Impact of Solar UV Radiation on Uptake of 15N-Ammonia and 15N-Nitrate by Marine Diatoms and Natural Phytoplankton

Biochem. Physiol. Pflanzen 187, 293-303 (1991) Gustav Fischer Verlag Jena

Impact of Solar UVRadiation on Uptake of1SN-Ammonia and lSN-Nitrate by Marine Diatoms and Natural Phytoplankton GUNTER DaHLER, ERIK HAGMEIER1), EVELYN GRIGOLEIT and KLAUS-DIETER KRAUSE Botanisches Institut der Universitat, Frankfurt a. M. and I) Biologische Anstalt Helgoland, FRG Key Term Index: nitrogen metabolism; marine diatoms, phytoplankton

Summary Natural marine phytoplankton populations and a community of cultured marine microalgae were tested in UV -transparent and non-transparent Plexiglas vessels after addition of 15N labelled inorganic nitrogen under natural light. Uptake rates of 15N-ammonia and 15N-nitrate were reduced after solar radiation at UV exceeding 590 J m- 2 d -I and in near surface waters of the North Sea. Assimilation of 15N_ ammonia was found to be more sensitive to enhanced UV levels than uptake of 15N-nitrate. Patterns of 15N label of several amino acids varied in dependence of UV influence and phytoplankton species. Results were discussed with reference to photo inhibitory effects and UV damages on nitrogen metabolism.

Introduction Solar radiation, its intensity, variability, and composition determines photosynthetic capacity and influences other processes like nitrogen metabolism of phytoplankton (FALKOWSKI 1984 and references therein). Since the introduction of the 15N tracer technique into marine research (DUGDALE and GOERING 1967) some insight into nitrogen metabolism of phytoplankton and other marine microalgae have been obtained (COLLOS and SLAWYK 1980; SYRETT 1981; ULLRICH 1983; WHEELER 1983 and references therein). Recent studies concerned the uptake and assimilation of ammonia and nitrate and the effect of ammonia on nitrate uptake (MAESTRINI et al. 1986; DORTCH 1990 and references therein). The use of 15N to estimate nitrogen uptake especially in long-term experiments was critically considered with reference to the effect of isotope dilution (DUGDALE and WILKERSON 1986; COLLOS

1987). DaHLER (1985) showed that the assimilation of nitrogen compounds by phytoplankton was affected by ultraviolet radiation. Nitrogen metabolism was found to be more sensitive to UV-B exposure than CO 2 fixation (DaHLER et al. 1987). Photoinhibitory effects on phytoplankton development under high light intensities were attributed to ambient solar UVB (280-315 nm) by JOKIEL and YORK (1984), while MASKE (1984) could demonstrate an inhibition of CO 2 uptake due to UV-A (315-400 nm). GUBERT et al. (1985) postulated differences in light sensitivity of winter and summer phytoplankton populations at the east coast of the U. S .. Salinity stress enhances the photoinhibition of photosynthesis (NEALE and Melis 1989). The ecological importance of photoinhibition in aquatic ecosystems at a basic photochemistry level has been summarized by NEALE (1987). Strategies of photo adaptation BPP 187 (1991) 4

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in connection with photosynthetic efficiency has been studied with the wide distributed Phaeocystis pouchetii isolated from Antarctica (PALMISANO et al. 1986) and from the North Sea (JAHNKE 1989). Marine phytoplankton near surface waters may be damaged by increased UV radiation. Damaging UV can penetrate to ecologically significant depths (SMITHS 1989 and references therein). The aim of this study was to supplement previous research on the uptake of 15N by testing different marine phytoplankton populations under light with and without the UV part of irradiance.

