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Regulations for ballast water treatment specify limits on the concentrations of living cells in discharge water. The vital stains fluorescein diacetate (FDA) and 5‐chloromethylfluorescein diacetate (CMFDA) in combination have been recommended for use in verification of ballast water treatment technology. We tested the effectiveness of FDA and CMFDA, singly and in combination, in discriminating between living and heat‐killed populations of 24 species of phytoplankton from seven divisions, verifying with quantitative growth assays that uniformly live and dead populations were compared. The diagnostic signal, per‐cell fluorescence intensity, was measured by flow cytometry and alternate discriminatory thresholds were defined statistically from the frequency distributions of the dead or living cells. Species were clustered by staining patterns: for four species, the staining of live versus dead cells was distinct, and live‐dead classification was essentially error free. But overlap between the frequency distributions of living and heat‐killed cells in the other taxa led to unavoidable errors, well in excess of 20% in many. In 4 very weakly staining taxa, the mean fluorescence intensity in the heat‐killed cells was higher than that of the living cells, which is inconsistent with the assumptions of the method. Applying the criteria of ≤5% false negative plus ≤5% false positive errors, and no significant loss of cells due to staining, FDA and FDA+CMFDA gave acceptably accurate results for only 8–10 of 24 species (i.e., 33%–42%). CMFDA was the least effective stain and its addition to FDA did not improve the performance of FDA alone.

An equation for the rate of photosynthesis as a function of irradiance introduced by T. T. Bannister included an empirical parameter b to account for observed variations in curvature between the initial slope and the maximum rate of photosynthesis. Yet researchers have generally favored equations with fixed curvature, possibly because b was viewed as having no physiological meaning. We developed an analytic photosynthesis-irradiance equation relating variations in curvature to changes in the degree of connectivity between photosystems, and also considered a recently published alternative, based on changes in the size of the plastoquinone pool. When fitted to a set of 185 observed photosynthesis-irradiance curves, it was found that the Bannister equation provided the best fit more frequently compared to either of the analytic equations. While Bannister's curvature parameter engendered negligible improvement in the statistical fit to the study data, we argued that the parameter is nevertheless quite useful because it allows for consistent estimates of initial slope and saturation irradiance for observations exhibiting a range of curvatures, which would otherwise have to be fitted to different fixed-curvature equations. Using theoretical models, we also found that intra- and intercellular self-shading can result in biased estimates of both curvature and the saturation irradiance parameter. We concluded that Bannister's is the best currently available equation accounting for variations in curvature precisely because it does not assign inappropriate physiological meaning to its curvature parameter, and we proposed that b should be thought of as the expression of the integration of all factors impacting curvature.

We discuss important sources of variability in sun-induced chlorophyll fluorescence and examine difficulties in deriving fluorescence data products from satellite imagery, with a focus on the MODerate-resolution Imaging Spectroradiometer (MODIS) sensor. Our results indicate that there are limitations in the present MODIS algorithms that could lead to biases in the interpretation of the fluorescence products across gradients of chlorophyll concentration. To avoid some of these limitations, we suggest replacing the calculation of absorbed radiation by phytoplankton (ARP) over a finite depth with integration over the entire water column, and including a term accounting for cellular reabsorption of fluoresced light. These suggestions are incorporated into two new algorithms, based on established bio-optical models for case 1 waters (most open ocean waters), to retrieve chlorophyll concentration and the quantum yield of fluorescence. We compare our results to the results using MODIS algorithms for two regions: one located off the coast of Central America, including the Costa Rica Dome, and the other in the Arabian Sea. The new algorithms provide a similar field for the quantum yield of fluorescence in the first region, while they provide a different and more uniform field in the second region. We suggest that this discrepancy originates from the use of the water leaving radiance at 412 nm in the MODIS standard algorithm, which is not used in our algorithm and can be problematic under certain environmental conditions (e.g., absorbing aerosols or highly scattering waters).

