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The use of flow cytometric applications to measure the effects of PAHs on growth, membrane integrity, and relative lipid content of the benthic diatom, Nitzschia brevirostris
Marine Pollution Bulletin xxx (2014) xxx–xxx
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The use of flow cytometric applications to measure the effects of PAHs on growth, membrane integrity, and relative lipid content of the benthic diatom, Nitzschia brevirostris April N. Croxton a,b,⇑, Gary H. Wikfors a, Richard D. Schulterbrandt-Gragg III b a b
Northeast Fisheries Science Center, NMFS, NOAA, 212 Rogers Avenue, Milford, CT 06460, USA School of the Environment, 1520 S. Bronough St., Florida A&M University, Tallahassee, FL 32307, USA
a r t i c l e
i n f o
Article history: Available online xxxx Keywords: Oil pollution Fluorescent probes Membrane permeability Microphytobenthos Algal cellular physiology Rapid method
a b s t r a c t This laboratory study measured the direct effects of three polycyclic aromatic hydrocarbon (PAH) compounds (naphthalene, pyrene, and benzo(a)pyrene) upon cell growth, membrane integrity, and BODIPY-stained lipid fluorescence intensity of the benthic diatom Nitzschia brevirostris using flow cytometry as an analysis tool. Previous field and laboratory studies have reported reductions in algal populations following PAH exposure, but specific, functional responses of the microalgae to these pollutants could not be revealed by cell numbers alone. Using flow-cytometric measurements, we confirmed that maximal cell densities in PAH-exposed diatom cultures were significantly lower compared to controls; however, we also discovered increases in lipids and cells with compromised membranes in PAH-exposed cultures. These results highlight new tools for measuring the direct effects of organic pollutants upon the physiology of taxa comprising microphytobenthic communities important in estuarine food webs. Published by Elsevier Ltd.
1. Introduction Originally used in clinical applications for immunology, hematology, and oncology, flow-cytometry has been used by researchers for over three decades as a technique to analyze phytoplankton (Berglund and Eversman, 1988; Olson et al., 1993; Mandy et al., 1995; Franqueira et al., 2000; Collier, 2004; Yentsch and Yentsch, 2008). Flow-cytometry provides rapid measurements of optical properties of single cells, allowing researchers to quantify phytoplankton populations and estimate growth rates and comparative chlorophyll a concentrations (Collier, 2000, 2004; Yentsch and Yentsch, 2008), as well as differentiate phytoplankton groups based upon accessory pigment fluorescence (Hofstraat et al., 1994). Microalgae, both planktonic and benthic, are a major component of aquatic ecosystems and are responsible for primary production and cycling nutrients throughout these environments (Christensen and Nyholm, 1984; Skoglund and Swackhamer, 1994; Lewis, 1995). Microphytobenthic communities are composed of a group of photosynthetic, eukaryotic algae and cyanobacteria that inhabit ⇑ Corresponding author at: Northeast Fisheries Science Center, NMFS, NOAA, 212 Rogers Avenue, Milford, CT 06460, USA. Tel.: +1 (203) 882 6552; fax: +1 (203) 882 6570. E-mail address: [email protected] (A.N. Croxton).
surface sediments (MacIntyre et al., 1996; Aberle-Malzahn, 2004). The microphytobenthic community is rarely acknowledged fully for its role in primary production in areas where phytoplankton abundance is low (Underwood and Kromkamp, 1999). These organisms also serve as sediment stabilizers, promoters of the transfer of nutrients between sediments and the water column, and food sources for deposit and suspension feeders, including bivalve species (MacIntyre et al., 1996; Aberle-Malzahn, 2004). The close proximity of microphytobenthic communities and sediments exposes this algal community to hydrophobic pollutants that may alter their physiological processes, thereby modifying their critical roles as primary producers and sediment stabilizers in the ecosystem. An example of a large event during which benthic algal communities were exposed to organic pollutants occurred in April 2012 when the Deepwater Horizon oil spill released several million barrels of oil into the productive waters of the Gulf of Mexico for nearly three months (Soniat et al., 2011). Polycyclic aromatic hydrocarbons (PAHs) are a group of hydrophobic organic compounds commonly found in aquatic ecosystems. These compounds are formed during industrial processes and can persist in the environment over a long period of time. Petroleum spills and improper disposal of used oil products are two sources of acute PAH contamination in aquatic environments (Holland et al., 2006). In aquatic ecosystems, hydrophobic
http://dx.doi.org/10.1016/j.marpolbul.2014.12.010 0025-326X/Published by Elsevier Ltd.
Please cite this article in press as: Croxton, A.N., et al. The use of flow cytometric applications to measure the effects of PAHs on growth, membrane integrity, and relative lipid content of the benthic diatom, Nitzschia brevirostris. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.12.010
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compounds are attracted to suspended particulate matter and lipid-rich biological membranes (Rubinstein et al., 1984). Particles with sorbed, hydrophobic substances settle to the seafloor where they are deposited onto the sediment surface. Aquatic surface sediments act as contaminant reservoirs for many environmental pollutants, including PAHs (Elroy et al., 2000). Microalgae associated with sediments are exposed to these organic pollutants and can potentially serve as vectors in the trophic transfer of these compounds as they are resuspended and filtered by bivalve species (Croxton et al., 2012). Extensive research has been conducted on the physiology of marine organisms exposed to hydrophobic pollutants (Anderson et al., 1981; Grundy et al., 1996; Fisher et al., 2000; Chu et al., 2002), but little data exist concerning the toxic effects of hydrophobic pollutants upon microphytobenthic communities located in these marine environments (Hook and Osborn, 2012). Several authors have studied the effects of specific PAH compounds and petroleum products on growth rates of both planktonic and benthic algae (Winters et al., 1976; Mahoney and Haskin, 1980; Nyholm, 1990; Moreno-Garrido et al., 2007). Findings from these studies indicate that hydrocarbon exposure can inhibit algal growth, but the degree of this inhibition varies between algal species. In contrast, Dunstan et al. (1975) observed stimulation of growth of phytoplankton species exposed to low-molecularweight hydrocarbons. Dunstan and colleagues suggested that the volatile fraction of low-molecular-weight hydrocarbons included the most biologically-active hydrocarbons, and this characteristic could explain the varied responses among algal species in various studies. The PAH compounds chosen for this study represent increasing molecular weights, with naphthalene having the lowest and benzo(a)pyrene having the highest molecular weight (Neff, 1979; Varanasi, 1989). Previous studies of microphytobenthic communities and petroleum products tend to highlight the effects of these compounds upon abundance, community structure, and grazing (Bennett et al., 1999, 2000; Piehler et al., 2003; Suderman and Thistle, 2004), but not physiology. These studies focused on trophic interactions between grazers and benthic communities located in polluted environments. Although these interactions are critical in understanding the overall effects of PAHs in an ecosystem, it is equally important to understand the direct effects of PAHs upon microphytobenthic algae. Chung et al. (2007) investigated the responses of the benthic microalga Chlorococcum meneghini to PAH-spiked sand and found that this exposure diminished cell densities and chlorophyll a concentrations. Similar studies also examined chlorophyll a concentrations and photosynthetic rates of PAH-exposed benthic algae (Bennett et al., 1999; Piehler et al., 2003). Earlier studies conducted by plant physiologists suggest that PAH compounds target plasma membranes and change permeability (Currier, 1951; Dallyn and Sweet, 1951; Currier and Peoples, 1954; Dunstan et al., 1975). Interactions between petroleum products, freshwater algae, and bacteria also have been investigated by several authors (Kauss and Hutchinson, 1977; Sikkema et al., 1994; McCann and Solomon, 2000; Hook and Osborn, 2012). Findings from these studies suggest that hydrocarbons are adsorbed onto biological membranes where they target lipid-containing structures (Kauss and Hutchinson, 1977; Weimburg et al., 1981; McCann and Solomon, 2000). The disruption of these structures alters lipid arrangement in membranes, causing the formation of pores and increasing the permeability of cells (McCann and Solomon, 2000; Chung et al., 2007). The aim of this study was to explore new methods for determining physiological responses of a representative microbenthic diatom to purified PAH compounds – to use flow-cytometric applications coupled with fluorescent probes to measure cell growth,
