Methodology to assess UVR effects on photosynthesis

11.2.1 Exposure of samples

In order to assess UVR effects on photosynthesis, three approaches for exposing algae to UVR are used. These include (1) natural solar radiation, modified by various filters that selectively screen off certain wavebands of radiation; (2) natural solar radiation which is supplemented with artificial UVR from lamps, and (3) fully artificial radiation, implying laboratory experiments. UVR experiments at their best require both that the target organisms are exposed to as realistic a light field as possible, and that high-quality measurements of radiation are obtained. The most realistic results are probably gained from experiments performed under natural solar radiation; artificial radiation sources, however, have also been shown to be very useful for studying mechanistic aspects of UVR responses.

11.2.1.1 In situ incubations

In this type of incubations, the samples are exposed to solar radiation in their natural habitat and at their natural in situ depth. To assess the effects of ambient UVR, this approach often involves three types of radiation fields, achieved by filters, i.e. PAR + UV-B + UV-A, PAR + UV-A, and only PAR (see section 11.2.3). Although in situ incubations will result in the most realistic responses, they certainly have the constraint of being conditioned by weather conditions. Therefore, comparatively few in situ studies on the effects of UVR on algal photosynthesis have been conducted, particularly in rough-weather areas, such as the Arctic [17] and Antarctica [18,19].

Phytoplankton can be exposed to an in situ field of radiation by using UV-transparent (see section 11.2.3) bottles hanging from a line or tubes placed in trays (Figure 1) which are incubated at different depths in the water column [18,20]. One disadvantage with this approach is that phytoplankton cells are kept at a fixed depth for the entire incubation period (e.g., few hours), thus receiving a constant proportion of the surface incident radiation. In the water column, however, cells are moving within the upper mixed layer (UML) and thus exposed to a variable field of irradiance [21] (see Chapter 4). So far, few studies have addressed the importance of mixing rates on the phytoplankton photosynthesis [21-23], and with the exception of the experiments performed by Marra [24] on the effects of PAR, we are not aware of such studies done under in situ conditions.

Fixed screens with different filtering capacities have been frequently used to study the in situ effect of ambient UVR on shallow-water benthic microalgae in streams [25], lakes [26] and marine habitats [27,28], In a four-month in situ experiment on UVR effects on MPB communities of a microtidal bay, Wulff et al. [29] used 80 x 80 cm screens placed in wooden frames that were pressed into the sediment. This type of field set-up, however, requires frequent cleaning and careful monitoring of the radiation field below the screens.

In the case of marine macrophytes (macroalgae and marine angiosperms), most of the in situ experimentation has been conducted in the intertidal zone, where access to growing plants is relatively easy [30-33]. Subtidal populations have received less attention due to the complications of working in situ at different depths, especially in high latitude zones [33,34]. Several authors [30-32,35] have investigated effects of UVR on macroalgal photosynthesis by incubating algae in their natural environment and monitoring daily variation in photosynthesis and irradiance under different radiation treatments using a similar set up as those described for MPB experiments (Figure 1). More recently,

Figure 1. Schematic representation of in situ incubation for phytoplankton and benthic algae. (A) General disposition of trays with tubes and filters for cutting off different portions of the solar spectrum; in the bottom a set up for benthic algae incubation is presented. (B) Close up of one tray containing duplicate quartz tubes for three different radiation treatments: PAB, unfiltered solar radiation; PA, PAR + UV-A and P, only PAR. (C) Transmission characteristics of various materials and filters used in photobiological experimentation.

Figure 1. Schematic representation of in situ incubation for phytoplankton and benthic algae. (A) General disposition of trays with tubes and filters for cutting off different portions of the solar spectrum; in the bottom a set up for benthic algae incubation is presented. (B) Close up of one tray containing duplicate quartz tubes for three different radiation treatments: PAB, unfiltered solar radiation; PA, PAR + UV-A and P, only PAR. (C) Transmission characteristics of various materials and filters used in photobiological experimentation.

however, efforts have been devoted to analyze in situ photosynthetic activity of subtidal algae. This experimental design has consisted of determining the effective quantum yield by using an underwater fluorometer [36-39]. An alternative approach has been to incubate marine plants for several days at their natural growth site, and after that period the algae were collected and the quantum yield measured using a non-submersible fluorometer [35,40-42],