The application of new molecular and genomic techniques to the ocean is driving a scientific revolution in marine microbiology. Discoveries range from previously unknown groups of organisms and novel metabolic pathways to a deeper appreciation of the fundamental genetic and functional diversity of oceanic microbes. The “oceanic genotype” represents only the potential biological capacity and sets an upper constraint on possible pathways and ecosystem rates. The realized structure and functioning of marine ecosystems, the “oceanic phenotype”, reflects the complex interactions of individuals and populations with their physical and chemical environment and with each other. A comprehensive exploitation of the wealth of new genomic data therefore requires a close synergy with interdisciplinary ocean research. Incorporating the information from environmental genomics, targeted process studies, and ocean observing systems into numerical models will improve predictions of the ocean's response to environmental perturbations. Integrating information from genes, populations, and ecosystems is the next great challenge for oceanography.

Autonomous underwater vehicles (AUVs) can map water conditions at high spatial (horizontal and vertical) and temporal resolution, including under cloudy conditions when satellite and airborne remote sensing are not feasible. Applications of this technology are numerous, and harnessing the full potential of AUV platforms for oceanographic research will require innovative sampling and data processing techniques, particularly in shallow littoral environments. We deployed a passive radiometer on a small AUV called the Remote Environmental Monitoring UnitS (REMUS; Hydroid) to demonstrate a novel method that uses one optical sensor at depth to characterize variability in underwater clarity and the constituents of coastal seawater (i.e., dissolved organic material, algal biomass, and other particles). This approach uses spectral differences between attenuation coefficients that are computed from ratios of downwelling irradiance measured at one depth assuming a constant shape, but not magnitude, for the solar irradiance spectrum at the surface. A spectral inversion model provides estimates of the absorption coefficients (m−1) of the constituents above the sensor. There is no requirement for sensors at the ocean surface using this approach and, compared to inversion methods for subsurface reflectance, the effects of bottom reflectance in shallow waters are minimal. Maps of biooptical properties at high spatial resolution demonstrate that our approach can be used for characterizing shallow and highly variable coastal waters. This simple and robust technique is also applicable to other in situ sampling platforms (e.g., gliders, moorings, Argo floats).

Size-fractionated chlorophyll concentration and phytoplankton absorption spectra were compared for a wide variety of natural communities. We found that, in general, when phytoplankton abundance increases, larger sizeclasses are added incrementally to a background of smaller cells. Natural phytoplankton communities from surface waters were explicitly characterized according to their dominant cell size and taxonomic group, and the relationships between this classification and the spectral shape of the phytoplankton absorption coefficient for the whole assemblage was described. By specifying the cell size of the dominant organism (pico-, ultra-, nano-, or microplankton), more than 80% of the variability in spectral shape of the phytoplankton absorption coefficient from 400 to 700 nm could be explained. This is a result of the strong covariation of the size of dominant organisms and several factors controlling the spectral shape of the phytoplankton absorption coefficient, such as pigment packaging and concentration of accessory pigments. Consequently, the shapes of phytoplankton absorption spectra can be reproduced using a spectral mixing model, where two spectra, representing the normalized phytoplankton absorption coefficients for the smallest and the largest cells found in our data set, are combined additively, using a single parameter to specify the complementary contribution of each. The differences between reproduced and measured spectra contain taxonomic and physiological information. This parameterization provides a simple tool for extracting ecological information from optical measurements. It can also be used in sensitivity analyses to describe the influence of the dominant cell size of phytoplankton on optical properties of surface waters. Changes in phytoplankton species composition are a central feature of marine ecosystem dynamics. Description and prediction of these changes are important goals to many fields in oceanography. In recent years, great effort has been made to understand how changes in phytoplankton species composition can affect optical properties of surface waters (e.g., Morel 1997; Kahru and Mitchell 1998; Stuart et al. 1998; Stramski et al. 2001). A major application of these results is the use of in situ optical instruments or remote sensing to observe variability of phytoplankton continuously or synoptically. This is a complicated topic because phytoplankton communities include species differing in size, shape, external and internal structures, and pigment composition. All these characteristics influence their interaction with the light field to some degree, so many factors must be considered to completely describe the optical properties of different communities of phytoplankton. A central goal is 1