membrane integrity, and lipid storage of a pure, microphytobenthic diatom culture exposed to naphthalene, pyrene, and benzo(a)pyrene (b(a)p) at experimentally-varied concentrations. 2. Materials and methods 2.1. Microphytobenthic diatom culture The benthic diatom, Nitzschia brevirostris (strain 0–1), obtained from the Milford Laboratory Microalgal Culture Collection, was used as the test alga to determine the effects of PAH compounds on growth, membrane integrity, and lipid storage. 2.2. Test compounds The PAH compounds naphthalene, pyrene, and benzo(a)pyrene were the environmental pollutants used to investigate possible effects upon the diatom. Naphthalene (99+%) and pyrene (98%) were purchased from Fisher Scientific; benzo(a)pyrene (97%) was purchased from Sigma. Reagent-grade acetone (Fisher Scientific) was used as the solvent carrier into which the water-insoluble compounds were dissolved before addition to the aqueous diatom culture. Acetone-dissolved PAHs were pipetted volumetrically into microalgal-culture media to achieve final concentrations of 10, 100, and 1000 lg L 1 for each PAH compound. The PAH concentrations selected represent a range of toxicity (e.g. low to high concentrations) found in natural settings (Fisher et al., 2000). 2.3. Experimental conditions N. brevirostris was transferred aseptically into 250-mL flasks containing Enriched Seawater Medium with Silicate (ESi; Ukeles, 1973) to achieve an initial cell density of 104 cells mL 1 in each flask. Control culture flasks (not exposed to PAH compounds), and acetone culture flasks (contained 1% of the solvent carrier) were prepared in addition to the PAH concentrations described above. All treatments were carried out in triplicate. Experimental flask cultures were incubated at 18 ± 1 °C with a light intensity of 300 lE m 2 s 1 PAR from cool-white fluorescent bulbs on a 12:12 h. light:dark cycle. Flask cultures were allowed to grow for 21 days and sub-sampled on days 0, 2, 5, 7, 9, 12, 14, 16, 19, and 21. 2.3.1. Fluorescent dyes Viability of diatom cells was measured using the fluorescent probe SYTOX Green (30 lM initial concentration, Invitrogen; Carlsbad, CA), which stains the nucleic acids of cells with compromised membranes. SYTOX-positive cells generally are considered to be dead. SYTOX emits fluorescence in the flow-cytometer FL1 channel (530 nm wavelength) wherein natural chlorophyll fluorescence in the 650-nm range does not interfere. Lipid storage in diatom cells was measured using the fluorescent stain BODIPY 493/503 (4,4Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene) (10 mg initial concentration, Invitrogen; Carlsbad, CA). BODIPY is a lipophilic fluorophore that emits fluorescence in the FL1 cytometer channel (530 nm wavelength). Without calibration using an independent method (e.g., chromatography), BODIPY fluorescence is recorded in arbitrary fluorescence values that allow contrasts between experimental treatments to be determined, but not quantitative measurements of lipid per cell. 2.3.2. Experimental procedures A 600-lL sample was taken from each triplicate culture flask immediately following the addition of PAH compounds for initial sampling. Algal cells and fluorescent probes were distributed into 5-mL polycarbonate tubes. A 200-lL sub-sample of cells and
Please cite this article in press as: Croxton, A.N., et al. The use of flow cytometric applications to measure the effects of PAHs on growth, membrane integrity, and relative lipid content of the benthic diatom, Nitzschia brevirostris. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.12.010
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2.4. Statistical analysis An analysis of variance (ANOVA) was performed to determine possible effects of acetone, naphthalene, pyrene, and benzo(a)pyrene, at three concentrations, upon division rates, membrane integrity, and lipid storage in N. brevirostris cultures. Multi-factor analysis of variance (MANOVA) was performed to determine combined effects of exposure treatments and exposure time on dependent variables. Tukey’s Honestly Significant Differences (HSD) test was performed on dependent variables to determine significance of contrasts between means. A multiple-range test was used to determine which dependent variable levels were significantly (P < 0.05) different for the experimental compounds. Significant differences among treatments were noted with alphabet superscripts in all tables and figures. All statistical analyses were performed using Statgraphics Plus (Manugistics, Rockville, MD) software. 3. Results Overall, the use of flow-cytometry coupled with fluorescent dyes was successful in measuring physiological changes in a benthic diatom species exposed to individual PAH compounds. Final population density, membrane stability, and lipid storage in N. brevirostris cultures were significantly (P < 0.05) affected by exposure treatments used in this study, but log-phase division rate was not. Significant (P < 0.05) interactions existed between treatments and duration of exposure. Log-phase division rates, calculated from Days 2–7 cell counts, were not significantly (P > 0.05) affected by PAH compounds at concentrations used in this study (Table 1). On Day 9, however, N. brevirostris maximal cell densities in stationary phase were significantly (P < 0.05) affected by contaminant treatments and duration of exposure (Fig. 3.1). Multiple-range analysis indicated that cell densities in naphthalene 1000 lg L 1 were significantly higher Table 1 Mean values of log-phase division rates calculated from Days 2–7 cell counts. Pvalue = 0.4732. PAH/concentration
Mean values
Standard error
Naphthalene 1000 lg/L Acetone Naphthalene 100 lg/L B(a)P 100 lg/L Control Naphthalene 10 lg/L B(a)P 10 lg/L B(a)P 1000 lg/L Pyrene 100 lg/L Pyrene 10 lg/L Pyrene 1000 lg/L
0.513 0.530 0.534 0.536 0.542 0.543 0.560 0.563 0.582 0.597 0.598
0.016 0.022 0.015 0.042 0.039 0.042 0.041 0.011 0.031 0.014 0.033
1.00E+07
Number of cells mL-1
10 lL of SYTOX, at a concentration of 0.1 mM, were mixed and incubated in the dark for 15 min. Counts of total diatom cells per mL were determined from counts of total, chlorophyll-containing particles (FL3 positive), and numbers of cells permeable to SYTOX (with compromised membranes) were determined as percentages of total diatom counts. A 200-lL sub-sample of algal cells and 10 lL of 0.1-mM BODIPY were mixed and incubated in the dark for 30 min. Following incubation, tubes were vortexed and analyzed on a BD Biosciences FACScan flow- cytometer (San Jose, CA). Biplots of diatom cells were generated using selected cytometer detector data (FL1 or FL3 in Windows Multiple Document Interface (WinMDI 2.8; Scripps Institute). Raw data obtained from WinMDI plots were imported into Microsoft Excel and analyzed as quantitative data for SYTOX and BODIPY fluorescence-intensity assays.
