Impact of natural and artificial UVB radiation on motility and growth rate of marine dinoflagellates

Jom-~lof J

ELSEVIER

Journal of Photochemistry and Photobiology B: Biology 27 (1995) 73-79

•

B:HIOLOGY

Impact of natural and artificial UVB radiation on motility and growth rate of marine dinoflagellates Tom Nielsen, Lars Olof Bj6rn, Nils G.A. Ekelund * Section of Plant Physiology, Lurid University, Box 117, S-22100 Lurid, Sweden Received 16 March 1994; accepted 27 July 1994

Abstract

The growth rates and motility of dinoflagellates were studied in the field in the presence or absence of UVB radiation, as well as in the laboratory under artificial radiation conditions. Photosynthetically active radiation (PAR, 400-700 nm) and UVB radiation showed large variations due to cloud cover and seasonal changes in natural daylight. In Swedish coastal water, UVB radiation was attenuated to about 10% of surface irradiance at a depth of 120 cm. There was no significant difference in the motility of two strains of Prorocentrum minimum (Atlantic, LAC4LI; Kattegat, LAC6KA83) kept in the water at different depths (35 and 120 cm) for 4 h, with or without natural solar UV radiation, except for a day with high UVB irradiance (1.2 W m-2), which decreased the motility at a depth of 35 cm for the two species). Simulated in situ experiments with 2 h natural daylight, with and without natural UV radiation (UVB, 1.6 W m-2), had a dramatic effect on the motility of Gyrodinium aureolum. Artificial UVB radiation from UV lamps (4 h, 2.72 kJ m -2 day -l, biologically effective UVB radiation, UVBaE ) in the laboratory decreased the motility of Heterocapsa triquetra (LAC20) by 56% and the two strains of P. minimum (Atlantic, LAC4LI; Kattegat, LAC6KA83) by 43% and 36% respectively; the growth was inhibited for all species, as well as for Amphidinium carterae (LACIKA83), when organisms were exposed to more than 0.7 kJ m -2 day -1 of UVBBE radiation. Keywords: Dinofiagellates; Growth rate; Motility; Phytoplankton; UVB radiation (280-320 nm)

1. Introduction

Recent field work has shown that an increase in UVB radiation (280-320 nm) is associated with a decrease in phytoplankton productivity [1,2]. UVB radiation affects different biological processes such as photosynthesis, nitrogen metabolism, growth rate, motility and orientation of phytoplankton [3-9]. Although UV radiation penetrates to significant depths in natural waters, most of the data on UVB effects on phytoplankton have been collected under laboratory conditions. One difficulty in the comparison of field work with laboratory experiments is the difference in spectral composition between natural daylight and lamp radiation both in the UV region and in the region 400-700 nm. UVB radiation has a negative effect on primary production, but so too does UVA radiation (320-400 nm). At the same time UVA may also cause photoreactivation [1,2,10,11]. The relationship between UVB, UVA and photosynthetically active radiation (PAR) is * Corresponding author.

1011-1344/95/$09.50 © 1995 Elsevier Science S.A. All rights reserved SSDI 1011-1344(94)07059-8

important for the biological response. PAR and UVA are almost unaffected by variations in column ozone, while UVB radiation changes with 03 [2]. The ratio of UVB irradiance to total irradiance is greater with 150 Dobson units (DU) of ozone than with 350 DU. Formerly, it was thought that UVB radiation was rapidly attenuated and would hardly affect aquatic organisms. However, recent investigations have shown that UVB radiation can be detected down to depths of 60-70 m in the Southern Ocean, but without any effect on the rates of primary production below 25 m. The ozone hole (150 DU) increases the effective penetration of UVB radiation into the water column by about 7 m [2]. Increased UVB radiation due to the decline in the concentration of stratospheric 03 has been observed in the Antarctic during the austral spring [2,12]. Even at temperate latitudes, during the cooler months UVB radiation may have increased by as much as 10% during the last few decades [13,14]. In this study, we examine the effects of radiation on the motility and growth rates of different phytoplankton organisms under natural daylight conditions in Sweden and in the laboratory.

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T. Nielsen et al. / J. Photochem. Photobiol. B: BioL 27 (1995) 73-79

2. Materials and methods

significance of the motility differences (P=0.05) was determined using the unpaired t-test.