In biological oceanography, it has been widely accepted that the maximum quantum yield of photosynthesis is influenced by nutrient stress. A closely related parameter, the maximum quantum yield for stable charge separation of PSII, (φPSII)m, can be estimated by measuring the increase in fluorescence yield from dark-adapted minimal fluorescence (Fo) to maximal fluorescence (Fm) associated with the closing of photosynthetic reaction centers with saturating light or with a photosynthetic inhibitor such as 3′-(3,4-dichlorophenyl)-1′,1′-dimethyl urea (DCMU). The ratio Fv/Fm (= (Fm− Fo)/Fm) is thus used as a diagnostic of nutrient stress. Published results indicate that Fv/Fm is depressed for nutrient-stressed phytoplankton, both during nutrient starvation (unbalanced growth) and acclimated nutrient limitation (steady-state or balanced growth). In contrast to published results, fluorescence measurements from our laboratory indicate that Fv/Fm is high and insensitive to nutrient limitation for cultures in steady state under a wide range of relative growth rates and irradiance levels. This discrepancy between results could be attributed to differences in measurement systems or to differences in growth conditions. To resolve the uncertainty about Fv/Fm as a diagnostic of nutrient stress, we grew the neritic diatom Thalassiosira pseudonana (Hustedt) Hasle et Heimdal under nutrient-replete and nutrient-stressed conditions, using replicate semicontinuous, batch, and continuous cultures. Fv/Fm was determined using a conventional fluorometer and DCMU and with a pulse amplitude modulated (PAM) fluorometer. Reduction of excitation irradiance in the conventional fluorometer eliminated overestimation of Fo in the DCMU methodology for cultures grown at lower light levels, and for a large range of growth conditions there was a strong correlation between the measurements of Fv/Fm with DCMU and PAM (r2 = 0.77, n = 460). Consistent with the literature, nutrient-replete cultures showed consistently high Fv/Fm (∼0.65), independent of growth irradiance. Under nutrient-starved (batch culture and perturbed steady state) conditions, Fv/Fm was significantly correlated to time without the limiting nutrient and to nutrient-limited growth rate before starvation. In contrast to published results, our continuous culture experiments showed that Fv/Fm was not a good measure of nutrient limitation under balanced growth conditions and remained constant (∼0.65) and independent of nutrient-limited growth rate under different irradiance levels. Because variable fluorescence can only be used as a diagnostic for nutrient-starved unbalanced growth conditions, a robust measure of nutrient stressed oceanic waters is still required.

It is well established that ultraviolet radiation (UVR, 280–400 nm) harms aquatic organisms. Reductions in productivity have been reported for phytoplankton, ichthyoplankton, and zooplankton in incubations exposed to UVR. It is difficult, however, to estimate the effects of UVR in natural waters. Quantitative assessments of UVR effects on aquatic organisms require high-resolution measurements of solar irradiance and its attenuation in the water, spectral weighting functions for biological effects, and realistic descriptions of the distributions and vertical movements of particles in the water column. Using experimentally determined biological weighting functions for UV-induced mortality along with measurement-based models of solar irradiance and of vertical distributions of embryos as influenced by mixing, we modeled UVR-induced mortality in the early life stages of two key species in the upper estuary and Gulf of St. Lawrence, Atlantic cod (Gadus morhua) and the planktonic copepod, Calanus finmarchicus. G. morhua embryos are insensitive to UVR, with an average daily survival of ~99% over numerous environmental conditions. C. finmarchicus are considerably more vulnerable, with an average survival of 90% ± 12% (SD). Lowest modeled daily survival was 59% under ambient ozone and 49% under 50% ozone loss. A sensitivity analysis allowed us to examine the relative influences of hydrographic variability, meteorological conditions, and ozone depletion on UVR-induced mortality in C. finmarchicus embryos. The modeled hydrographic and meteorological conditions are a representative range of natural variability for the St. Lawrence region during the 1997 field season, with the exception of extreme ozone depletion (50%). Effects are expressed as relative change of survival normalized to survival under a reference simulation. Similar to other studies, water column mixing and water clarity have the most significant influence on embryo survivorship, with a 3%–80% increased chance of survival when in static, compared with mixed waters, and a 3%–46% increased chance of survival when in the darkest, compared with the clearest waters. Cloudy skies increase survivorship between 1%–30%, and ozone depletion of 50% can decrease survivorship by 9%. On average, ozone depletion decreases survival by 3% and of the factors considered has the smallest influence on mortality of C. finmarchicus embryos.