1.00E+06
Control Acetone Nap-10 Nap-100 Nap-1000 Pyr-10 Pyr-100 Pyr-1000 Bap-10 Bap-100 Bap-1000
1.00E+05
1.00E+04
0
2
5
7
9
12 14 16 19 21
Days Fig. 3.1. Growth curve of PAH-exposed N. brevirostris. Error bars are not included in the graph for clarity purposes.
than control cultures (Table 2). Cell densities in naphthalene 10 and 100 lg L 1 treatments were significantly lower than control treatment cell densities on Day 21 (Table 2). Analyses of membrane stability of experimentally-exposed diatoms were performed using SYTOX fluorescence on Days 0, 5, 9, 12, and 21. Naphthalene 1000 lg L 1 – treated cultures had significantly more cells with compromised membrane integrity (P < 0.05) than control treatments immediately after exposure on Day 0 (Table 3). Diatom cells in the naphthalene 1000 lg L 1 treatment, however, maintained membrane integrity more effectively (P < 0.05) than cultures in the acetone, pyrene 100 and 1000 lg L 1, b(a)p 10 and 100 lg L 1 treatments on Day 9 (Table 3). On Day 12, diatoms in the naphthalene 100 lg L 1 treatment maintained membrane integrity significantly more (P < 0.05) than all other experimental treatments in the study. All naphthalene treatments maintained membrane integrity significantly more (P < 0.05) than b(a)p 100 and 1000 lg L 1 treatments on Day 21. Control cultures had significantly more dead (P < 0.05) cells than all treatments on Day 21, but also more live cells than most other treatments. Membrane stability did not differ between cultures on Day 5, in log phase (P > 0.05). Both treatment and length of exposure had significant (P < 0.001) effects upon lipid content in N. brevirostris cultures. Increases in lipids over time were observed in all exposure treatments. Cultures in the pyrene 100 lg L 1 treatment had significantly more lipid than all other exposure treatments, and control cultures had significantly less lipid than PAH-exposed culture treatments (Fig. 3.2). Culture treatments had higher lipid on Day 0 than any other day in the experiment, because a stationary-phase culture was used as inoculum, and the least amount of lipid on Day 9 at the end of the log phase (Fig. 3.3). MANOVA was also used to compare lipid fluorescence during the exponential (Day 0–9) and stationary (Day 12–21) phases of diatom growth in exposed treatments. Results from this analysis
Please cite this article in press as: Croxton, A.N., et al. The use of flow cytometric applications to measure the effects of PAHs on growth, membrane integrity, and relative lipid content of the benthic diatom, Nitzschia brevirostris. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.12.010
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A.N. Croxton et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx Table 2 Mean values of maximal cell densities on Days 9, 12, and 21. Alphabet superscripts indicate homogeneous mean groups. Experimental treatment
Day 9 Mean P-value = 0.0051
Day 12 Mean P-value = 0.3872
Day 21 Mean P-value = 0.0002
Acetone B(a)P 10 lg/L B(a)P 100 lg/L B(a)P 1000 lg/L Control Naphthalene 10 lg/L Naphthalene 100 lg/L Naphthalene 1000 lg/L Pyrene 10 lg/L Pyrene 100 lg/L Pyrene 1000 lg/L
1.52 106cd 1.06 106ab 1.21 106abc 1.39 106abc 1.17 106abc 1.46 106bc 1.35 106abc 1.92 106d 1.57 106cd 1.31 106abc 1.03 106a
1.01 106 9.89 105 8.29 105 6.81 105 9.23 105 6.50 105 7.95 105 1.07 106 8.91 105 7.94 105 5.63 105
8.02 105abc 7.82 105abc 1.25 106c 1.13 106c 2.04 106d 5.70 105ab 3.99 105a 1.13 106bc 7.42 105abc 7.29 105abc 9.30 105abc
Table 3 Mean values of percentages of cells with compromised membrane integrity on Days 0, 5, 9, 12, and 21. Alphabet superscripts indicate homogeneous mean groups. Experimental treatment
Day 0 Mean P-value = 0.0001
Day 5 Mean P-value = 0.0507
Day 9 Mean P-value = 0.0412
Day 12 Mean P-value = 0.0095
Day 21 Mean P-value = 0.0000
Acetone B(a)P 10 lg/L B(a)P 100 lg/L B(a)P 1000 lg/L Control Naphthalene 10 lg/L Naphthalene 100 lg/L Naphthalene 1000 lg/L Pyrene 10 lg/L Pyrene 100 lg/L Pyrene 1000 lg/L
5.33 103ab 1.26 104cd 1.50 104de 1.49 104de 7.86 103abc 1.03 104bcd 4.70 103a 1.81 104e 1.18 104cd 1.00 104bcd 1.17 104cd
2.28 105 1.63 105 1.56 105 1.19 105 2.29 105 1.13 105 1.19 105 2.01 105 6.92 104 2.13 105 2.14 105
4.73 105bcd 5.62 105d 5.42 105cd 3.82 105abcd 1.99 105ab 3.46 105abcd 2.51 105abc 1.51 105a 2.06 105a 5.21 105cd 5.15 105cd
7.51 105bc 6.84 105b 1.08 106c 1.00 106bc 6.64 105b 7.69 105bc 3.10 105a 8.49 105bc 6.75 105b 7.22 105b 8.76 105bc
3.42 105cd 2.40 105abc 3.79 105d 3.20 105bcd 6.88 105e 1.30 105a 1.54 105a 1.50 105a 2.05 105ab 1.98 105ab 2.52 105abc
Fig. 3.2. Mean values of BODIPY lipid fluorescence intensity per experimental treatment over the exposure period. Error bars represent standard error of mean values. Letters indicate significant differences among treatments.