2.1. Organisms and culture conditions The marine dinoflagellates Heterocapsa triquetra (LAC20), Amphidinium carterae (LAC1KA83), Gyrodinium aureolum Hulburt (Ba6), Prorocentrum minimum (Atlantic, LAC4LI) and Prorocentrum minimum (Kattegat, LAC6KA83) were isolated from Kattegat by Dr. Edna Granrli (Department of Marine Ecology, Lund). The cells were grown in f/2 medium (modified by Dr. Granrli) in 100 ml Erlenmeyer flasks [15]. For control treatment, glass flasks were used, while quartz glass was used for treatments with UVB radiation. The cells were grown in growth rooms at 19 °C with a 16 h-8 h light-dark cycle. The control cultures were grown in white light with an irradiance of 19 W m -2 (400-700 nm). The Edenmeyer flasks were placed at a 45 ° angle to the light.

2.2. Determination of growth The organisms used for the determination of the specific growth rate were cultured for 7 days. Cell counts were made with a haematocytometer at the start and after 7 days. Knowing the initial number of cells (No) and the number of cells at the end of the period (N), the specific growth rate can be calculated by the formula tz--k x log(N/No)/t where t is the time in days and k = 3.3222. For the growth curves, the cells were counted every day and exponential growth was observed from the beginning of the control experiments to the tenth day after inoculation (data not shown). The specific growth rate represents the number of population doublings per day. The experiments were replicated at least five times.

2.3. Measurement of motility Determinations of motility were carried out with organisms grown under different light conditions (control and UVB radiation). The motility was determined with a fully automatic, computer-controlled video analysis system. The program was written by Professor D.-P. H~ider and coworkers using algorithms developed for cell detection and tracking [16]. An IR-sensitive video camera (Ikegami, CCD) was mounted over a Nikon Optiphot microscope (4 x objective), and the built-in lamp was used in combination with an IR transmitting cut-off filter (RG 780 nm, 2 mm, Schott and Gen., Mainz, Germany) to produce the monitoring radiation. The organisms were introduced into a fiat glass cuvette (7 mm × 20 mm x 1 ram) and the cuvette was placed under the microscope. The motility of the cells was calculated from tests of 1000 organisms and replicated at least three times. The

2.4. Experimental procedure 2.4.1. Artificial light conditions (1) Control PAR (400-700 nm, 19 W m -2) light was obtained from four 400 W metal halogen lamps (Power Star HQIL, Osram). (2) Treatment conditions consisted of control plus 1, 2 and 4 h UVB radiation per day from one or two 40 W sunlamps (FS 40, Westinghouse Elec. Corp. Lamp Div., Bloomfield, NJ, USA). Cellulose acetate (CA) film (thickness, 0.13 mm) was preburnt for 72 h at a distance of 1 m from the four UV lamps in order to minimize the change in its filter properties, and was used to remove the shorter wavelength components not encountered in nature. In comparison, the UVB experiments by Ekelund [9] were made without CA, The data for PAR and UVB radiation are shown in Table 1. 2.4.Z Natural sunlight experiments (1) Plankton directly from the culture room were placed in Plexiglas (PMMA-Acrylplast GS 233, 4 mm, R6hm GmbH, Germany) cuvettes (20 cm x 20 cm) with no transmission of UVB radiation or Plexiglas (Acrylplast GS 2458, 4 mm, R6hm GmbH, Germany) cuvettes which transmit UVB radiation. The cuvettes were cooled with water; the temperature in the cuvettes was kept at 17-20 °C. The cuvettes were placed on a tilting table on the roof of the building of the Section of Plant Physiology in Lund (55.7°N, 13.4°E). The values for the different light regimes are given in Table 2. (2) Glass and quartz bottles were placed on a holder (maximum, 8 bottles) at 90° to the water surface for 4 h. The holders were kept at 35 and 120 cm below the water surface of the Gullmar Fjord close to the Marine Biological Station at Kristineberg (58.2°N, 11°E). The light conditions for the different experimental days above the water surface are shown in Table 1. Table 1 Natural sunlight (field, motility studies) and artificial light (laboratory, growth studies) conditions (unweighted irradiance) for the different experiments Field/laboratory

Field 4 August 1992 8 August 1992 10 August 1992 Laboratory

PAR (W m -2)

UVB (W m -z)

200 224 155 19 19

1.1 1.2 0.6 0.31 (1 lamp) 0,57 (2 lamps)

