CHLOROPHYLL SPECIFIC ABSORPTION COEFFICIENTS AND THE IMPACT OF PHYTOPLANKTON TAXONOMIC GROUP OF SURFACE WATERS IN THE NORTHEASTERN GULF OF MEXICO

The chlorophyll-specific absorption coefficient at 440 nm (aph(440)) of surface water in the Northeastern Gulf of Mexico varied by a factor of 7 (0.02-0.15 m mg) with the of chlorophyll-a concentration of 0.06-12.25 mg m. In general, lower values of aph(440) (<0.06 m mg) were observed in the inshore particularly in the major river mouths. During summer, lower values of aph(440) were also observed offshore associated with low-salinity waters of the Mississippi River plume. Higher values of aph(440) (>0.1 m mg) were otherwise observed outside the river plumes in the outer shelf and slope, where lower chlorophyll-a concentration occurred. Based on phytoplankton taxonomic groups, the average value of aph(440), of microphytoplankton group was significantly lower than that of nanophytoplankton and picophytoplantkon groups, suggesting that an increase in cell optical size (pigment packaging) resulted in decreasing aph(440) values. The relationship between aph(440) and chlorophyll-a concentration was also not linear, indicating pigment composition played an important role in determining aph(440) variability.


INTRODUCTION
The phytoplankton absorption coefficient per unit of chlorophyll concentration (chlorophyll-specific absorption coefficient, a * ph (λ) is a key factor when modeling light propagation within the ocean and ocean color (e.q., Carder et al., 1986;Gordon et al., 1988;Morel, 1988,), carbon fixation by phytoplankton (Kiefer and Mitchell, 1983), the contribution of phytoplankton to the total absorption coefficient of seawater, as well as for modeling marine primary production (Morel and Andre, 1991;Carder et al., 1995;Sakshaug et al., 1997;Ishizaka, 1998).
Variability in the magnitude and spectral shape of a * ph (λ) can be attributed to three factors: (1) packaging effect i.e., pigments packed into stacks of shelfshaded chloroplasts are less efficient in absorbing light per unit pigment mass than an optically thin solution (Kirk, 1994), ( 2) pigment composition and (3) cell size.
An increase in pigment packaging can occur either as cell size increases or the internal concentration of pigments increases (Morel and Bricaud, 1981;Kirk, 1994;Lohrenz et al., 2003).Differences in phytoplankton species, as well as variation within a species grown under different environmental conditions (e.g., growth irradiance), also causes variability in a * ph (λ) due to pigmentation and packaging effects (Morel and Bricaud, 1986;Bricaud et al., 1988;Mitchell and Kiefer, 1988a;Berner et al., 1989, Stramski andMorel 1990;Fujiki and Taguchi, 2002).
Several investigators have reported an increase in a * ph (λ) with a decrease in chlorophyll concentration (Carder et al., 1986(Carder et al., , 1991;;Bricaud and Stramski, 1990;Cleveland, 1995;Bricaud et al., 1998Bricaud et al., , 1995)).They interpreted this result mainly due to packaging effect or a general trend of decreasing cell size with decreasing chlorophyll concentration.However, the above trend can instead be attributed solely to the increasing contribution of accessory pigments in waters with low chlorophyll concentration, with no consideration of packaging effect (Wozniak and Ostrowska, 1990;Bricaud et al., 1995;Sakshaug et al., 1997;Ciotti et al., 1999).Carder et al. (1991) also suggested that chlorophyll dependency of a * ph (λ) could differ between regions such as subtropical and temperate regions due to the typical differences in cell size, light, and nutrient regimes.In contrast, Hoepffner and Sathyendranath (1992) reported that there was no dependency of a * ph (440) on chlorophyll concentration in the Gulf of Maine and Georges Bank regions within the range of 0.05-2.5 mg m -3 chlorophyll, although that could all be considered part of the same region.
The Northeastern Gulf of Mexico (NEGOM) encompasses a wide variety of ecosystems which are influenced by a combination of nutrient-rich input from rivers and estuaries, coastal upwelling, vertical mixing (Muller-Karger, 2000;Gilbes et al., 1986), and the Loop Current (Huh et al., 1981;Muller-Karger et al., 2001;Weisberg and He, 2003).These processes exert strong influences on the phytoplankton abundance and distribution in the NEGOM.To date, a * ph (λ) variability has not been studied in the NEGOM region.
The objective of this study was to determine the seasonal and spatial variation of the chlorophyll-specific absorption coefficient of phytoplankton and its relationship with phytoplankton community structure based on pigment composition in the NEGOM.
Water samples (0.5-5.0 L) were collected from the outflow of a flowthrough system that pumped at a rate of 10 liters/minute from a hull depth of about 3-m, which passed water via a 10liter debubbler and mixing chamber.The samples were immediately filtered aboard ship using Whatman GF/F filters (0.7 um pore size) under low vacuum pressure (<0.5 atm).The volume of water filtered varied between ~0.05 and 5.0 liters depending on the concentration of pigmented particles in the sample.Filtering was discontinued once the filter displayed sufficient color to the naked eye.Each filter pad was folded and placed into a 2.0 ml sterile Nalgene cryogenic vial, and stored in liquid N 2  Near-surface water samples for pigment analyses were collected from one of twelve 10-L Niskin bottles operated with a Sea-Bird SBE 911 profiling system used to collect conductivity-temperature-depth (CTD) profiles.Pigment sampling and analyses, including the calibration of a fluorometer on the flow-through system, were conducted by Texas A&M University.Detailed description of data collection and analysis methods can be found in Qian et al. (2003).