showed that lipid mean values were not significantly (P > 0.05) different between exposure treatments during the exponential phase, but PAH-exposed treatments had significantly (P < 0.001) more lipid than control cultures in the stationary phase. Significant (P < 0.001) decreases in lipid were observed on Day 9 during the end of the exponential growth phase, and on Day 16 (P < 0.001) in late stationary phase.
4. Discussion The adaptation of flow-cytometric applications from the biomedical field to environmental science is still developing as fluorescent, physiological dyes are added to natural pigment
Fig. 3.3. Mean values of BODIPY lipid fluorescence intensity per treatment day. Mean lipid content was decreased significantly on Day 9, the end of log phase, compared to other days. Error bars represent standard error of mean values. Letters indicate significant differences among treatments.
fluorescence and cell size and internal complexity variables used in earlier studies. In the present study, multiple analyses were performed rapidly with small sample requirements and simple sample preparation protocols. Inclusion of fluorescent dyes for membrane integrity and relative lipid content revealed that specific PAH compounds, naphthalene, pyrene, and b(a)p, had different effects upon cell densities and lipid storage in N. brevirostris cultures during a 21-day exposure. The inhibition or stimulation of test variables varied with PAH compound and concentration of exposure. Final, stationary-phase cell densities in naphthalene 1000 lg L 1 culture treatments were significantly higher than final cell densities in control treatments. Division rates of N. brevirostris cells were not significantly altered by PAH compounds. Increases in lipid content
Please cite this article in press as: Croxton, A.N., et al. The use of flow cytometric applications to measure the effects of PAHs on growth, membrane integrity, and relative lipid content of the benthic diatom, Nitzschia brevirostris. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.12.010
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were observed in some PAH-exposed culture treatments in stationary phase. N. brevirostris cells in pyrene at 100 lg L 1 had significantly more lipid fluorescence than all other experimental treatments. Results suggest that PAH concentrations used in this exposure study did not exceed threshold levels for lethal effects of any compound investigated. Dunstan et al. (1975) found that a concentration of 104 lg L 1 was the growth-inhibition threshold for marine algae exposed to xylene, benzene, and toluene. Marine algal species exposed to those compounds displayed varied responses. Similarly, varied responses among compounds and concentrations also were observed in the present study. Several field and laboratory studies have described reductions in cell densities of both planktonic and benthic algae exposed to petroleum products (Carman et al., 1997; Megharaj et al., 2000; Piehler et al., 2003; Moreno-Garrido et al., 2007). PAH compounds selected for this study were representative of low, medium, and high toxicity levels, intended to examine algal responses following exposure to a broad spectrum of compounds. Earlier research on plant physiology suggests that low-molecular-weight PAHs are biologically more active as a result of their volatile nature (Dunstan et al., 1975). A reduction in cell densities in naphthalene treatments, therefore, was anticipated in this study. This hypothesis was proven correct for low and intermediate concentrations; however, maximal cell-density results revealed a stimulatory effect on diatom cell growth by naphthalene at the highest concentration (1000 lg L 1). Cell membrane integrity of diatoms in the experimental treatments overall remained intact during the 21 day exposure. On Day 0, a relatively high percentage of diatom cells in the naphthalene 1000 lg L 1 treatment had compromised membranes as the fluorescent stain SYTOX was able to penetrate cells in this culture. By Day 12, however, membranes of cells in this culture recovered stability at a higher percentage than all other treatments in the study. The ability of PAH-exposed cells to maintain membrane stability further indicates that this diatom species can serve as a vector of PAH trophic transfer. Hook and Osborn (2012) reported an increase in SYTOX fluorescence in algal cells exposed to dispersants and the chemically-enhanced water accommodated fraction (CWAF), indicating membrane damage. In the present study, on Day 21 control cultures had a higher mean percentage of SYTOXpositive cells than all other treatments, especially the lower molecular weight PAH, naphthalene. Lipid fluorescence intensity in this diatom also appeared to be stimulated by the PAH compounds in stationary phase. Lipid mean values for all PAH-exposed treatments were significantly higher than control treatments. It appears that the increase in lipid content is a cellular response to exposure to PAH compounds, possibly indicating a need for more energy to maintain cell growth or alternately diluting the PAH compound within a larger cellular lipid pool. Overall, there was an expected increase in lipid level as cultures entered stationary phase. In summary, the use of flow cytometry provided a more comprehensive analysis of how individual PAHs with varying molecular weights modified both growth and physiology of a diatom species. Results from this study suggest both inhibitory and stimulatory responses of a diatom culture to exposure to individual PAH compounds. These findings suggest the potential for some microphytobenthic diatoms to grow in PAH-contaminated environments and subsequently to pass assimilated PAHs to grazing organisms. This hypothesis is supported by results described in work by Megharaj et al. (2000) which reported both reductions and increases in biomass of algal populations in diesel-contaminated sediments. Hook and Osborn (2012) exposed a diatom species, Phaeodactylum tricornutum, to weathered oil, dispersed weathered oil, and a hydrocarbon-based dispersant and observed
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variable sensitivity in growth from exposure to the weathered and dispersed weathered oil. Carman et al. (1997) also observed an increase in algal biomass following exposure to diesel contamination in the field. This increase was believed to be a result of a reduction in grazer populations that were diminished by exposure to contaminated sediments. Nevertheless, continued studies examining additional environmental factors including the effects of PAH mixtures and increased concentrations, are also needed to fully understand the potential effects of PAHs on this ecologicallyimportant community. We conclude that flow-cytometric tools will facilitate detection of sub-lethal effects upon primary producers that will be relevant to trophic transfer of pollutants in the ecosystem.