75

T. Nielsen et al. / J. Photochem. Photobiol. B." Biol. 27 (1995) 73-79 t_

"rable 2 Effect of natural sunlight conditions ifi Lund on 23 July 1992 for 2 h on the motility of the dinoflagellate G. aureolum PAR (W m -2) Indoors ,~crylplast GS 233 outdoors) /~crylplast GS 2458 outdoors)

UV (W m -2)

19 346

Speed ( ~ m s -I)

Motile cells (%)

159 84

90 7

0

0

100 t

80 60 .m

40

°m

346

1.6

"7, 20 ,.d"

L 5. Light measurements

Natural and artificial PAR and UVB radiation in ~ir were measured with a spectroradiometer (model 42, Optronics Lab., Orlando, FL, USA) (using a Teflon diffuser for 250-360 nm) interfaced with a Hewlett Packard 85 computer. Light measurements were performed at noon under different weather conditions: clear days (0-1/8 clouds) and cloudy days (7-8/8 clouds). PAR and UVB radiation in water were measured with ~n underwater sensor (Lambda Inst. Corp. UWQ2431";'905) and a broad-band UVB sensor (Delta-T Devices lad., Cambridge CB5 0F_J, UK), which was kept in a waterproof box with a Plexiglas (Acrylplast GS 2458, z- mm, R6hm GmbH, Germany) window transmitting UVB radiation. The spectral response curve for the broad-band UVB sensor is shown in Fig. I(A). The broad-band UVB sensor was calibrated against the ~pectroradiometer (Fig. I(B)) in air by variation of the UVB radiation in natural sunlight. The UVB values ~rom different depths were not corrected for the diflerences in transmission at different wavelengths in water, the different water types due to the amounts of chlorophyll and other particles, or the angle at which l he light penetrates the water (due to the refraction at the water surface). The UVB values from the tmderwater measurements (Fig. 2) should therefore be considered as semiquantitative. The biologically effective radiation (UVBBE) was based on the generalized plant action spectrum to UVB radiation [17,18]. The action spectrum was normalized :o unity at 300 nm. Figs. 3(A) and 3(B) [19] show the "action" curve which is obtained by multiplying the daylight spectrum, point by point, by the plant action :,pectrum. The "integral of action" is then obtained by :,umming the values for "action" for all wavelengths. Fhe two curves with and without ozone depletion will :herefore give different values of the "integrated biologically effective radiation". From the measurements of the spectral irradiance, we computed the expected percentage photosynthesis .'ate not inhibited by UV using the model of Cullen .'t al. [10] and their absolute action spectrum for the ~ensitivity of Prorocentrum micans. Spectral irradiance

A

m

0 280

300

320

34O

Wavelength (nm)

0.08

B ~"

•

0.06

•

0.04

;~

0.02

0.00 0.0

0.5

W m

1.0 .2

z 1.5

(Spectroradiometer)

Fig. 1. Spectral response curve for the broad-band U V B sensor according to the manufacturer (A) and a calibration curve for the broad-band sensor obtained by comparison with the spectroradiometcr

(~). values from 286 to 360 nm were used, since the action spectrum at longer wavelengths could not be read from the graph.

3. Results

The natural light conditions at the Marine Biological Station in Kristineberg exhibit large variations between sunny days and cloudy days both in the visible and UV regions (Figs. 4(A) and 4(B)). The penetration of PAR and UVB radiation into the water shows that PAR is highest at about 13 h (solar noon) and that UVB radiation is more constant between 11 and 15 h compared with PAR (Figs. 2(A) and 2(B)). The attenuation of UVB radiation is very rapid and at 120 cm only about 10% is left. The effect of UVB radiation on the growth rate was studied over 7 days in the laboratory under PAR and 2 h UVB radiation (UVBBE of 0, 0.46, 0.68, 0.93 and 1.37 kJ m -2 day-1) per day. This resulted in a decrease in growth rate for all species (Fig. 5). The UVB radiation for 2 h was used to simulate natural light conditions

76

7". Nielsen et al. / J. Photochem. Photobiol. B: Biol. 27 (1995) 73-79

A

~

0.60 \ ,9[--~...--" .°°-"

2OO

\

°-,.....

°.°-"

Plant action spectrum ~

Daylight without

/

. ..........

.°""

0.40

~cm A

180 I

A

-.