Materials and Methods Phytoplankton samples for our experiments were partly composed from cultured algae kept in the Botanisches Institut der Universitat Frankfurt (Ditylum brightwellii (West) Gron., Lithodesmium variabile Takano, Odontella sinensis (Grev.) Gron., Thalassiosira punctigera (Castr. Fryxell, Simonsen et Hasle) and Thalassiosira rotula Meun.) or in the Biologische Anstalt Helgoland (Rhizosolenia delicatula Cleve, Skeletonema costatum (Grev.) Cleve, Thalassiosira rotula Meun., Prorocentrum micans Ehrenb. and Scrippsiella faeroeense (Pauls.) Balech et Soares. Natural communities tested were collected at the experimental days near Helgoland by M.B. "Aade", containing mainly the diatoms Cerataulina bergonii Perag., Chaetoceros spec., Coscinodiscus spec., Rhizosolenia setigera Brightw., colonies of the haptophycean Phaeocystis pouchetii (Harlot) Lagerh. and the dinoflagellate Ceratium fusus (Ehrenb.) Dujard. In another series of experiments impact of UV -B radiation on inorganic nitrogen uptake by natural phytoplankton were studied under laboratory conditions (UV lamps, Philips TL 40112 and cut-off filters WG 305). Samples were collected in the German wadden sea area of List; Sylt near the harbour. Isolated Phaeocystis pouchetii grown as unialgal culture was used for the UV experiments, too. Phytoplankton samples were washed with filtered sea water, resuspended in fresh sea water and transferred into special Plexiglas vessels, UV-transparent (Rahm GmbH, Darmstadt no. 2458) and 700

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BPP 187 (1991) 4

Frankfurt under controlled conditions with constant artificial light (see Figure 1 for UV part of radiation). After exposures, plankton samples were collected on Whatman filters (GF/C, 47 mm jil) and dried at 70°C. Further procedures to measure 15N contents followed the DUMAS method, as described by DOHLER and ROSSLENBROICH (1981), using an atomic emission spectrometer Statron NOI-5 (Zeiss, Jena). Results are expressed as relative frequency ("enrichment") of 15N in the samples or as pg 15N assimilated per cell. Total carbon and nitrogen contents of phytoplankton were determined by an analyzer of Carlo Erba (model 1106). Chlorophyll a was measured in 90% aceton extracts of phytoplankton according to SCOR-UNESCO (1966). Data ofpH, salinity, temperature, inorganic phosphorus, ammonia, nitrate and nitrite in Helgoland sea water (used for resuspension of phytoplankton in experiments) were kindly made available by P. MANGELSDORF, Biologische Anstalt Helgoland. In Frankfurt, incoming radiation in UV and visual ranges was measured by an equipment of Optronic Lab. Inc., USA obtained from Prof. Dr. M. TEVINI, Karlsruhe. Intensities in the UV range were weighted according to CALDWELL (1971) by a function considering the effect on plants. During our experiments in Helgoland, only total incoming radiation was registered on the institute's roof by a pyranometer (type Robitzsch-Fuess). A very rough idea about UV intensities in the depths of sample exposures can be derived from local transparency estimations (Secchi disk) and by conversions found in literature (CALKINS and THORDARDOTTIR 1982): Incoming global radiation between 104 and 335 J cm- 2 h- 1could probably result in 65-21OJ m- 2 h- 1UV -B atthe sea surface, in42-137 J m- 2 h- 1atO.17 mand 3.3-1O.5Jm- 2 h- 1 at 1.17m.

Results In Frankfurt, a community of diatom species (pure cultures) were exposed for 2 days to natural light in August 1986. Solar UV dosis varied between 193 and 608 J m- 2 d- 1whereas white light intensities changed from 870 to 2660 kJ m- 2 d- 1. Uptake of15N-ammonia and 15N_ nitrate by the assemblage of marine diatoms is summarized in Fig. 2 at different light conditions. In general, ammonia is incorporated in larger quantities than nitrate and uptake continues during the 8 hours of the experiments. A reduction of assimilation in UV transparent vessels compared to controls could be found from UV dosis of 590 J m- 2 d- 1 upwards (data not shown). Uptake of nitrate increased slightly at higher light intensities, while ammonia utilization was reduced in the range tested (Fig. 2B). Parallel experiments led to similar results; However, statistic was not possible due to the variation in environmental conditions. Chlorophyll a content increased at 360 J m- 2 d- 1but was reduced at higher UV doses. Growth of the diatom species responded differently: Thalassiosira rotula and Odontella sinensis proved to be most sensitive while growth of Ditylum brightwellii increased. Lithodesmium variabile was affected least of all tested diatoms by UV (data not shown). In Helgoland, phytoplankton of pure cultures and natural populations from net hauls were tested in May and August 1987. Figure 3 presents data on 15N-ammonia uptake by natural phytoplankton at different depths. In general, a diminution of 15N-ammonia assimilation was observed in the UV transparent cuvettes compared to results from phytoplankton in vessels not transparent to UV. High global light intensities led to significantly lower assimilation rates independent of UV transparencies. Relatively low levels of ambient solar radiation resulted in a reduction of the uptake rates. However, a contradictory behaviour was found during the uptakeofI5-N-nitrate by natural plankton one day later (Fig. 4). A damaging effect of ambient solar radiation on nitrate utilization was found at higher dose, only. Here, higher BPP 187 (1991) 4