Absorption Measurements
Absorption spectra for total particulate (phytoplankton and detritus, a p (λ)) and detritus (a d (λ)) was determined by the quantitative filter technique (Yentsch, 1962;Kiefer and SooHoo, 1982).Prior to analysis, sample and reference filter pads were allowed to thaw slowly at room temperature for about 5-10 minutes prior to being placed in a dark petri dish and moistened with a drop of Milli-Q water.The moist sample and reference filter pads were placed on individual glass plates (diameter=2.4cm) in a custom-made diffuse transmissometer box.Prior to each scan, the filters were slid one at a time over a tungsten-halogen light source that shone through a blue long-pass/cut-off filter and a quartz glass diffuser.Using a custom made, 512-channel spectroradiometer (~350-850nm), the transmittances of the sample filter (T sample (λ)) and the reference filter (T reference (λ)) were measured three times.These measurements were averaged and used to obtain the optical densities of total particulate matter (OD p (λ)) as shown below.
The sample filter was then soaked with ~40-50ml of hot 100% methanol for 10-15 minutes in the dark to extract phytoplankton pigments (Kishino et al., 1985;Roesler et al., 1989;Bissett et al., 1997).Transmittances of the extracted filter (T detritus (λ)) and the reference filter were once again measured three times, and the optical density of detritus (OD d (λ)) calculated.
Optical densities were calculated as follows: The absorption coefficients of particulate matter (a p (λ)) and detritus (a d (λ)), were calculated as follows: where "l" is the geometric pathlength equal to the volume of seawater filtered divided by the effective filtration area of the filter (Πr 2 , r=0.0215/2 m), and β is the pathlength amplification or "β factor" (Butler, 1962).The β factor is an empirical formulation defined as the ratio of optical to geometric pathlength that corrects for multiple scattering inside the filter.In this study, an average of two published β factor formulations (Bricaud and Stramski, 1990;Nelson et al., 1993) was used as follows: (3) 0.5 Spectra with OD p (675) less than 0.04 were omitted from this study (Bissett et  al., 1997) to minimize artifacts due to uncertainty in the β factor (Mitchell and Kiefer, 1988a;Bricaud and Stramski, 1990;Cleveland and Weidemann, 1993;Nelson et al., 1993;Moore et al., 1995;Lohrenz, 2000).All other spectra were set to zero at 750 nm or above to correct for residual scattering caused by nonuniformity in wetness between the sample and reference filters or for stray light.
Using Eqs.2a and 2b, the phytoplankton absorption coefficient, a ph (λ), were calculated as follows: Fluorometric chlorophyll and phaeopigment concentrations were determined on the filtrate of phytoplankton pigments extracted from the sample filter with hot 100% methanol using a Turner 10-AU-005 fluorometer according to the methods of Holm-Hansen and Riemann (1978).Finally a ph (λ) was converted to chlorophyll-a specific absorption coefficient (a * ph (λ)) dividing by chlorophyll-a concentration.
The local maxima of the phytoplankton absorption coefficient spectra near 440 nm were usually observed in the range of 440+5 nm, respectively.Therefore, the magnitude of phytoplankton and chlorophyll-specific absorption coefficients at 440 nm was obtained by averaging spectra from the above range of wavelengths.
During summer cruises, relatively high biomass proportions of microphytoplankton were also observed in the offshore region (Figure 2) along the Mississippi River plume.It seems that relatively high biomass proportions of microphytoplankton were associated with low salinity (Nababan, 2005).This trend is consistent with those shown in Qian et al. (2003) in which they applied different methods to quantify the abundance and distribution of diatoms in the same region.
Relatively high values of biomass proportion for nanophytoplankton (chromophytes nanoflagellates and cryptophytes) were observed particularly offshore, and were relatively low inshore.Relatively high biomass proportion for nanophytoplankton were associated with low chlorophyll-a concentrations.During summer, it appeared that relatively low nanophytoplankton biomass proportions extended offshore following the Mississippi River plume (Figure 3).
The abundance of the picophytoplankton group (cyanobacteria, prochlorophytes and green flagellates) was mixed along the NEGOM but relatively high biomass proportions were only observed in late spring (Sp-99) and summer (Su-98, Su-99, Su-00) over the outer shelf and slope (Figure 4) in mostly oligotrophic waters since this group is a nitrogen fixer phytoplankton.This pattern was also consistent with the observation conducted by Qian et al.  (2003).