To assess the potential impacts of ozone depletion on photosynthesis in the Southern Ocean, we need to know more about effects of ultraviolet radiation (UV) on phytoplankton in Antarctic waters, where, in addition to variable stratospheric ozone, temporal and regional differences in vertical mixing might influence photosynthesis and photoacclimation of phytoplankton assemblages. Toward this end, we quantified the responses to UV of Antarctic phytoplankton in the Weddell-Scotia Confluence during the austral spring of 1993. Experimental results on spectral sensitivity of photosynthesis were fit statistically to a model that incorporated uninhibited photosynthesis as a function of photosynthetically available radiation (PAR), wavelength-dependence of inhibition, and the kinetics of photosynthesis during exposure to UV. In contrast to previous results on UV-induced photoinhibition in a diatom culture at 20°C, natural phytoplankton from open waters of the Antarctic showed no ability to counter UV-induced inhibition of photosynthesis during exposures of 0.5-4 h: the rate of photosynthesis declined exponentially as a function of cumulative exposure, and inhibition was not reversed during incubations for up to 3.5 h under benign conditions. The results suggest that nonlinear exposure-response relationships are necessary for modeling UV-dependent photosynthesis in the surface mixed layer of the springtime Weddell-Scotia Confluence. Consequently, we modified our laboratory-based model of photosynthesis and photoinhibition to describe photoinhibition as a nonlinear function of biologically weighted cumulative exposure to damaging irradiance. The model described ∼90% of the spectrally dependent experimental variation in photosynthetic rate, and yielded six biological weighting functions (BWFs) for phytoplankton in the Weddell-Scotia Confluence. Assemblages from different stations showed substantial variability in sensitivity to UV. Tolerance of UV was generally highest in assemblages from shallower mixed layers, which presumably had experienced higher irradiance, including UV, prior to sampling. The BWFs of assemblages that seemed acclimated to low irradiance showed the highest sensitivity to UV yet seen for Southern Ocean phytoplankton. The pattern of UV sensitivity was consistent with acclimation, but also with selection against less tolerant species.

Concerns about harmful algal blooms (HABs) have grown in recent years. There is a pressing need for robust, quantitative, and cost-effective methods to detect and characterize algal blooms. Critical applications of these methods include long-term monitoring of coastal waters to indicate the degree to which present trends of HABs and human activities are linked, early warning systems to protect aquaculture sites and to signal the need for further investigations, and systems to characterize synoptically the distributions and physiological state of phytoplankton in an oceanographic context. Because phytoplankton strongly influence the fate of light in the ocean, optical methods are well suited for HAB applications. Passive optical systems can measure ocean color or the penetration of solar irradiance in surface waters; both properties can be related to the constituents of natural waters, including phytoplankton. The sensors measure radiometric quantities: consequently, rigorous calibration is possible and measurements can be compared between sites and over long periods of time. One passive optical system-a radiometer buoy-is shown to be useful for characterizing biological variability in surface waters over scales from minutes to months. A red tide was easily observed in measures of ocean color from the buoy; sensors for downwelling irradiance detected a subsurface bloom. Some optical instruments use artificial illumination to determine optical properties such as the coefficients of absorption and scattering. These measures can be made continuously in situ and effectively related to phytoplankton. Several types of fluorometers can be used to characterize the abundance, pigmentation, and physiological state of phytoplankton. All of these optical technologies should be useful for the study of HABs, but biological interpretations of many optical measurements need further testing.

C. R. BOOTH AND J. H. MORROW, Biospherical Instruments, San Dlego, CA, USA THOMAS P. COOHILL, Ultraviolet Consultants, Bowling Green, KY, USA JOHN J. CULLEN, Dalhousie University, Halifax, Nova Scotla, Canada JOHN E. FREDERICK, University of Chicago, IL, USA DONAT-P. HADER, University of Erlangen-Nurnberg, Germany OSMUND HOLM-HANSEN, Scripps Institution of Oceanography, La Jolla, CA, USA WADE H. JEFFREY, University of West Florida, Pensacola, FL, USA DAVID L. MITCHELL, M.D. Anderson Cancer Center, Smithvllle, TX, USA PATRICK J. NEALE, Smithsonian Environmental Research Center, Edgewater, MD, USA IGOR SOBOLEV, Chemical and Polymer Technology, Inc., Orinda, CAI USA JAN VAN DER LEUN, University of Utrecht, The Netherlands ROBERT C. WORREST, CIESIN, Washington, DC, USA