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Fig. 2. Utilization of15N-ammonia (110 11M) and 15N-nitrate (330 11M) by an assemblage of marine diatoms. Unialgal cultures of the marine diatoms (Dithylum brightwellii, Lithodesmium variabile, Odontella sinensis, Thalassiosira punctigera and Thalassiosira rotula) were exposed to natural light conditions in vessels transparent (1) and non-transparent to UV (2, controls). Rates were expressed as 15N enrichment. Light intensities during 2 days: A 400-750 nm: 95,4'103 mW m- 2 (2580 kJ m- 2 d- 1); 280-320nm: 21,8'103 mW m- 2 (590 J m- 2 d- 1); August 8-9,1986. B 400-750 nm: 76,08'103 mW m- 2 (2035 kJ m- 2 d- 1 280-320 nm: 13,5.103 mW m- 2 (362 J m- 2 d- 1); August 11-12, 1986. The weighting function according CALDWELL (1971) has been used for calculating the UV dose. Further details in Material and Methods. Fig. 3. Uptake of 15N-ammonia (96 atom%, final concentration 100 11M) by natural phytoplankton in different depths, May 21, 1987. Autotropic organisms: 3281640 cells dm- 3 , with 94% Phaeocystis pouchetii, 5.1 % Rhizosolenia setigera, and low numbers of Coscinodiscus spec., Chaetoceros spec., Cerataulina bergonii and Ceratium fusus. Chlorophyll a: 79.9 ng per mg phytoplankton carbon; C/N ratio 6.93. Nutrients in experimental water: pol-: 0.24, NHt: 3.04m N0 3 : 39.57, NOi: 0.99/tM. Salinity: 29.4%0, pH 7.5, water temperature: 8.3°C. Incubation depths: I: 17, II: 117 cm. Times of exposures 12.00-13.00. Global radiation at the surface: 36,9 .104 mW m- 2 h- 1 (133 J cm- 2 h- 1). Results from UV transparent vessels in open bars, from UV non-transparent vessels in hatched bars.

light intensities (Fig. 4A) resulted in higher uptake rates and the protecting effect on the vessels not transparent to UV was indicated only near the surface. These results indicate less sensitivity of nitrate assimilation to enhanced light intensities compared to ammonia uptake. The ammonia content of the natural water used in the experiment was low (1.37 /lM) and should not have influenced nitrate uptake of the phytoplankton. In another series of experiments a community of several algae from pure cultures was 296

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Fig. 4. Uptake of 15N-nitrate (95 atom%, final concentration 100!JM) by natural phytoplankton in different depths, May 22, 1987. Autotrophic organisms: 310160 cells dm- 3 , with 60% Phaeocystis pouchetii, 21.7% Rhizosolenia setigera, 7.7% deratiumfusus and lower numbers of Chaetoceros spec., Coscinodiscus spec., Cerataulina bergonii, Nitzschia seriata, Dinophysis acuminata. Chlorophyll a: 67 ng per mg phytoplankton carbon; CIN ratio 5.83., Nutrients in experimental water: pol-: 0.21, NHt: 1.37, NO): 36.37, NOi: 0.99IlM. Salinity: 28.0%0, pH 7.2, water temperature: 9.2°C. Incubation depths: I: 17, IT: 117 cm. Times of exposures A: 11.00-12.00, B: 13.45-15,20. Global radiation at the surface: A: 58.0·104 mW m- 2 h- 1 (209 J cm- 2 h- 1), B: 28,88,104 mW m- 2 h- 1 (104 J cm- 2 h- 1). Results from UV-transparent vessels in open bars, from UV non-transparent vessels in hatched bars.