Acknowledgements This work was performed under appointment to the National Oceanic and Atmospheric Administration, Education Partnership Program with Minority Serving Institutions, Graduate Sciences Program. Additional funding was provided by the Northeast Fisheries Science Center, Milford Laboratory. The authors would like to thank Jennifer Alix for her technical assistance.
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The use of flow cytometric applications to measure the effects of PAHs on growth, membrane integrity, and relative lipid content of the benthic diatom, Nitzschia brevirostris April N. Croxton a,b,⇑, Gary H. Wikfors a, Richard D. Schulterbrandt-Gragg III b a b
Northeast Fisheries Science Center, NMFS, NOAA, 212 Rogers Avenue, Milford, CT 06460, USA School of the Environment, 1520 S. Bronough St., Florida A&M University, Tallahassee, FL 32307, USA
a r t i c l e
i n f o
Article history: Available online xxxx Keywords: Oil pollution Fluorescent probes Membrane permeability Microphytobenthos Algal cellular physiology Rapid method
a b s t r a c t This laboratory study measured the direct effects of three polycyclic aromatic hydrocarbon (PAH) compounds (naphthalene, pyrene, and benzo(a)pyrene) upon cell growth, membrane integrity, and BODIPY-stained lipid fluorescence intensity of the benthic diatom Nitzschia brevirostris using flow cytometry as an analysis tool. Previous field and laboratory studies have reported reductions in algal populations following PAH exposure, but specific, functional responses of the microalgae to these pollutants could not be revealed by cell numbers alone. Using flow-cytometric measurements, we confirmed that maximal cell densities in PAH-exposed diatom cultures were significantly lower compared to controls; however, we also discovered increases in lipids and cells with compromised membranes in PAH-exposed cultures. These results highlight new tools for measuring the direct effects of organic pollutants upon the physiology of taxa comprising microphytobenthic communities important in estuarine food webs. Published by Elsevier Ltd.
1. Introduction Originally used in clinical applications for immunology, hematology, and oncology, flow-cytometry has been used by researchers for over three decades as a technique to analyze phytoplankton (Berglund and Eversman, 1988; Olson et al., 1993; Mandy et al., 1995; Franqueira et al., 2000; Collier, 2004; Yentsch and Yentsch, 2008). Flow-cytometry provides rapid measurements of optical properties of single cells, allowing researchers to quantify phytoplankton populations and estimate growth rates and comparative chlorophyll a concentrations (Collier, 2000, 2004; Yentsch and Yentsch, 2008), as well as differentiate phytoplankton groups based upon accessory pigment fluorescence (Hofstraat et al., 1994). Microalgae, both planktonic and benthic, are a major component of aquatic ecosystems and are responsible for primary production and cycling nutrients throughout these environments (Christensen and Nyholm, 1984; Skoglund and Swackhamer, 1994; Lewis, 1995). Microphytobenthic communities are composed of a group of photosynthetic, eukaryotic algae and cyanobacteria that inhabit ⇑ Corresponding author at: Northeast Fisheries Science Center, NMFS, NOAA, 212 Rogers Avenue, Milford, CT 06460, USA. Tel.: +1 (203) 882 6552; fax: +1 (203) 882 6570. E-mail address: [email protected] (A.N. Croxton).
surface sediments (MacIntyre et al., 1996; Aberle-Malzahn, 2004). The microphytobenthic community is rarely acknowledged fully for its role in primary production in areas where phytoplankton abundance is low (Underwood and Kromkamp, 1999). These organisms also serve as sediment stabilizers, promoters of the transfer of nutrients between sediments and the water column, and food sources for deposit and suspension feeders, including bivalve species (MacIntyre et al., 1996; Aberle-Malzahn, 2004). The close proximity of microphytobenthic communities and sediments exposes this algal community to hydrophobic pollutants that may alter their physiological processes, thereby modifying their critical roles as primary producers and sediment stabilizers in the ecosystem. An example of a large event during which benthic algal communities were exposed to organic pollutants occurred in April 2012 when the Deepwater Horizon oil spill released several million barrels of oil into the productive waters of the Gulf of Mexico for nearly three months (Soniat et al., 2011). Polycyclic aromatic hydrocarbons (PAHs) are a group of hydrophobic organic compounds commonly found in aquatic ecosystems. These compounds are formed during industrial processes and can persist in the environment over a long period of time. Petroleum spills and improper disposal of used oil products are two sources of acute PAH contamination in aquatic environments (Holland et al., 2006). In aquatic ecosystems, hydrophobic
http://dx.doi.org/10.1016/j.marpolbul.2014.12.010 0025-326X/Published by Elsevier Ltd.
