al., 1984; Buma et al., 1991). Unlike chl a, fucoxanthin, an accessory pigment (Goericke and Welschmeyer, 1992), responded with increasing concentration at the low light intensity already after 2

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cold-water forms exist, there is a risk that current UV-B intensities have an impact on primary production and phytoplankton biomass. The light regime and light intensity, however, are much more uniform in laboratory studies than in nature, so care should be taken when laboratory results obtained from UV-B experiments are extrapolated to natural plankton communities, hence this hypothesis remain speculative and needs further investigation.

Acknowledgements

We thank Tom Nielsen for kindly providing the light equipment and for helping with ¨

the light measurements, Louise Schluter, Morten Søndergaard, and Kim Gustavson for the critical reading of the manuscript, Kristian Møller Christensen and Winnie Martinsen for technical assistance, and Kathleen Gail Jensen for linguistic corrections. This work was supported by the Danish Environmental Research Programme, the Danish Research Academy, an EU MAST III contract (MAS3-CT96-0053-PHASE), an EU MAST III contract (MAS3-CT97-0154-MIDAS), and by VKI. [RW]

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repaired by a time-dependent process stimulated by UV-A and PAR or by dark repair

(Quesada et al., 1995; Ekelund, 1996; Gieskes and Buma, 1997). Work by Behrenfeld et

al. (1992) has suggested that the changes in cell volume upon UV-B exposure are a

reversible process. In the present experiment, the cell volume in the UV-B-exposed algal

cells decreased by the end of the dark period (Fig. 2B), but increased immediately when

the light was switched on again, which supports the theory raised by Behrenfeld et al.

(1992). The UV-B exposed algal cells were, however, not exposed to UV-A so the

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Fig. 6. The pigment concentrations per algal cell in Emiliania huxleyi culture grown at four different light conditions. The figures from A–E each represent a pigment, chl a chlorophyll a; fuco, fucoxanthin; 199-hex, 199-hexanolyloxyfucoxanthin; DD, diadinoxanthin; DT, diatoxanthin. The horizontal bar indicates the dark period from 16 to 24 h. Abbreviations as in Fig. 1.

potential repair mechanisms would either be due to PAR light or dark repair. The

balance between UV-B exposure and the concurrent intensities of UV-A and PAR,

mainly determined by the vertical mixing of the water column, is therefore of

importance for the UV-B-damage caused to plankton assemblages. Jeffrey et al. (1996)

revealed that photoproducts (i.e. cyclobutane pyrimidine dimers, CPD) induced in

natural bacterial plankton by UV-B exposure, accumulated in the surface water during

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calm weather, while in windy periods with strong vertical mixing, CPD was evenly

distributed in the water column. An accumulation of enlarged algal cells in the surface

water during calm weather, in addition to reduced food supply because of lack of cell

division, would have serious consequences for phytoplankton grazers and may alter the

interactions between organisms in the entire food web. Although cell enlargement upon

UV-B exposure has been confirmed in several laboratory studies (Karentz et al., 1991;

Van Donk and Hessen, 1995; Buma et al., 1996), this kind of abnormal-looking cell has

not been reported as currently observed in the surface of the ocean layers.

14 22

The uptake of

C was also affected by UV-B doses

.2.7 kJ m

UV-B

(Fig. 3A),

but the effect was not as pronounced as the potential signs of DNA damage. The

UV-B-exposed algal cells continued to take up

C even though cell division had

stopped, but the incorporation rate of

C was reduced (Fig. 4) and corresponded to the

incorporation rate measured in the algal cells exposed to low PAR (Fig. 3A). The

artificial sun-lamps used as the source of UV-B in this study, however, produced

irradiance more damaging to DNA than to photosynthesis, in contrast to UV-B in nature,

which can explain the different response in uptake of

C and cell morphology.