exposed to field conditions in Helgoland. The lowest uptake rates of 15N-ammonia were found in the sample under the highest light intensity (Fig. 5AI). Here, near the surface, the influence of UV radiation is likely to enhance the reductive effect of strong light. The other surface sample (Fig. 5BI) received less radiation, the 15N uptake was higher and the difference between results from micro algae in UV-transparent and non-transparent vessels less pronounced. In samples with medium light intensities at 117 cm depth (All and B II) the relation seems to be inverse. This might be due to the transparency of the UV-transparent vessels. In these experiments with cultured algae 15N uptake with ammonia (Fig. 4) was also higher than utilization of nitrate. Assimilation of 15N-nitrate by an assemblage of cultured microalgae was significantly lower than that of 15N-arnmonia. The uptake rate in UV non-transparent vessels was higher than in UV-transparent Plexiglas in near surface waters (data not shown). Figures 3 - 5 show results from 4 days in May. There are 3 more days with experiments in May and 6 in August 1987. During research in August, incoming radiation was reduced by clouds and exposure times were prolonged. Parallels with 15N-ammonia and 15N-nitrate on August 19, 21, 23 and 24 confirmed the preference for ammonia assimilation by the algae. Summarizing results from May and August concerning UV influence in near-surface samples of phytoplankton: While a reduced uptake of 15N~labelled compounds by phytoplankter in transparent vessels was found in BPP 187

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Fig. 5. Uptake of15N-ammonia (96 atom%, final concentration 100 !1M) by cultured microalgae at different depths, May 24, 1987. Community composition (3700000 cells dm- 3 ): 53 .3% Skeletonema costatum, 29 .1 % Thalassiosira rotula, 8.9% Prorocentrum micans, 8.1 % Rhizosolenia delicatula, 8 % Odontella sinensis and 6% Scrippsiella fa eroeense. Chlorophyll a : 101.4 ng per mg phytoplankton carbon ; CIN ratio 5.63 . Experimental water : salinity 30.3 % , pH 8.0. Incubation depths : I : 17, II : 117 cm. Times of exposures A: 13.45-14.50, B: 17.30-18.30. Global radiation atthe surface : A: 81.1 . 104 mW m- 2 h- I (292 Jcm- 2 h- I ), B : 54.7 . 104 mW m- 2 h- I (197 Jcm- 2 h- I ) . Results from UV-transparent vessels in open bars, from UV non-transparent vessels in hatched bars .

14 cases, there was no distinct difference between parallels (in UV-transparent vessels) in analyses. The reason for it was the different light conditions during these field experiments; therefore , statistic calculations not are possible. Additionally, we studied the 15N incorporation into several amino acids of natural phytoplankton (dominated by C eratium fusus) and of pure cultures of Skeletonema costatum under natural conditions (Table 1). Major parts of 15N label could be found in glumatine of both samples tested. A relatively high 15N enrichment was measured also in aspartic acid and asparagine, while only low values could be detected in alanine and glutamic acid. A reduction of 15N incorporation into alanine and glutamic acid in samples exposed in quartz bottles was obvious . In addition to the studies near Helgoland we have carried out laboratory experiments with phytoplankton collected from the Wadden Sea at the harbour of Li st, Sylt in August 1989. The natural phytoplankton populations were exposed to UV-B in UV-transparent cuvettes . The impact of UV -B on the uptake of inorganic nitrogen was investigated during irradiance up to 3 h (Figs . 6and 7). Generally, a damaging effect could be observed after 30 or 60 min UV -B radiation. The utilization of 15N-ammonia and 15N-nitrate of phytoplankton containing mainly Phaeocystis pouchetii (> 95%) was very sensitive to UV-B radiation (Fig. 6) . A practically total inhibition of 15N-nitrate uptake has been found . The same results were obtained using phytoplankton under similar field conditions within the next 5 days, Pure 298

BPP 187 (1991) 4

Table 1. J5N incorporation into several amino acids of natural phytoplankton (90% Ceratium fusus) arul of an unialgal culture of Skeletonema costatum. Samples were exposed in different depths (1, 3, and 6 m) in glass (G) and quartz (Q) bottles. Results are expressed as percentage 15N enrichment (a) and as percentage of total 15N incorporated (b) . Incubation of samples for 2 h with ISN14Cl (96 atom%, final concentration 1 mM), average light intensities : 665 W m- 2 at 1 m, 133 W m- 2 at 3 m and 67 W m- 2 at 6 m depth. 1m

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cultures of Phaeocystis pouchetii showed the same response to UV-B exposure than the natural phytoplankton (data not shown). Short-term experiments indicate on a high sensitivity of 15N-nitrate uptake by phytoplankton consisting of Phaeocystis whereas a diminution in phytoplankton containing Ceratium and Prorocentrum was observed after 2 h UV -B irradiance (Fig. 7). Assimilation of 15N-ammonia was significantly higher and more sensitive to UV-B than that of 15N-nitrate.