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compounds are attracted to suspended particulate matter and lipid-rich biological membranes (Rubinstein et al., 1984). Particles with sorbed, hydrophobic substances settle to the seafloor where they are deposited onto the sediment surface. Aquatic surface sediments act as contaminant reservoirs for many environmental pollutants, including PAHs (Elroy et al., 2000). Microalgae associated with sediments are exposed to these organic pollutants and can potentially serve as vectors in the trophic transfer of these compounds as they are resuspended and filtered by bivalve species (Croxton et al., 2012). Extensive research has been conducted on the physiology of marine organisms exposed to hydrophobic pollutants (Anderson et al., 1981; Grundy et al., 1996; Fisher et al., 2000; Chu et al., 2002), but little data exist concerning the toxic effects of hydrophobic pollutants upon microphytobenthic communities located in these marine environments (Hook and Osborn, 2012). Several authors have studied the effects of specific PAH compounds and petroleum products on growth rates of both planktonic and benthic algae (Winters et al., 1976; Mahoney and Haskin, 1980; Nyholm, 1990; Moreno-Garrido et al., 2007). Findings from these studies indicate that hydrocarbon exposure can inhibit algal growth, but the degree of this inhibition varies between algal species. In contrast, Dunstan et al. (1975) observed stimulation of growth of phytoplankton species exposed to low-molecularweight hydrocarbons. Dunstan and colleagues suggested that the volatile fraction of low-molecular-weight hydrocarbons included the most biologically-active hydrocarbons, and this characteristic could explain the varied responses among algal species in various studies. The PAH compounds chosen for this study represent increasing molecular weights, with naphthalene having the lowest and benzo(a)pyrene having the highest molecular weight (Neff, 1979; Varanasi, 1989). Previous studies of microphytobenthic communities and petroleum products tend to highlight the effects of these compounds upon abundance, community structure, and grazing (Bennett et al., 1999, 2000; Piehler et al., 2003; Suderman and Thistle, 2004), but not physiology. These studies focused on trophic interactions between grazers and benthic communities located in polluted environments. Although these interactions are critical in understanding the overall effects of PAHs in an ecosystem, it is equally important to understand the direct effects of PAHs upon microphytobenthic algae. Chung et al. (2007) investigated the responses of the benthic microalga Chlorococcum meneghini to PAH-spiked sand and found that this exposure diminished cell densities and chlorophyll a concentrations. Similar studies also examined chlorophyll a concentrations and photosynthetic rates of PAH-exposed benthic algae (Bennett et al., 1999; Piehler et al., 2003). Earlier studies conducted by plant physiologists suggest that PAH compounds target plasma membranes and change permeability (Currier, 1951; Dallyn and Sweet, 1951; Currier and Peoples, 1954; Dunstan et al., 1975). Interactions between petroleum products, freshwater algae, and bacteria also have been investigated by several authors (Kauss and Hutchinson, 1977; Sikkema et al., 1994; McCann and Solomon, 2000; Hook and Osborn, 2012). Findings from these studies suggest that hydrocarbons are adsorbed onto biological membranes where they target lipid-containing structures (Kauss and Hutchinson, 1977; Weimburg et al., 1981; McCann and Solomon, 2000). The disruption of these structures alters lipid arrangement in membranes, causing the formation of pores and increasing the permeability of cells (McCann and Solomon, 2000; Chung et al., 2007). The aim of this study was to explore new methods for determining physiological responses of a representative microbenthic diatom to purified PAH compounds – to use flow-cytometric applications coupled with fluorescent probes to measure cell growth,
membrane integrity, and lipid storage of a pure, microphytobenthic diatom culture exposed to naphthalene, pyrene, and benzo(a)pyrene (b(a)p) at experimentally-varied concentrations. 2. Materials and methods 2.1. Microphytobenthic diatom culture The benthic diatom, Nitzschia brevirostris (strain 0–1), obtained from the Milford Laboratory Microalgal Culture Collection, was used as the test alga to determine the effects of PAH compounds on growth, membrane integrity, and lipid storage. 2.2. Test compounds The PAH compounds naphthalene, pyrene, and benzo(a)pyrene were the environmental pollutants used to investigate possible effects upon the diatom. Naphthalene (99+%) and pyrene (98%) were purchased from Fisher Scientific; benzo(a)pyrene (97%) was purchased from Sigma. Reagent-grade acetone (Fisher Scientific) was used as the solvent carrier into which the water-insoluble compounds were dissolved before addition to the aqueous diatom culture. Acetone-dissolved PAHs were pipetted volumetrically into microalgal-culture media to achieve final concentrations of 10, 100, and 1000 lg L 1 for each PAH compound. The PAH concentrations selected represent a range of toxicity (e.g. low to high concentrations) found in natural settings (Fisher et al., 2000). 2.3. Experimental conditions N. brevirostris was transferred aseptically into 250-mL flasks containing Enriched Seawater Medium with Silicate (ESi; Ukeles, 1973) to achieve an initial cell density of 104 cells mL 1 in each flask. Control culture flasks (not exposed to PAH compounds), and acetone culture flasks (contained 1% of the solvent carrier) were prepared in addition to the PAH concentrations described above. All treatments were carried out in triplicate. Experimental flask cultures were incubated at 18 ± 1 °C with a light intensity of 300 lE m 2 s 1 PAR from cool-white fluorescent bulbs on a 12:12 h. light:dark cycle. Flask cultures were allowed to grow for 21 days and sub-sampled on days 0, 2, 5, 7, 9, 12, 14, 16, 19, and 21. 2.3.1. Fluorescent dyes Viability of diatom cells was measured using the fluorescent probe SYTOX Green (30 lM initial concentration, Invitrogen; Carlsbad, CA), which stains the nucleic acids of cells with compromised membranes. SYTOX-positive cells generally are considered to be dead. SYTOX emits fluorescence in the flow-cytometer FL1 channel (530 nm wavelength) wherein natural chlorophyll fluorescence in the 650-nm range does not interfere. Lipid storage in diatom cells was measured using the fluorescent stain BODIPY 493/503 (4,4Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene) (10 mg initial concentration, Invitrogen; Carlsbad, CA). BODIPY is a lipophilic fluorophore that emits fluorescence in the FL1 cytometer channel (530 nm wavelength). Without calibration using an independent method (e.g., chromatography), BODIPY fluorescence is recorded in arbitrary fluorescence values that allow contrasts between experimental treatments to be determined, but not quantitative measurements of lipid per cell. 2.3.2. Experimental procedures A 600-lL sample was taken from each triplicate culture flask immediately following the addition of PAH compounds for initial sampling. Algal cells and fluorescent probes were distributed into 5-mL polycarbonate tubes. A 200-lL sub-sample of cells and
Please cite this article in press as: Croxton, A.N., et al. The use of flow cytometric applications to measure the effects of PAHs on growth, membrane integrity, and relative lipid content of the benthic diatom, Nitzschia brevirostris. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.12.010
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2.4. Statistical analysis An analysis of variance (ANOVA) was performed to determine possible effects of acetone, naphthalene, pyrene, and benzo(a)pyrene, at three concentrations, upon division rates, membrane integrity, and lipid storage in N. brevirostris cultures. Multi-factor analysis of variance (MANOVA) was performed to determine combined effects of exposure treatments and exposure time on dependent variables. Tukey’s Honestly Significant Differences (HSD) test was performed on dependent variables to determine significance of contrasts between means. A multiple-range test was used to determine which dependent variable levels were significantly (P < 0.05) different for the experimental compounds. Significant differences among treatments were noted with alphabet superscripts in all tables and figures. All statistical analyses were performed using Statgraphics Plus (Manugistics, Rockville, MD) software. 3. Results Overall, the use of flow-cytometry coupled with fluorescent dyes was successful in measuring physiological changes in a benthic diatom species exposed to individual PAH compounds. Final population density, membrane stability, and lipid storage in N. brevirostris cultures were significantly (P < 0.05) affected by exposure treatments used in this study, but log-phase division rate was not. Significant (P < 0.05) interactions existed between treatments and duration of exposure. Log-phase division rates, calculated from Days 2–7 cell counts, were not significantly (P > 0.05) affected by PAH compounds at concentrations used in this study (Table 1). On Day 9, however, N. brevirostris maximal cell densities in stationary phase were significantly (P < 0.05) affected by contaminant treatments and duration of exposure (Fig. 3.1). Multiple-range analysis indicated that cell densities in naphthalene 1000 lg L 1 were significantly higher Table 1 Mean values of log-phase division rates calculated from Days 2–7 cell counts. Pvalue = 0.4732. PAH/concentration
Mean values
Standard error
Naphthalene 1000 lg/L Acetone Naphthalene 100 lg/L B(a)P 100 lg/L Control Naphthalene 10 lg/L B(a)P 10 lg/L B(a)P 1000 lg/L Pyrene 100 lg/L Pyrene 10 lg/L Pyrene 1000 lg/L
0.513 0.530 0.534 0.536 0.542 0.543 0.560 0.563 0.582 0.597 0.598
0.016 0.022 0.015 0.042 0.039 0.042 0.041 0.011 0.031 0.014 0.033
1.00E+07
Number of cells mL-1
10 lL of SYTOX, at a concentration of 0.1 mM, were mixed and incubated in the dark for 15 min. Counts of total diatom cells per mL were determined from counts of total, chlorophyll-containing particles (FL3 positive), and numbers of cells permeable to SYTOX (with compromised membranes) were determined as percentages of total diatom counts. A 200-lL sub-sample of algal cells and 10 lL of 0.1-mM BODIPY were mixed and incubated in the dark for 30 min. Following incubation, tubes were vortexed and analyzed on a BD Biosciences FACScan flow- cytometer (San Jose, CA). Biplots of diatom cells were generated using selected cytometer detector data (FL1 or FL3 in Windows Multiple Document Interface (WinMDI 2.8; Scripps Institute). Raw data obtained from WinMDI plots were imported into Microsoft Excel and analyzed as quantitative data for SYTOX and BODIPY fluorescence-intensity assays.