Cultures of Rhizosolenia formosa H. Peragallo were studied to assess whether or not physiological and optical characteristics of this large diatom were consistent with the ability to migrate vertically in the open ocean. Time-course experiments examined changes in chemical composition and buoyancy of R. formosa during nitrate (N)–replete growth, N starvation, and recovery. Cells could maintain unbalanced growth for at least 53 h after depletion of ambient nitrate. Increases in C:N and carbohydrate: protein ratios observed during N starvation reversed within 24 h of reintroduction of nitrate to culture medium. Buoyancy was related to nutrition: Upon N depletion, the percentage of positively buoyant cells decreased to 4% from 11% but reverted to 9% within 12 h of nitrate readdition. Cells took up nitrate in the dark. Nitrogen-specific uptake rates averaged 0.48 d−1; these rates were higher than N-specific growth rates (0. 15 d−1), indicating the potential for luxury consumption of nitrate, which can be stored for later use. Measurements of photosynthesis vs. irradiance, chlorophyll-specific absorption (aph*(λ)), and pigment composition showed that cells may be adapted for growth under a wide range of irradiances. Values of aph*(λ) were lower for N-depleted cells than for N-replete cells, and N-depleted cells had higher ratios of total carotenoids to chlorophyll a. Aggregation of chloroplasts was more pronounced in N-depleted cells. These are possibly photoprotective mechanisms that would be an advantage to N-depleted cells in surface waters. Compounds that absorb in the ultraviolet region were detected in N-replete cells but were absent in N-depleted cultures. Overall, these results have important implications for migrations of Rhizosolenia in nature. Cells may survive fairly long periods in N-depleted surface waters and will continue to take up carbon; then they can resume nitrate uptake and revert to positive buoyancy upon returning to deep, N-rich water. Uncoupled uptake of carbon and nitrogen during migrations of Rhizosolenia is a form of new production that may result in the net removal of carbon from oceanic surface waters.

Experiments on a marine diatom, Thalassiosira pseudonana (Hustedt) clone 3H, demonstrate that under moderate photon flux densities (75 [mu]mol quanta [center dot] m[sup [minus]2][center dot]s[sup [minus]1]) of visible light inhibition of photosynthesis by supplemental ultraviolet (UV) radiation (UV-B: 280-320 nm) is well described as a hyperbolic function of UV-B irradiance for time scales of 0.5-4 h. Results are consistent with predictions of a recently developed model of photosynthesis under the influence of UV and visible irradiance. Although net destruction of chlorophyll occurs during a 4-h exposure to UV-B, and the effect is a function of exposure, the principal effect of UV-B is a decrease in chlorophyll-specific photosynthetic rate. The dependence of photoinhibition on dosage rate, rather than cumulative dose, and the hyperbolic shape of the relationship are consistent with net photoinhibition being an equilibrium between damage and repair. The ratio of damage to repair is estimated by a mathematical analysis of the inhibition of photosynthesis during exposures to UV-B. A nitrate-limited culture was much more sensitive to UV-B than were the nutrient-replete cultures, but the kinetics of photoinhibition were similar. The analysis suggests that the nutrient-limited culture was much more sensitive than the nutrient-replete cultures because repair or turnover ofmore » critical proteins associated with photosynthesis is inhibited. An inhibitor of chloroplast protein synthesis was used to suppress repair processes. Photoinhibition by UV-B was enhanced, and inhibition was a function of cumulative dose, as expected if damage were not countered by repair. The fundamental importance of repair processes should be considered in the design of field experiments and models of UV-B effects in the environment, especially in the context of vertical mixing. Repair processes must also be considered whenever biological weighting functions are developed. 69 refs., 6 figs., 3 tabs.« less

Ocean high-nutrient, low-chlorophyll waters are characterized principally by the persistence of major nutrients at the sea surface. This condition indicates control of autotrophic production by something other than NO{sub 3} or PO{sub 4}, but the nature of this control is at present unresolved. The range of hypotheses to explain the high-nutrient condition is illustrated by the grazing hypothesis (specific growth rates of phytoplankton are maximal and environmental stability allows development of a balanced food web that maintains low standing crops of phytoplankton) and the iron hypothesis (standing crop of plankton is constrained by availability of Fe: if more Fe were available, the standing crop of phytoplankton would increase and NO{sub 3} would be depleted, despite grazing). The iron hypothesis has been examined experimentally in the subarctic and equatorial Pacific and in Antarctic waters. In each environment, Fe enrichment enhanced the field yield of phytoplankton and that enrichment of high-nutrient waters with Fe would change the species composition of phytoplankton and food-web interactions, thereby enhancing utilization of NO{sub 3}. The magnitude of this enhancement cannot be predicted with confidence.