Variation in Chlorophyll-Specific Absorption Coefficient
The chlorophyll-specific absorption coefficient at 440 nm was found to be highly variable in the NEGOM region for all cruises (Figure 5). a * ph (440) varied by about a factor of 7 ranging from 0.02 to 0.15 m 2 mg -1 for the chlorophyll-a range of 0.06-12.25 mg m -3 over the study period (Figure 5).Variability of a * ph (673) was less pronounced and only varied by a factor 2. For chlorophyll-a range of 0.06-12.25 mg m -3 over the study period, a * ph (673) ranged from 0.0100 to 0.0248 m 2 (mg chl) -1 with the average value of 0.0175 m 2 (mg chl) -1 .
Near-surface spatial and temporal variability of a * ph (440) for seven cruises is presented in Figure 6.There was a general trend of increasing a * ph (440) with distance from shore where chlorophyll-a concentration also decreased.During summer cruises, relatively low a * ph (440) was also observed in the outer shelf and slope of the western NEGOM (Figure 6) and higher a * ph (440) values (~0.15 m 2 mg -1 ) were observed only in the middle shelf off Florida.Relatively low a * ph (440) Figure 2. Spatial Distribution of Biomass Proportion of Cell Size Marker for Microphytoplankton.See Table 1 for Cruise Identification.and Bricaud, 1981; Carder et al., 1986,  1999; Morel and Bricaud, 1986; Bricaud  et al., 1988; Mitchell and Kiefer, 1988a;  Berner et al., 1989; Stramski and Morel  1990; Kirk, 1994; Fujiki and Taguchi,  2002; Lohrenz et al., 2003).The spatial distribution of a * ph (440) also followed salinity patterns (Nababan, 2005).Low salinity, with a higher nutrient content, may have played a role in selecting for species with low a * ph (440).However, there was no statistical relationship between surface nutrient concentrations (NO 2 +NO 3 +NH 4 +Urea) and a * ph (440), likely because nutrients are consumed as fast as they are supplied.
To examine the effect of taxonomic groups on a * ph (440) in the NEGOM, an index was computed based on pigment composition as per Vidussi et  al., (2001) as discussed in previous section.For each major Biomass Proportion (BP>0.5), the a * ph (440) values Nababan were grouped into the three corresponding cell size markers.Scatter plots of a * ph (440) and Biomass Proportion of cell size markers are presented in Figure 7.Although scatter is high, it seems that biomass proportion of nanophytoplankton and picophytoplankton have a positive correlation with a * ph (440).While, microphytoplankton showed negative correlation suggesting higher abundance of microphytoplankton lowered the value of a * ph (440) (Figure 7).The average value of a * ph (440) corresponding to the major biomass proportion of microphytoplankton was also significantly lower than that of nanophytoplankton and picophytoplantkon groups (Table 2).These results strongly suggest that an increase in cell size resulted in decrease a * ph (440) values or increase in pigment packaging.