Discussion Results obtained from phytoplankton and communities of algal cultures showed that uptake of nitrogenous compounds in samples exposed close to the sea surface was mostly reduced in UV -transparent vessels when compared to the uptake in vessels not transparent to UV (Figs. 3-5). It is known that enhanced levels of UV-B can damage the nitrogen metabolism of rnicroalgae, especially of marine diatoms (DOHLER 1985, 1988). Similar results were obtained from phytoplankton populations exposed to UV -B under laboratory conditions (Figs. 6-7). Generally , uptake of 15N-ammonia was more affected by UV-B radiation than that of 15N-nitrate. Acontradictionary response was observed with phytoplankton consisting of Phaeocystis (Fig. 6) or using pure cultures of Phaeocystis. An explanation for the higher sensitivity of ammonia uptake to UV -B , compared to that of nitrate, cannot yet be given . The different response of 15N-ammonia uptake to 15N-nitrate indicates a different uptake system of both inorganic compounds (see WHEELER 1983). The transport system of ammonia might be directly affected by UV irradiance or via a damage to the regulation BPP 187 (1991) 4

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mechanism of the surge uptake. On the other hand, photoinhibition may be caused by influences ofUV radiation and as well of high white light intensities (NEALE 1987). Several authors have indications of the importance of UV-B (DaHLER 1985; HALLDAL 1967, WORREST et al. 1981) to growth and metabolic processes of phytoplankton. Photoinhibitory effects caused by UV -A on the carbon metabolism of phytoplankton could be demonstrated by BUHLMANN et aI. (1987) and MASKE (1984). Our results presented in this paper cannot distinguish between inhibitions by UV -A and UV -B. However, it could be shown that UV -B inhibits assimilation of inorganic nitrogen compounds of marine diatoms (DaHLER and STOLTER 1986). Enhanced levels of UV -B resulted in an increase of glutamine (DaHLER 1985). It is known that glutamine may act as an effector in the inhibition of nitrate assimilation (RIGANO et aI. 1980). The data of 15N incorporation into amino acids indicate an influence ofUV radiation of the glutamine synthetase/glutamate synthease (GS/GOGAT) pathway (Table 1) , which is identical with findings of other authors (DaHLER 1985 ; FALKOWSKI 1984). 15N enrichment in alanine of algae exposed in quartz bottles was significantly reduced . This can be attributed to the impact of solar UV radiation on key enzymes. A possible explanation is an inhibition of the alanine aminotransferase activity; a reduction by UV-B radiation up to 90% was found. The diminution of 15N incorporation into glutamic acid observed in Skeletonema cells 300

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indicates an inhibition of glutamate synthase. This is also in agreement with other results (DOHLER 1988), where a reduction up to 19% was measured. Summarizing, ultraviolet irradiance can affect the key enzymes of nitrogen metabolism. This interpretation could be supported by studies with synchronized Synedra (DOHLER 1990; DOHLER and I. BIERMANN 1991 in preparation). In natural environments, however, phytoplankton cells are mostly not forced to stay in one depth as in our experiments. The transition from high to low light intensities and vice versa within relatively short time ranges from 1 to 10 h may be important for metabolic processes (LEWIS et al. 1984). MORTAIN-BERTRAND et al. (1987) found an influence of different light periods on the carbon pathway of Skeletonema costa tum, using the 14C tracer technique and enzymatic tests : The rate of ~-carboxylation varied in dependence of the dark! light cycle. KULLENBERG (1982) considered the role of vertical mixing in the impact of UV radiation on marine plankton organisms which may have different sensitivities, response and recovery times. Further experiments should elucidate the many of questions still unanswered.

Acknowledgements Thanks are expressed to the Bundesministerium fUr Forschung und Technologie, Bonn and the Gesellschaft fUr Strahlen- und Umweltforschung, Miinchen for their support ofthis work. We want to thank also all collaborators of the Biologische Anstalt Helgoland and of the Wattenmeer Station List, Sylt, for helping us. BPP 187 (1991) 4

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Received May 21, 1990; revised form accepted February 13, 1991 Authors' addresses: Prof. Dr. G. Dohler, Johann-Wolfgang-Goethe-Universitat Frankfurt a. M., Botanisches Institut, Siesmayerstr. 70, Postfach 111932, W -6000 Frankfurt a. M. 11. Dr. E. HAGMEIER., Biologische Anstalt Helgoland, W - 2192 Helgoland.

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