Experimental assessment of UV effects on temperate marine phytoplankton when exposed to variable radiation regimes

Phytoplankton samples were collected at Bahı́a Engaño, Chubut, Argentina (438S, 658W) at different times of the year to assess the combined effect of ultraviolet radiation (UVR, 280–400 nm) and vertical mixing (i.e., the depth of the upper mixed layer, UML) on photosynthesis. Samples were exposed to fixed and fluctuating radiation regimes in an illuminated chamber at 158C (photosynthetically available radiation [PAR] 5 66 W m22; UV-A 5 15.3 W m22; UV-B 5 0.7 W m22), receiving either PAR 1 UVR or PAR only. A comparison between fixed and rotating systems showed that when ZUML/ZEu 5 0.6 (i.e., 60% of the euphotic zone [Eu] was mixed), only postbloom assemblages (codominated by nanoplanktonic flagellates and diatoms [Chaetoceros spp.]) were affected significantly by UVR. Integrated carbon fixation values during preand postbloom periods were higher under mixed conditions than under fixed irradiances. However, during the bloom (dominated by the microplanktonic diatom Odontella aurita), phytoplankton exposed to fluctuation radiation regimes had lower integrated carbon fixation. When postbloom samples were exposed to different mixing conditions, integrated UVR-induced inhibition reduced carbon fixation by 11–13% when ZUML/ZEu 5 0.6, whereas when ZUML/ZEu 5 0.91, carbon fixation increased by 7–12%. The differences in responses observed between prebloom, bloom, and postbloom samples can be attributed to a number of factors, such as the light history of cells, taxonomic composition, and size structure of the community and most probably reflect the different inhibition kinetics of these assemblages. Phytoplankton are normally exposed to fluctuating radiation regimes in their natural environment. In a specific geographic location, radiation levels reaching the Earth’s surface, both photosynthetically available radiation (PAR, 400– 700 nm) and ultraviolet radiation (UVR, 280–400 nm), vary throughout the year, mainly because of changes in solar zenith angle (Madronich 1993). Natural radiation fluctuations also occur within temporal scales ranging from days to minutes because of changes in cloudiness and ozone conditions (Lubin and Jensen 1995) as well as mixing depth (Denman and Gargett 1983), potentially affecting organisms in different ways. Phytoplankton, whose movement in the water column mostly depends on turbulent motions, experience significant fluctuations in radiation regimes because of changes in the depth of the upper mixed layer (UML), which take place mainly as a result of variations in weather conditions, such as sun heating, wind stress, or storm activity (Neale et al. in press). The most obvious effects of fluctuating radiation regimes on phytoplankton include changes in their photoacclimation (Falkowski and Wirick 1981; Cullen and Lewis 1988), with 1 Corresponding author ([email protected]). Acknowledgments We thank L. Sala for his help with 14C analysis and Fundación Playa Unión for logistic support. H. Zagarese (U. N. Comahue, Argentina) provided comments and suggestions in early versions of this manuscript. We thank two anonymous reviewers for their comments and suggestions, which greatly improved our paper. This work was supported by Agencia Nacional de Promoción Cientı́fica y Tecnológica—ANPCyT/BID (Project PICT97 07-00000-02206), The Third World Academy of Sciences—TWAS (Project 98-036 RG/BIO/LA), and Fundación Antorchas (Project A-13622/1-100). This is Contribution 37 of Estación de Fotobiologı́a Playa Unión. concomitant variations in P-E parameters, fluorescence yield, or cellular chemical composition (Marra 1978; Denman and Gargett 1983; Cullen and Lewis 1988). Although most of these studies have considered the responses of organisms under variable PAR regimes, it is now recognized that fluctuating UVR levels such as those produced by mixing can also affect the performance and fitness of aquatic organisms (Helbling et al. 1994; Neale et al. 1998a,b, in press; Zagarese et al. 1998; Huot et al. 2000; Xenopoulos et al. 2000; Köhler et al. 2001). Very few studies have addressed the combined effects of variable UVR regimes and vertical mixing on phytoplankton photosynthesis (Helbling et al. 1994; Neale et al. 1998a,b, in press; Köhler et al. 2001). In the Atlantic coastal area, very few studies have determined on-site phytoplankton marine primary productivity rates (Charpy and Charpy-Roubaud 1980; Buma et al. 2001; Helbling et al. 2001a). On the Patagonia coast, research has just started to evaluate the effects of UVR on phytoplankton (Villafañe et al. 2001) and determining its effect on photosynthesis and DNA material (Buma et al. 2001; Helbling et al. 2001a); however, none of these studies have specifically addressed the effect of fluctuating radiation regimes on these processes. These types of studies are of great importance for this region, where aquatic organisms are normally exposed to changes in radiation regimes, especially by the alternation of strong winds with calm periods, which ultimately affects the depth of the UML. In this area, dense phytoplankton blooms develop during winter, characterized by the dominance of the diatom Odontella aurita, that sustain a very rich coastal fishery (Villafañe et al. 1991). Therefore, the aim of this study is to determine the effects of variable UVR and PAR on the integrated primary production of natural phytoplankton populations characteristic of coastal Patagonian waters. 