Conclusions
Variability of a * ph (440) was more pronounced within a cruise than among the seasons.It varied by about a factor of 7, i.e. a * ph (440) ranged from 0.02 to 0.15 m 2 mg -1 for the range of 0.06-12.25 mg m -3 chlorophyll-a concentration.In general, lower values of a * ph (440) were observed inshore particularly in the major rivers mouth regions.Higher a * ph (440) were observed offshore, associated with high salinity, and lower chlorophyll-a, except during summer when lower a * ph (440) were also observed offshore to the west of about 85°W in the low salinity Mississippi plume.Mean values of a * ph (440) in waters dominated by microphytoplankton (diatoms) were significantly lower than in waters with nanophytoplankton and picophytoplankton communities, indicating particle size (packaging effect) play a role in determining a * ph (440) variability.The relationship between a * ph (440) and chlorophyll-a concentration was also not linear, indicating pigment composition also play a role in determining a * ph (440) variability.
Variability of a * ph (673) was less pronounced and only varied by a factor 2, i.e. a * ph (673) ranged from 0.0100 to 0.0248 m 2 (mg chl) -1 with average value of 0.0175 m 2 (mg chl) -1 for the range of 0.06-12.25 mg m -3 chlorophyll-a concentration.

Figure 1 .
Figure 1.Study area: the NEGOM Region, Encompassing the Area between 27.3 -30.7°N and 82.6 -89.6°W.The map shows the 10, 20, 100, 200, 500, and 1000 m bathymetric contours and cruise transect lines (solid line).Closed triangles were CTD stations from which discrete water samples for light absorption coefficients and pigments analyses were taken.Rectangle area in the Gulf of Mexico inset is the area of the study.

Figure 3 .
Figure 3. Spatial Distribution of Biomass Proportion of Cell Size Marker for Nanophytoplankton.SeeTable 1 for Cruise Identification.

Figure 5 .
Figure 5. Near-surface Chlorophyll-specific Absorption Spectra Measured on the NEGOM Region during Summer, Spring and Fall Seasons between 1998 and 2000.See Table1for cruises identification.Note the different scales on y-axis.

Figure 6 .
Figure 6.Near-surface Distribution of the Chlorophyll-specific Absorption Coefficient at 440 nm in the NEGOM.White dots show location of water sample collections.See Table1for cruise identification.

Figure 7 .*
Figure 7. Scatter Plots of a * ph (440) and Biomass Proportion per Algal Group for All Seven NEGOM Cruises.

:
This research was supported by MMS Cooperative Agreement 1335-01-97-CA-30857 and by NASA grant NAG5-10738 awarded to Dr. Frank E. Muller-Karger, USF.I would like to express my appreciation to Drs.Frank E. Muller-Karger and Douglas C. Biggs for their scholarly advise, critical reviews and stimulating ideas for completing this research.

Table 1 .
Cruise Identifiers and Dates.
Table 1 for Cruise Identification.