.~

E t-

120cm

J

I

I

I

A

E

E

c "o

0.20

I

_.

",-............

0.00

(J p. 2

°m

"o

L,.

T,.

surface

0.60 ~\ ~

Plant action spectrum

D

m

\

o

Daylight with

~

/

35 cra 1 '''~"

"~""

~

..

0.20

~

i I

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

0 11

I

12

I

I

13

14

,----,/

"

120era

/-,"",,,

~

Action

15

T i m e o f d a y (h) Fig. 2. Irradiance values of PAR (400-700 rim) (A) and UVB radiation (280-320 nm) (B) above the water surface and at 35 and 120 cm depths in the Gullmar Fjord close to the Marine Biological Station at Kristineberg (58.2°N, 11.3°E).

when the UVB irradiance is highest during the 4 h around noon. With 4 h artificial UVB radiation over 7 days, there was no growth of the phytoplankton. UVB radiation from UV lamps for 4 h (UVBBE of 2.72 kJ m -2 day -1) decreased the motility by 56% for H. triquetra and by 36% and 43% for the two strains of P. minimum (Kattegat and Atlantic respectively) (Table

3). The field experiments at different depths (35 and 120 cm) did not show any significant (t-test, P<0.05) variations between glass and quartz bottles for the two strains of P. minimum, except for P. minimum (Kattegat) at 35 cm on 8 August 1992, where the organisms in the glass bottles showed higher speed (Table 3). Indoor measurements of the speed were performed to check the behaviour of the phytoplankton organisms before the experiments were started. The experiments with G. aureolum showed a dramatic effect on the speed and the number of motile cells on a sunny day in Lund both with and without natural solar UVB radiation. However, the motility of the UVBtreated cells was affected more strongly (Table 2).

260

300

320

340

Wavelength (nm) Fig. 3. "Plant action spectrum" together with the daylight spectrum on a clear day (June 15) without ozone depletion (A) and with a calculated 30% ozone depletion (B) in Lund, Sweden. The "action" curve has been obtained by multiplying the daylight spectrum by the "plant action spectrum". The "action" curves are magnified ( × 100) in order to show the differences.

4. Discussion

UVB radiation could only be detected down to about 1-2 m and no effect of natural UVB radiation on the motility of the dinoflagellates was discernible at this depth. These results indicate that plankton at a depth of more than about 1 m will be less affected by UVB radiation than organisms constantly remaining very close to the surface of the water column. At the latitude investigated, large seasonal and daily (due to clouds) changes occur in the UVB dose. These large variations make it more difficult to replicate experiments outdoors than under the more constant light conditions in the laboratory, and the two experimental conditions do not give the same numerical results (Table 3). For example, 2.72 kJ m-2 day-t UVBsE (4h artificial UVB per day) in the laboratory decreases the motility of P. minimum (Kattegat) by 56%, but with 1.8 kJ m -2 day -1 UVBBE in the field the motility decreases by 26% (at a depth of 35 cm, 8 August 1992). Unweighted UVB and PAR in the field were 1.2 and 224 W m-2 respectively, while the corresponding values in the

7". Nielsen et aL /J. Photochem. Photobiol. B: Biol. 27 (1995) 73-79

1.2

A 1.0 Clear day

0.8 0.6

N

? J

0.4 0.2 360

;J

460

560

660

°m

t_ t_ i.m

0.4

B

t_

0~ 0.2

//I"'-'-'"'"

0,1

/¢Heavy 0.0 260

280

300

clouds (8/8)

320

340

360

Wavelength (nm) !~ig. 4. Spectral irradiance of visible (A) and UV (B) radiation on t:lear and cloudy days above the water surface at latitude 58.2°N, i 1.3°E.

~

0.0

0

1~ o)

-0.2

~0~4

.

Ac.rt.rl.

" ~

~ triq~tra

~.

06

0.0

P.