In previous work, we evaluated the effects of ultraviolet (UV = 280–400 nm) radiation on the early life stages of a planktonic Calanoid copepod (Calanus finmarchicus Gunnerus) and of Atlantic cod (Gadus morhua). Both are key species in North Atlantic food webs. To further describe the potential impacts of UV exposure on the early life stages of these two species, we measured the wavelength-specific DNA damage (cyclobutane pyrimidine dimer [CPD] formation per megabase of DNA) induced under controlled experimental exposure to UV radiation. UV-induced DNA damage in C. finmarchicus and cod eggs was highest in the UV-B exposure treatments. Under the same spectral exposures, CPD loads in C. finmarchicus eggs were higher than those in cod eggs, and for both C. finmarchicus and cod embryos, CPD loads were generally lower in eggs than in larvae. Biological weighting functions (BWF) and exposure response curves that explain most of the variability in CPD production were derived from these data. Comparison ...

A model of UV-induced DNA damage in oceanic bacterioplankton was developed and tested against previously published and novel measurements of cyclobutane pyrimidine dimers (CPD) in surface layers of the ocean. The model describes the effects of solar irradiance, windforced mixing of bacterioplankton and optical properties of the water on net DNA damage in the water column. The biological part includes the induction of CPD by UV radiation and repair of this damage through photoreactivation and excision. The modeled damage is compared with measured variability of CPD in the ocean: diel variation in natural bacterioplankton communities at the surface and in vertical profiles under different wind conditions (net damage as influenced by repair and mixing); in situ incubation of natural assemblages of bacterioplankton (damage and repair, no mixing); and in situ incubation of DNA solutions (no repair, no mixing). The model predictions are generally consistent with the measurements, showing similar patterns with depth, time and wind speed. A sensitivity analysis assesses the effect on net DNA damage of varying ozone thickness, colored dissolved organic matter concentration, chlorophyll concentration, wind speed and mixed layer depth. Ozone thickness and mixed layer depth are the most important factors affecting net DNA damage in the mixed layer. From the model, the total amplification factor (TAF; a relative measure of the increase of damage associated with a decrease in ozone thickness) for net DNA damage in the euphotic zone is 1.7, as compared with 2.1‐2.2 for irradiance weighted for damage to DNA at the surface.

A comparison is made of the in vivo Chl a fluorescence per cell measured by the flow cytometer (1c,,,) and dark-adapted bulk fluorescence measured in a standard field fluorometer for the marine cryptomonad Chroomonas sp. (clone Chang 2). The bulk fluorescence protocol estimated the levels of the minimum (F,) and maximum (F’,,) fluorescence yields that are exhibited depending on the rcdox state of the photosystem II reaction center. Both I;0 and F,,, are known to be functions of cell irradiance history. During the illumination of control samples at growth irradiance (40 pmol quanta m-* s-l), F,, F,,,, and F,,, (EPICS V) all increased by about the same proportion. After exposure to photoinhibiting irradiance (1,700 pmol quanta m-2 s-l), F,,,,, decreased and F0 increased. Parameters of the photosynthesis-irradiance curve verified that photoinhibition had occurred, indicating less activity at all irradiances. In contrast to bulk fluorescence measurements, relative changes in F,,, in response to strong-irradiance treatment were much smaller than changes in FO, and F,,,,,,. We conclude that this is because FM is intermediate between F. and F,,,,,. Multiple regression analyses suggest that, under the flow cytometry conditions used, F,, exhibits -20% enhancement above F,,, i.e. an average of 20% of the increase from F. to F,,,. Time scales of photosystem II primary photochemistry are consistent with this amount of fluorescence enhancement occurring over the residence time of the cell in the laser beam. These results suggest caution in using oversimplified interpretations of Fcyt. The enhancement effect should also be considered in other instances where fluorescence is excited by a brief saturating flash, for example, some types of in situ fluorometers.