CICEET Progress Report for the period 3/16/04 through 9/15/04

Project Title: Advanced Laser Fluorescence (ALF) Technology for Estuarine and Coastal Environmental Biomonitoring
Principal Investigator(s): Alexander Chekalyuk, Kenneth Moore, David White, and Dwayne Porter

Objectives
To determine and optimize ALF technical solutions with regard to specifics of coastal and estuarine aquatic environments

Tasks to meet objectives
Task # 2: Optimization of ALF technology for pigment assessment and taxonomic monitoring in coastal and estuarine waters
Task # 3: Optimization of pump-during-probe (PDP) technology for assessment of algal physiological status in coastal and estuarine waters
Task # 3: Optimization of CDOM fluorescence assessment in coastal and estuarine waters

Accomplishments
Scheduled Tasks:

• Laboratory technological tests and optimization
• Initial ALF field tests at the Chesapeake Bay NERR site in Virginia (CBNERRVA, June 2004)
• Field measurements at the North Inlet ­ Winyah Bay (NI-WB) NERR site (SC, July 2004)
• Data processing and initial analysis
• Specification of ALF instrumental solutions and ordering hardware for the prototype ALF system

Progress on Tasks
ALF field measurements at the Chesapeake Bay in Virginia and North Inlet ­ Winyah Bay NERR sites
The initial ALF field tests were conducted in close collaboration with the Virginia Institute of Marine Science (VIMS) in June 2004 (see Figure 1). Water samples were collected with a boat at 7 selected locations in the York River and Chesapeake Bay in the vicinity of the Chesapeake Bay in Virginia NERR (CBNERRVA) sites (see Figure 2 and Figure 3). In particular, the Goodwin Island CBNERRVA Reserve Site is close to station #1; the Catlett Island CBNERRVA Reserve Site is adjacent to station #4. The Data Flow flow-through monitoring system was operated on a boat along the York River to provide supporting underway measurements of chlorophyll-a (Chl-a) concentration, turbidity (NTU), water temperature (respectively presented in green panels in Figure 2), dissolved oxygen, and salinity. Laser fluorescence measurements were conducted in the laboratory of VIMS with time delay not exceeding 2-3 hours after sample collection. The house-built Laser Phytoplankton Analyzer (LPA, see the lower left picture in Figure 1) was utilized to provide hyperspectral emission measurements at 5 excitation wavelengths (473, 532, 640, 650 and 666 nm) and pump-during-probe (PDP) fluorescence induction assessments of phytoplankton physiological status. The Laser Excitation-Emission Matrix (LEEM, see the upper right picture in Figure 1) fluorometer provided hyperspectral emission measurements at selected excitation wavelengths in the blue-green spectral area (409, 435, 450, 460, 478, 495, 525, and 532 nm). The water samples were filtered for the HPLC pigment analysis and fluorescence chlorophyll assessments. Some filtrates were measured with the LPA and LEEM systems to assess CDOM pedestal contributions and potential presence of weak phycocyanin fluorescence in the original sample spectra.

The second field campaign was conducted in close collaboration with Belle W. Baruch Institute for Marine and Coastal Science (University of South Carolina) at the North Inlet ­ Winyah Bay (NI-WB NERR) in July 2004 (see Figure 4). Water samples were collected at 10 selected locations in the North Inlet and Winyah Bay on July 27-29 (see Figure 5 and Figure 6). Laser fluorescence measurements were conducted with the LPA and LEEM fluorometers (see lower pictures in Figure 4) in the Marine Laboratory of the Belle W. Baruch Institute (upper left picture in Figure 4). The collected water samples were filtered for the HPLC pigment analysis, chlorophyll extraction measurements, and microscopic taxonomic determinations.

Detailed interpretation of the observed spatial and temporal variability in fluorescence signatures will be conducted after thorough analysis of HPLC pigment measurements and results of microscopic taxonomic analysis. Nonetheless, the preliminary analysis of specific spectral features of the acquired data provided a basis for identifying and optimizing some key ALF technological and instrumental solutions.

Task # 2: Optimization of ALF technology for pigment assessment and taxonomic monitoring in coastal and estuarine waters

• Spatial and temporal variability in the LPA and LEEM spectral patterns of laser-induced fluorescence were analyzed at the NERR sites to choose optimal excitation wavelengths.
• Algorithms to account for potential adverse effects of intensive elastic scattering and broadband fluorescence pedestal were identified and selected.
• Blue (405 nm) and green (409 nm) laser excitation wavelengths were selected as optimal excitation wavelengths for the prototype ALF system.
• Utilization of compact hyperspectral CCD spectrometer was determined to be an optimal technological solution for laser fluorescence analysis in coastal and estuarine environments.