1.00E+06
Control Acetone Nap-10 Nap-100 Nap-1000 Pyr-10 Pyr-100 Pyr-1000 Bap-10 Bap-100 Bap-1000
1.00E+05
1.00E+04
0
2
5
7
9
12 14 16 19 21
Days Fig. 3.1. Growth curve of PAH-exposed N. brevirostris. Error bars are not included in the graph for clarity purposes.
than control cultures (Table 2). Cell densities in naphthalene 10 and 100 lg L 1 treatments were significantly lower than control treatment cell densities on Day 21 (Table 2). Analyses of membrane stability of experimentally-exposed diatoms were performed using SYTOX fluorescence on Days 0, 5, 9, 12, and 21. Naphthalene 1000 lg L 1 – treated cultures had significantly more cells with compromised membrane integrity (P < 0.05) than control treatments immediately after exposure on Day 0 (Table 3). Diatom cells in the naphthalene 1000 lg L 1 treatment, however, maintained membrane integrity more effectively (P < 0.05) than cultures in the acetone, pyrene 100 and 1000 lg L 1, b(a)p 10 and 100 lg L 1 treatments on Day 9 (Table 3). On Day 12, diatoms in the naphthalene 100 lg L 1 treatment maintained membrane integrity significantly more (P < 0.05) than all other experimental treatments in the study. All naphthalene treatments maintained membrane integrity significantly more (P < 0.05) than b(a)p 100 and 1000 lg L 1 treatments on Day 21. Control cultures had significantly more dead (P < 0.05) cells than all treatments on Day 21, but also more live cells than most other treatments. Membrane stability did not differ between cultures on Day 5, in log phase (P > 0.05). Both treatment and length of exposure had significant (P < 0.001) effects upon lipid content in N. brevirostris cultures. Increases in lipids over time were observed in all exposure treatments. Cultures in the pyrene 100 lg L 1 treatment had significantly more lipid than all other exposure treatments, and control cultures had significantly less lipid than PAH-exposed culture treatments (Fig. 3.2). Culture treatments had higher lipid on Day 0 than any other day in the experiment, because a stationary-phase culture was used as inoculum, and the least amount of lipid on Day 9 at the end of the log phase (Fig. 3.3). MANOVA was also used to compare lipid fluorescence during the exponential (Day 0–9) and stationary (Day 12–21) phases of diatom growth in exposed treatments. Results from this analysis
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A.N. Croxton et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx Table 2 Mean values of maximal cell densities on Days 9, 12, and 21. Alphabet superscripts indicate homogeneous mean groups. Experimental treatment
Day 9 Mean P-value = 0.0051
Day 12 Mean P-value = 0.3872
Day 21 Mean P-value = 0.0002
Acetone B(a)P 10 lg/L B(a)P 100 lg/L B(a)P 1000 lg/L Control Naphthalene 10 lg/L Naphthalene 100 lg/L Naphthalene 1000 lg/L Pyrene 10 lg/L Pyrene 100 lg/L Pyrene 1000 lg/L
1.52 106cd 1.06 106ab 1.21 106abc 1.39 106abc 1.17 106abc 1.46 106bc 1.35 106abc 1.92 106d 1.57 106cd 1.31 106abc 1.03 106a
1.01 106 9.89 105 8.29 105 6.81 105 9.23 105 6.50 105 7.95 105 1.07 106 8.91 105 7.94 105 5.63 105
8.02 105abc 7.82 105abc 1.25 106c 1.13 106c 2.04 106d 5.70 105ab 3.99 105a 1.13 106bc 7.42 105abc 7.29 105abc 9.30 105abc
Table 3 Mean values of percentages of cells with compromised membrane integrity on Days 0, 5, 9, 12, and 21. Alphabet superscripts indicate homogeneous mean groups. Experimental treatment
Day 0 Mean P-value = 0.0001
Day 5 Mean P-value = 0.0507
Day 9 Mean P-value = 0.0412
Day 12 Mean P-value = 0.0095
Day 21 Mean P-value = 0.0000
Acetone B(a)P 10 lg/L B(a)P 100 lg/L B(a)P 1000 lg/L Control Naphthalene 10 lg/L Naphthalene 100 lg/L Naphthalene 1000 lg/L Pyrene 10 lg/L Pyrene 100 lg/L Pyrene 1000 lg/L
5.33 103ab 1.26 104cd 1.50 104de 1.49 104de 7.86 103abc 1.03 104bcd 4.70 103a 1.81 104e 1.18 104cd 1.00 104bcd 1.17 104cd
2.28 105 1.63 105 1.56 105 1.19 105 2.29 105 1.13 105 1.19 105 2.01 105 6.92 104 2.13 105 2.14 105
4.73 105bcd 5.62 105d 5.42 105cd 3.82 105abcd 1.99 105ab 3.46 105abcd 2.51 105abc 1.51 105a 2.06 105a 5.21 105cd 5.15 105cd
7.51 105bc 6.84 105b 1.08 106c 1.00 106bc 6.64 105b 7.69 105bc 3.10 105a 8.49 105bc 6.75 105b 7.22 105b 8.76 105bc
3.42 105cd 2.40 105abc 3.79 105d 3.20 105bcd 6.88 105e 1.30 105a 1.54 105a 1.50 105a 2.05 105ab 1.98 105ab 2.52 105abc
Fig. 3.2. Mean values of BODIPY lipid fluorescence intensity per experimental treatment over the exposure period. Error bars represent standard error of mean values. Letters indicate significant differences among treatments.