11.2.1.2 Simulated in situ incubations

Considering the practical difficulties of in situ incubations, outdoor incubations in temperature-controlled containers (e.g., on deck of research vessels, or in flow-through systems on land sites) have been used as an alternative approach. This incubation method is suitable for both short-term (hours) and long-term (days-weeks) experiments carried out with microalgae [43-47], as well as with macroalgae [32,48-54]. This set up is often used for determining a worst-case scenario, as samples are exposed to surface (i.e., maximum) incident irradiance. Therefore, neutral density filters are often used to approximately simulate the attenuation of solar radiation in the water column. These filters, however, do not mimic the differential spectral attenuation that actually occurs in the water column (Chapter 3), and samples are generally exposed to higher UV-B/UV-A/PAR ratios than they would normally experience. It is particularly important to approach realistic ratios between UV-B, UV-A and PAR, as DNA repair mechanisms depend on those ratios [55,56] (see Chapter 9).

In contrast to phytoplankton, simulated in situ incubations imply fairly realistic light conditions for MPB in the intertidal or littoral zone. This is particularly true when incubating intact sediment cores, as the sediment will provide natural refuges for benthic microalgae, such as motile diatoms and cyanobacteria [46,47,57]. Similar approaches have also been used for hard substrata, often involving colonization of artificial substrata [58].

11.2.1.3 Supplemented UV-B or UVR

As with several UV experiments carried out with terrestrial organisms [59,60], experimental treatments on aquatic organisms have included the enhancement of ambient UV-B. In some cases, these treatments simulate ozone depletion events. Such experiments, in which natural solar radiation is enhanced by artificial UVR, have been done with phytoplankton [61-63] and micro-phytobenthos [64]. A few studies have included simultaneously exclusion and enhancement of UV-B [47,65]. A shortcoming, however, in the majority of experiments using elevated levels of UV-B, has been the use of fixed levels of UV-B for few hours per day. Moreover, the levels of enhancement have varied greatly, from moderate (~20%) to ca. 100% above ambient, often resulting in unnatural ratios between PAR and UVR, thus making comparisons between experiments difficult. As mentioned before, it is crucial for ecologically relevant studies that the spectral composition of the radiation is realistic [66]. One way to achieve this is to provide additional UV-B so that it mirrors the natural dose curve, as has been used in terrestrial studies [67]. This is possible with a system in which the intensity given is controlled by a computer system linked to a UV-B sensor that continuously measures ambient UV-B levels. This type of set-up allows the simulation of low levels of enhanced UV-B (5-20%) as observed during ozone depletion events, and has been used to study the UV-B response of both MPB [65,68] and phytoplankton [63],

364 V.E. VILLAFAÑE, K. SUNDBÁCK, F.L. FIGUEROA AND E.W. HELBLING 11.2.1.4 Artificial radiation

Various artificial radiation sources have been used to assess UVR effects on aquatic autotrophic organisms. An assorted number of them are commercially available, such as fluorescent and halogen lamps. So far, most studies carried out with artificial radiation sources have been done with the main objective to determine the impact of UVR at fixed irradiances [69], or in combination with neutral density screens and cut-off filters to obtain biological weighting functions (BWFs) [70,71]. In order to determine the sensitivity of intertidal and subtidal algae, Dring et al. [72,73] used a solar simulator, in which different levels of ozone reduction can be arranged, and Róttgers [74] had used a similar system to address the response of phytoplankton cultures to changes in UVR. However, it has been found very difficult to mimic the solar radiation spectrum in these types of experiments; in fact very few of the light sources can give reliable results in photobiological research [75]. Moreover, one should be extremely cautious when extrapolating the results obtained in this way to the natural environment.

11.2.2 Materials and filters

A combination of different materials and filters are normally used to separate different wavebands of the incident irradiance spectrum. In most of the experiments conducted either in the field or in the laboratory, it is customary to use tubes or vessels made of a material transparent to UVR, such as Quartz, Plexiglas, or Teflon. There are many types of filters that are broadly used in photobiological research, ranging from "film type" filters, such as Ultraphan, Folex, Mylar-D, and acetate, to "glass type" filters such as Schott, Hoya and Oriel. Representative spectra of the transmission characteristics of commonly used filters and materials are shown in Figure 1. In general, the materials are long pass filters, and thus they screen off the energy of the lower wavelengths. However, there are filters that allow the energy of just a portion of the spectrum to pass, as is the case of the UG11 filters (see Figure 1).