~

/

~

"

~

minimum (All.) 0.5

1.0

UV-BaE (kJ m - 2 d a y - 1

1.5

)

Fig. 5. Growth rate (doublings per day) with UYB radiation each day for 1 week corresponding to UVt3BE radiation of 0.46, 0.68, 0.93 and 1,37 kJ m -2 day -~. Error bars indicate the standard error (SE).

laboratory studies were 0.57 and 19 W m -z. These differences in response between field and laboratory conditions indicate that UVB radiation and PAR (and UVA radiation, not measured in this work) are important when studying the effects of UVB radiation. The present work shows that the motility of P. minimum (Kattegat) near the water surface is affected after 4 h when UVB and PAR exceed 1 and 200 W m -2

77

respectively. Other environmental effects which could affect the speed of the organisms include the difference in temperature between field and laboratory experiments. In the present work, the temperature in the laboratory was 19-20 °C and in the field 16--17 °C. The motility of G. aureolum was strongly impaired by natural sunlight with and without UV radiation (after only 2 h) (Table 2). The dramatic effect on the motility of G. aureolum could be a shock effect on transfer from low light conditions (19 W m -2) to natural sunlight. In this experiment, there was no absorption by dissolved organic matter and the levels of PAR and UVB were high as it was a clear day and mid-summer. This reduction in motility may be due to photoinhibition which has been observed for Amphidinium carterae exposed to PAR of 150/~mol m - 2 s- 1 (no UV radiation) [20]. Short high UVB exposure is more damaging than longer exposure times of lower irradiance for the inhibition of photosynthesis [21]. The difficulties in simulating natural light regimes make it important to use a proper weighting factor, which takes into account the wavelength dependence of biolbgical action. Generally, the lower the wavelength, the more effective the radiation. Several action spectra have been used, e.g. DNA [22], the generalized plant action spectrum [18] and chloroplast photoinhibition [23]. However, no weighting spectrum has been determined for our organisms. Therefore, for the present work, the generalized plant action spectrum has been used together with the action spectrum for Prorocentrum micans [10]. The action spectrum for P. micans describes the inhibition of photosynthesis by UV radiation, and this weighting function is not necessarily correct when studying the effect of UVB radiation on the motility of phytoplankton. In order to obtain a correct weighting factor, an action spectrum for the inhibition of motility should be determined for the organism under study. Cullen et al. [10] concluded that the irradiance, not the daily UV exposure (irradiance × duration), determines the degree of inhibition of photosynthesis in dinoflagellates, i.e. the duration of exposure is not important provided that it exceeds 30 min. On the other hand, it is clear from our experiments that the growth rate depends on the daily exposure time. For the motility experiments, no data are available to determine whether exposure or irradiance is the most important factor. The comparison made (Table 3) with the model of Cullen et al. [10] for photosynthesis shows no significant deviation for daylight conditions, while the sensitivity under artificial conditions is much greater than that predicted by the model. The deviation may be due to a different action spectrum for the inhibition of motility or to the low PAR in the laboratory experiments. The penetration of UVB radiation into natural waters varies according to the concentration of chlorophyll

78

T. Nielsen et aL / J. Photochem. PhotobioL B: Biol. 27 (1995) 73-79

Table 3 Effect of 4 h artificial U V B radiation (0.57 W m -2 unweighted irradiance or 2.72 kJ m -2 day-J UVBaE) and natural sunlight conditions in Kristineberg on the motility (tz m s -x + S E ) of different dinoflagellates. Significant differences (t-test) between control and U V and glass and quartz bottles are marked with an asterisk. To compare the relative inhibition of motility vs. photosynthesis, the action spectrum for photosynthesis of Prorocentrum micans presented by Cullen et al. [10] and their model for the inhibition of photosynthesis were used Laboratory (organism)

Control

LIV

H. triquetra P. minimum (Katt.) P. minimum (Atl.)

274 4- 14 137 4- 7 114 4- 6

120 + 8* 88 4- 8* 65 4- 5*

Field (Depth, organism, date)

Glass

Quartz

100 × quartz/ glass

Expected for photosynthesis (% not inhibited)

114+29 158 + 24 126+ 13

9 2 + 11 117-1-17" 138+ 13

81 + 2 3 74 + 16 110+ 15

77 73 82

141 + 13 1264-8

108-¢- 2 132+ 11

77 4- 7 105 4-11

77 82

95 4- 9 122 4- 5 182 4-10

106 4-19 120 4- 10 135 4- 25

112 4- 23 98 4- 9 74 4-14

136 + 14 161 4-16 144-t-2

102 4- 7 1624-5 1334-8

75 4- 9 101 4-11 924-6

35 cm P. minimum 4 August 8 August 10 August 35 cm P. minimum 4 August 10 August 120 cm P. minimum 4 August 8 August 10 August 120 cm P. minimum 4 August 8 August 10 August

Not inhibited (%)

44 64 57

Expected for photosynthesis (% not inhibited) 79 79 79

(Katt.)