showed that lipid mean values were not significantly (P > 0.05) different between exposure treatments during the exponential phase, but PAH-exposed treatments had significantly (P < 0.001) more lipid than control cultures in the stationary phase. Significant (P < 0.001) decreases in lipid were observed on Day 9 during the end of the exponential growth phase, and on Day 16 (P < 0.001) in late stationary phase.
4. Discussion The adaptation of flow-cytometric applications from the biomedical field to environmental science is still developing as fluorescent, physiological dyes are added to natural pigment
Fig. 3.3. Mean values of BODIPY lipid fluorescence intensity per treatment day. Mean lipid content was decreased significantly on Day 9, the end of log phase, compared to other days. Error bars represent standard error of mean values. Letters indicate significant differences among treatments.
fluorescence and cell size and internal complexity variables used in earlier studies. In the present study, multiple analyses were performed rapidly with small sample requirements and simple sample preparation protocols. Inclusion of fluorescent dyes for membrane integrity and relative lipid content revealed that specific PAH compounds, naphthalene, pyrene, and b(a)p, had different effects upon cell densities and lipid storage in N. brevirostris cultures during a 21-day exposure. The inhibition or stimulation of test variables varied with PAH compound and concentration of exposure. Final, stationary-phase cell densities in naphthalene 1000 lg L 1 culture treatments were significantly higher than final cell densities in control treatments. Division rates of N. brevirostris cells were not significantly altered by PAH compounds. Increases in lipid content
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were observed in some PAH-exposed culture treatments in stationary phase. N. brevirostris cells in pyrene at 100 lg L 1 had significantly more lipid fluorescence than all other experimental treatments. Results suggest that PAH concentrations used in this exposure study did not exceed threshold levels for lethal effects of any compound investigated. Dunstan et al. (1975) found that a concentration of 104 lg L 1 was the growth-inhibition threshold for marine algae exposed to xylene, benzene, and toluene. Marine algal species exposed to those compounds displayed varied responses. Similarly, varied responses among compounds and concentrations also were observed in the present study. Several field and laboratory studies have described reductions in cell densities of both planktonic and benthic algae exposed to petroleum products (Carman et al., 1997; Megharaj et al., 2000; Piehler et al., 2003; Moreno-Garrido et al., 2007). PAH compounds selected for this study were representative of low, medium, and high toxicity levels, intended to examine algal responses following exposure to a broad spectrum of compounds. Earlier research on plant physiology suggests that low-molecular-weight PAHs are biologically more active as a result of their volatile nature (Dunstan et al., 1975). A reduction in cell densities in naphthalene treatments, therefore, was anticipated in this study. This hypothesis was proven correct for low and intermediate concentrations; however, maximal cell-density results revealed a stimulatory effect on diatom cell growth by naphthalene at the highest concentration (1000 lg L 1). Cell membrane integrity of diatoms in the experimental treatments overall remained intact during the 21 day exposure. On Day 0, a relatively high percentage of diatom cells in the naphthalene 1000 lg L 1 treatment had compromised membranes as the fluorescent stain SYTOX was able to penetrate cells in this culture. By Day 12, however, membranes of cells in this culture recovered stability at a higher percentage than all other treatments in the study. The ability of PAH-exposed cells to maintain membrane stability further indicates that this diatom species can serve as a vector of PAH trophic transfer. Hook and Osborn (2012) reported an increase in SYTOX fluorescence in algal cells exposed to dispersants and the chemically-enhanced water accommodated fraction (CWAF), indicating membrane damage. In the present study, on Day 21 control cultures had a higher mean percentage of SYTOXpositive cells than all other treatments, especially the lower molecular weight PAH, naphthalene. Lipid fluorescence intensity in this diatom also appeared to be stimulated by the PAH compounds in stationary phase. Lipid mean values for all PAH-exposed treatments were significantly higher than control treatments. It appears that the increase in lipid content is a cellular response to exposure to PAH compounds, possibly indicating a need for more energy to maintain cell growth or alternately diluting the PAH compound within a larger cellular lipid pool. Overall, there was an expected increase in lipid level as cultures entered stationary phase. In summary, the use of flow cytometry provided a more comprehensive analysis of how individual PAHs with varying molecular weights modified both growth and physiology of a diatom species. Results from this study suggest both inhibitory and stimulatory responses of a diatom culture to exposure to individual PAH compounds. These findings suggest the potential for some microphytobenthic diatoms to grow in PAH-contaminated environments and subsequently to pass assimilated PAHs to grazing organisms. This hypothesis is supported by results described in work by Megharaj et al. (2000) which reported both reductions and increases in biomass of algal populations in diesel-contaminated sediments. Hook and Osborn (2012) exposed a diatom species, Phaeodactylum tricornutum, to weathered oil, dispersed weathered oil, and a hydrocarbon-based dispersant and observed
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variable sensitivity in growth from exposure to the weathered and dispersed weathered oil. Carman et al. (1997) also observed an increase in algal biomass following exposure to diesel contamination in the field. This increase was believed to be a result of a reduction in grazer populations that were diminished by exposure to contaminated sediments. Nevertheless, continued studies examining additional environmental factors including the effects of PAH mixtures and increased concentrations, are also needed to fully understand the potential effects of PAHs on this ecologicallyimportant community. We conclude that flow-cytometric tools will facilitate detection of sub-lethal effects upon primary producers that will be relevant to trophic transfer of pollutants in the ecosystem.
Acknowledgements This work was performed under appointment to the National Oceanic and Atmospheric Administration, Education Partnership Program with Minority Serving Institutions, Graduate Sciences Program. Additional funding was provided by the Northeast Fisheries Science Center, Milford Laboratory. The authors would like to thank Jennifer Alix for her technical assistance.
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