11.2.3 Variables measured and experimen tal approaches

Various experimental approaches have been used to evaluate the impact of UVR on different cell processes (Figure 2). The evolution of oxygen [76,77] and incorporation of radiocarbon [20,78] have been widely used not only to determine the productivity of a water body, but also to assess the impact of UVR [18,22,30,43,49,79-84]. In addition, oxygen microsensors [85] have been shown to be practical tools for high-resolution measurements of UVR effects in sediments and microfilms [46,64], particularly in combination with optical microsensors measuring UVR [16].

In recent years, pulse amplitude modulated (PAM) chlorophyll fluorescence associated with the photosystem II (PS II) has become a useful tool for evaluation of photosynthesis [86-90]. In fact, chlorophyll fluorescence can be an

Figure 2. Diagram of a eukaryotic algal cell indicating different processes that could be influenced by UVR and that directly or indirectly affect the photosynthetic process.

indicator of different functional levels in photosynthesis, such as photon capture by light-harvesting pigments, primary light reactions, thylakoid electron transport reactions, dark-enzymatic stroma reactions and slow regulatory feedback processes [91]. The relationship between oxygen evolution and chlorophyll fluorescence in different organisms has also been demonstrated [92,93]. Photo-synthetic activity has been estimated as chlorophyll fluorescence in macroalgae growing in a variety of water bodies, as in the Arctic [40,94], Antarctic [33,95], North Sea [72], Chinese Sea [96], Mediterranean Sea [31,50,51,97], tropical [98] or Patagonian [99,100]. Taking into account the differences in photosynthetic organization between macroalgae and higher plants, an optimization of the PAM instrumentation has been needed to measure accurately the low chlorophyll fluorescence emission of macroalgae [88,101]. Furthermore, the presence of phycobilisomes in the light-harvesting system of red algae results in generally lower fluorescence values than that measured in green- and brown-algae [101]. Due to the increased sensitivity of the PAM fluorescence instrumentation in recent years, this technique has been also used to study UV-B effects on M PB [65], as well as to address UVR effects on phytoplankton [74,102,103].

Studies on the effects of UVR upon phytoplankton have been conducted using both natural communities and monospecific cultures [17,18,21,22,43,44,81-84,103-113]. The exposure of samples has included in situ [19,44,81,111] and simulated in situ incubations [21,43,44,82], as well as the use of artificial radiation [83,104]. Short-term studies have been generally performed in periods of less than one day, implying that no acclimation has been allowed, and, hence, some of the observed effects represent the worst-case scenario. Still, the majority of UVR studies on phytoplankton photosynthesis have been done using this approach, and they provide a base of comparison among species and different ecosystems. Long-term experiments (i.e., days, weeks), on the other hand, are a preferable choice when making predictions about the effects of UVR on an ecological scale; however, relatively few studies have been performed using this approach [83,107,110,114-118].

The response of benthic microalgae to UVR has mainly been assessed by studying natural or semi-natural communities in situ, or in outdoor experimental flumes (see references below), although laboratory experiments have also been made [119,120]. Basically, two types of studies have been conducted: (1) experiments where communities have been allowed to colonize on hard substrata [58,121] and, (2) experiments where intact natural communities in sediments have been studied [29,47]. These two approaches differ in the aspect that the former allows UVR to exert a selective pressure during early growth and succession, which is not the case when studying already established, dense communities with no or little net growth. These two approaches also differ in the choice of target variables. While photosynthetic rate (14C incorporation, oxygen microprofiles) and photochemistry (PAM) have been monitored for MPB in sediments, accrual of biomass (as chl-a, or algal cells) has been the most commonly measured variable, particularly in long-term experiments on periphyton on hard substrata (see references in section 11.4).

Finally, the impact of UV-B radiation on marine macrophytes has been mostly conducted on individual species and not on the whole community. The criteria to select species for experimentation/analyses have varied: (a) they are key species due to their contribution to primary production, or because they create a habitat for other marine plants and invertebrates, as the seagrass Posidonia oceanica in the Mediterranean Sea [42,122], Laminaria beds in the North Sea [123], or Macrocystis on the Pacific coast of California [4], (b) they represent a high share of macroalgal biomass in the ecosystem, as Ulva in eutrophic coastal waters [124] and, (c) they are commercially important as Porphyra sp., Gelidium sequipedale, Macrocystis pyrifera or Chondrus crispus [30,38,125,126].

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