1649 UV effects under variable radiation Materials and methods Sampling site—Studies were carried out with natural phytoplankton populations collected at Bahı́a Engaño, Chubut, Argentina (438S, 658W) from March to December 2000 at a coastal station denominated Egi. This station, located at the mouth of the Chubut River estuary, was found to be highly variable with regard to its physical and biochemical characteristics (Villafañe et al. 1991; Helbling et al. 1992a); thus, a continuous monitoring of phytoplankton species composition, chlorophyll a (Chl a), UV-absorbing compounds, surface water temperature, and conductivity was carried out every 10–20 d since 1999. In addition, vertical profiles of temperature and irradiance were determined during several cruises using a submersible ELDONET filter radiometer (Real Time Computers), with sensors for UV-B (280–315 nm), UV-A (315–400 nm), PAR (400–700 nm), temperature, and depth. Data from these profiles were used to calculate attenuation coefficients for solar radiation and to estimate the depth of the UML (i.e., ZUML) based on the temperature data from the profiling radiometer and conductivity data obtained with a Horiba probe. Experiments to determine the effects of UVR on phytoplankton photosynthesis under a variable radiation field were carried out during prebloom, bloom, and postbloom conditions. Samples were collected during high tide with an acid-clean (1 N HCl) carboy, dispensed into a plastic container, and immediately taken to the laboratory at Estación de Fotobiologı́a Playa Unión (EFPU), where experiments were conducted as described below. Experimentation—Because the study area is highly variable in terms of wind speed and duration (thus conditioning the depth of the UML), two different approaches were implemented to study the combined effects of UVR and vertical mixing on phytoplankton photosynthesis. In the first type of experiments, samples collected at different times of the year were exposed to a variable irradiance field, as if 60% of the euphotic zone (Eu) was mixed (i.e., ZUML/ZEu 5 0.6). In these experiments, we wanted to establish a common level of comparison (i.e., similar mixing and irradiance conditions) for natural phytoplankton populations that had different light histories and acclimations to solar radiation. These experiments were done during March (prebloom), June (bloom), and December (postbloom) 2000. In another set of experiments, samples with the same light history and acclimation (i.e., the same phytoplankton population) were exposed to increasing levels of mixing (ZUML/ZEu 5 0.6, 0.76, and 0.91), thus simulating a deepening of the UML. These experiments were conducted during spring (early December 2000), which is the windy season in the study area. An experimental device, similar to that described in Helbling et al. (1994) with two systems, one fixed and one rotating, was used in these experiments. Both systems had various layers of neutral-density screens that allowed attenuation of incident radiation (from 100% to either 6, 3, or 1.5%), thus approximately simulating the irradiance field received by cells in different portions of the euphotic zone (although they did not mimic the differential attenuation of UVR and PAR in the water column). Both systems were placed inside an illuminated chamber (see below); the samples in the fixed system were incubated at the same irradiance level during the whole incubation period, whereas the samples in the rotating system were gradually moved to the next irradiance level every 30 min. The experiments with ZUML/ZEu 5 0.6 lasted 5 h for a complete rotation (five levels of irradiance), whereas the experiments with ZUML/ZEu ratios of 0.76 and 0.91 lasted 6 and 7 h (six and seven levels of irradiance, respectively). Samples in each irradiance level were exposed to either UVR 1 PAR (samples in quartz tubes) or to PAR only (samples in quartz tubes covered with Ultraphan UV Opak [Digefra]), 50% transmission at 395 nm. A total of four tubes (duplicate samples for PAR 1 UVR and PAR) were exposed in each irradiance level in each system. For all experiments, surface phytoplankton samples were placed in 50-ml quartz tubes, inoculated with labeled sodium bicarbonate (see below), and incubated at 158C in an illuminated chamber (Sanyo model MLR 350) with five Q-Panel UVA 340 lamps and 10 Phillips daylight lamps, which provided irradiances of 66, 15.3, and 0.7 W m22 for PAR, UVA, and UV-B, respectively. In our experimental device, these irradiances represented the 100% level. Analysis and measurements—Photosynthetic rates were determined using the technique described in Holm-Hansen and Helbling (1995). Briefly, samples were incubated with 5 m Ci of NaHCO3 (0.185 MBq), and after the incubation period, they were filtered onto Whatman GF/F glass fiber filters (25 mm diameter), put in 7-ml scintillation vials, and exposed to HCl fumes overnight. Then, the samples were dried and counted using standard liquid scintillation techniques. For Chl a analysis, an aliquot of 100 ml of sample was filtered onto Whatman GF/F filters (25 mm) and the photosynthetic pigments extracted in absolute methanol (HolmHansen and Rieman 1978). Chl a determination was done by fluorometric methods (Holm-Hansen et al. 1965). Chl a concentration in the pico-nanoplankton fraction (,20 mm diameter) was determined as previously described, except that the sample was pre-filtered through a 20-mm Nitext mesh. The fluorescence of the methanolic extract was measured in a Turner Designs fluorometer (model TD 700) before and after acidification, and Chl a concentration was calculated from these readings. The fluorometer was calibrated using pure Chl a from Anacystis nidulans (Sigma C6144). We also determined the presence of UV-absorbing compounds in the samples. For this analysis, an aliquot of 500– 1,000 ml of water sample was filtered onto Whatman GF/F filters (47 mm), and the photosynthetic pigments and UVabsorbing compounds were extracted overnight at 48C in 7 ml of absolute methanol. The estimation of concentration of these UV-absorbing compounds (Helbling et al. 1996) was done by peak analysis (Microcal Origin Software) of the scans (250–750 nm) with a Hewlett Packard spectrophotometer (model 8453E). Floristic analyses were made of phytoplankton samples fixed with buffered formalin (final concentration in the sample, 0.4%). Samples were settled overnight using 10or 25ml cylinders, and qualitative and quantitative analyses were done with an inverted microscope. 1650 Barbieri et al. Fig. 1. General characteristics of the study area during the year 2000 as a function of time (day of the year). (A) Daily doses of PAR (400–700 nm); (B) daily doses of UV-A (315–400 nm) and UV-B (280–315 nm). Units are kJ m22. (C) Phytoplankton concentration, as estimated by Chl a (mg L21), and percentage of nanoplanktonic cells. The three horizontal lines indicate the time of sampling of prebloom, bloom, and postbloom assemblages and