The exudation of organic matter by algal cells has been suggested to be an active

release of excess photosynthates that accumulate when carbon fixation exceeds

incorpo-ration into new cell material (Fogg, 1983). In accordance with this assumption, the %

DOC excretion has been found to be positively correlated to light stress, i.e. both high

and low light intensities (Zlotnik and Dubinsky, 1989). In the present study, however,

the lowest % DOC excretion was observed in the algal cells exposed to light stress

situations, i.e. UV-B radiation and low light intensity (Fig. 3C). This result remains

unexplainable, but the % DOC excretion levels were in the order 0–2% for all light

treatments, which is negligible compared to levels previously reported, e.g. 5–50%

(Zlotnik and Dubinsky, 1989), and up to 90% (Fogg et al., 1965). The DO C was

correlated to the PO C (Fig. 3) and was not biomass dependent (compare Fig. 2A and

Fig. 3C), which implies that the exudation could not solely be explained by passive

diffusion, as suggested by Bjørnsen, (1988).

The carotenoid / chl a ratio has been implied to increase upon UV-B exposure (Goes et

al., 1994; Quesada et al., 1995; Roos and Vincent, 1998). In the present study, there was

no difference in the carotenoid / chl a ratio of UV-B-exposed algae and cells exposed to

HL and ML, while the algae exposed to low light exhibited a lower ratio (Fig. 5C).

Assuming that HL and ML are light saturating conditions for E. huxleyi in this study, the

observed variations in the carotenoid / chl a ratio indicate that the ratio reflect the

response to light in general rather than UV-B alone. There was no decrease in any

pigment concentration in the UV-B-exposed algal cells during the light period (Fig. 6),

implying that there was no photobleaching of the pigments (Nielsen and Ekelund, 1993;

¨

Donkor and Hader, 1996).

The chl a content per cell surprisingly peaked at the highest light intensities (Fig. 6A),

probably because of an endogenous circadian rhythm. Since the algae had been adapted

to the light cycle for four days, the chl a content seemed to be related to an endogenous

circadian rhythm rather than to the current light regime (Post et al., 1984; Buma et al.,

1991). Unlike chl a, fucoxanthin, an accessory pigment (Goericke and Welschmeyer,

1992), responded with increasing concentration at the low light intensity already after 2

(4)

h of incubation and decreased during the dark period in all cultures (Fig. 6B). Another

pigment, the marker pigment for the algal group prymnesiophytes, 199-hex, does not

seem to be of photosynthetic importance, in contradiction to the suggestion by Haxo

(1985), since the lowest concentration was found in the low light intensity (Fig. 6C).

Our results is in accordance with Van Leeuwe and Stefels (1998), who found that the

light-harvesting pigment fucoxanthin, was transformed into 199-hex in the marine

prymnesiophyte Phaeocystis sp., when the algal cells was shifted from low light to high

light intensity. It does not seem, however, that 199-hex is as important for light

protection as diadinoxanthin, which was the only pigment where the highest

con-centration was found in the algal cells exposed to UV-B radiation (Fig. 6E).

To summarize, we found termination of cell division, changes in cell volume, and

14 14 14

inhibition of uptake (PO C) and excretion (DO C) of

C at UV-B doses

representa-tive for the top layer of clear open oceans (Behrenfeld et al., 1993). Since E. huxleyi is a

common bloom-forming algae with a wide geographic distribution, i.e. both warm- and

cold-water forms exist, there is a risk that current UV-B intensities have an impact on

primary production and phytoplankton biomass. The light regime and light intensity,

however, are much more uniform in laboratory studies than in nature, so care should be

taken when laboratory results obtained from UV-B experiments are extrapolated to

natural plankton communities, hence this hypothesis remain speculative and needs

further investigation.

Acknowledgements

We thank Tom Nielsen for kindly providing the light equipment and for helping with

¨

the light measurements, Louise Schluter, Morten Søndergaard, and Kim Gustavson for

the critical reading of the manuscript, Kristian Møller Christensen and Winnie Martinsen

for technical assistance, and Kathleen Gail Jensen for linguistic corrections. This work

was supported by the Danish Environmental Research Programme, the Danish Research

Academy, an EU MAST III contract (MAS3-CT96-0053-PHASE), an EU MAST III

contract (MAS3-CT97-0154-MIDAS), and by VKI.

[RW]

References