(Atl.)

(Katt.)

(Atl.)

and dissolved organic material (DOM) [24]. Therefore plankton in productive waters at temperate latitudes with more seasonal variations in UVB radiation are less exposed to UVB radiation compared with phytoplankton populations from clear tropical waters. Phytoplankton from tropical waters are more tolerant to UV radiation than Antarctic phytoplankton [1]. The maximum irradiance from just below the surface at the equator (65°S and 55.7°N) varies from 1.6, 0.7 to 1.0 W m -2 nm- 1 (non-weighted), respectively which means that, even under the Antarctic ozone hole, the irradiance will not reach the same level as at the equator [11]. The large variation in natural light conditions, the short exposure times and the scarce data make it difficult to estimate the consequences of UVB radiation on phytoplankton communities at temperate latitudes. However, the present report shows that phytoplankton at temperate latitudes are negatively affected when exposed to short periods of UVB radiation. In addition, the comparison between different weighting spectra has shown the importance of using the correct action spec-

trum when comparing the effects of UV radiation on different parameters. It will be necessary in future studies to determine the correct action spectra for the processes under investigation in order to estimate meaningful UVB effects on phytoplankton. This work confirms that field work must be performed for more accurate predictions of UVB effects on natural phytoplankton and emphasizes the difficulty in predicting outdoor responses from laboratory experiments with artificial radiation.

Acknowledgements We wish to thank Mrs. Lena Carlsson for technical assistance and Dr. Janet Bornman for help with the manuscript. This work was financed by a grant from the Swedish Environmental Protection Agency (EUproject) and by a grant from the Hierta-Retzius Foundation.

T. Nielsen et al. / Z Photochem. Photobiol. B: Biol. 27 (1995) 73-79

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[13] J.B. Kerr and C.T. McElroy, Evidence for large upward trends of ultraviolet-B radiation linked to ozone depletion, Science, 262 (1993) 1032-1034. [14] D.-P. H~ider and R.C. Worrest, Effects of enhanced solar ultraviolet radiation on aquatic ecosystems, Photochem. Photobiol., 53 (1991) 717-725. [15] R.L.L. Guillard, Culture of phytoplankton for feeding marine invertebrates, in W.L. Smith and M.H. Chanley (eds.), Culture o f Marine Invertebrates Animals, Plenum, New York, 1975, pp. 29-60. [16] D.-P. H~ider and M. Lebert, Real time computer-controlled tracking of motile microorganisms, Photochem. Photobiol., 42 (1985) 509-514. [17] M.M. Caldwell, Solar UV irradiation and the growth and development of higher plants, in A.C. Giese (ed.), Photophysiology, Academic Press, New York, 1971, pp. 131-171. [18] R.W. Thimijan, H.R. Cams and L.E. Campbell, Radiation sources and related environmental control for biological and climatic effects of UV research (BACER), in Final Report, Environmental Protection Agency, BACER Program, Washington DC, 1978. [19] L.O. Bj6rn and T.M. Murphy, Computer calculation of solar ultraviolet radiation at ground level, Physiol. V~g., 23 (1985) 555-561. [20] G. Samuelsson and K. Richardson, Photoinhibition at low quantum flux densities in a marine dinoflagellate (Amphidinium carterae), Mar. Biol., 70 (1982) 21-26. [21] J.J. Cullen and M.P. Lesser, Inhibition of photosynthesis by ultraviolet radiation as a function of dose and dosage rate: results for a marine diatom, Mar. Biol., 111 (1991) 18.3--190. [22] R.B. Setlow, The wavelengths of sunlight effective in producing skin cancer: a theoretical analysis, Proc. Natl. Acad. Sci. USA, 71 (1974) 3363-3366. [23] L.W. Jones and B. Kok, Photoinhibition of chloroplast reactions. I. Kinetics and action spectrum, Plant Physiol., 41 (1966) 1589--1606. [24] R.C. Smith and S.B. Baker, Penetration of UV-B and biologically effective dose-rates in natural waters, Photochem. Pt, otobiol., 29 (1979) 311-323. J.B. Kerr and C.T. McElroy, Evidence for large upward trends of ultraviolet-B radiation linked to ozone depletion, Science, 262 (1993) 1032-1034.