Task # 3: Optimization of PDP technology for assessment algal physiology in coastal and estuarine waters

• Potential distortions in PDP induction measurements produced by non-chlorophyll background fluorescence and elevated elastic scattering in NERR waters were analyzed.
• A miniature TTL modulated blue laser (405 nm) was chosen as an optimal excitation source for PDP measurements. Along with PDP measurements, the laser can also be efficiently used for fluorescence assessments of CDOM and Chl-a concentrations.

Task # 4: Optimization of CDOM fluorescence assessment in coastal and estuarine waters

• Spatial and temporal variability in laser-induced fluorescence patterns of NERR waters was thoroughly studied with OPO excitation in the 410-500 nm range.
• The potential of utilizing super-bright LED blue excitation at 440 nm for CDOM assessment in estuarine and coastal waters was evaluated.
• A miniature blue laser (405 nm) was chosen as an excitation source of CDOM fluorescence in the ALF system (see above).

Difficulties Encountered
The use of 405 nm for PDP excitation will minimize a potential problem with the elastic scattering pedestal during the PDP measurements; on the other hand, the 405 nm excitation may result in a tale pedestal contribution from CDOM fluorescence in the spectral region of Chl-a fluorescence that may affect accuracy of PDP assessment of phytoplankton physiology. The utilization of broadband hyperspectral emission measurements along with 405 nm excitation will be evaluated to account for the CDOM pedestal contribution.

Though significant changes in FEX_5 algal signatures measured with LPA excitation were observed, high intensive and variable elastic scattering, which is typical for coastal and estuarine waters, may affect FEX_5 patterns measured with red laser excitation (640-667 nm) due to its close spectral location to the Chl fluorescence band (685 nm). Selecting excitation wavelengths in blue spectral regions may significantly reduce the adverse effect of elastic scattering. We have already acquired a representative FEX_n data measured with both blue and red excitation, which will be thoroughly analyzed with regard to observed variability in pigment and taxonomic composition assessed from HPLC and microscopic analysis. Potential correlation of FEX patterns with intensity of elastic scattering will be also studied.

Anticipated Success in Meeting Project Objectives in Scheduled Project Period
We do not anticipate any significant problems with meeting the project objectives in the scheduled project period.

Preliminary Data
Some results of preliminary data analysis are presented in Figure3, Figure 5, and Figure 6. Overall, the acquired fluorescence data provides for quite comprehensive characterizations of the bio-environmental conditions in the surveyed areas. Significant, ~10-fold spatial and temporal variability was observed in Chl-a and CDOM abundances that were assessed from fluorescence intensities of these constituents, normalized to water Raman scattering (red and blue bars, respectively, in the panel inserts). The laser fluorescence assessments were well correlated with independent supporting measurements. For example, the lower left panel in Figure 2 presents the results of regression analysis between laser fluorescence assessments, CHL_O/R_O, and Data Flow measurements of Chl-a concentrations (Rsq = 0.80). The correlation analysis has revealed that laser measurements can provide additional useful information. In particular, intensity of laser elastic scattering showed high correlation with water turbidity measured by the Data Flow flow-through system (Rsq = 0.88, see the lower right panel insert in Figure 2). The variable fluorescence, Fv/Fm, measured with the PDP LPA sensor, indicated relatively poor physiological status of phytoplankton at the CBNERRVA sites late in June (Fv/Fm ~ 0.33-0.41; the highest Fv/Fm magnitude, 0.55, was observed in the sample taken at VIMS pier in the York River ­ see Figure 3). The magnitude of photosynthetic absorption cross-section, Sigma, also showed 30% spatial variability in the surveyed area, which reflects respective spatial changes in algal photosynthetic capacity. The blue 5-bar patterns, FEX_5, in Figure 3 represent the relative efficiencies of Chl-a fluorescence excitation at 5 laser wavelengths (473, 532, 640, 650 and 666 nm ­ left to right in the FEX_5 patterns) measured with the LPA. As evident from Figure 3, the fluorescence excitation (FEX) signatures showed significant spatial variability that reflect respective changes in accessory pigments and, potentially, taxonomic composition. Of particular interest is the unusually high FEX efficiency at 640 nm in the York River mouth (see the 3rd bar in FEX_5 patterns at stations 1A and 2A in Figure 3), which may indicate significant abundance of phycocyanin-containing cyanobacteria. We have not observed this feature during our earlier FEX_5 measurements in the upper Chesapeake Bay, Delaware Bay and Delaware River. Our hyperspectral emission measurements with LPA and LEEM systems also indicated elevated concentration of phycoerythrin, a biomarker pigment for phycobilin containing algal groups. The acquired LEEM data also showed significant changes in fluorescence excitation patterns measured in the blue-green spectral region.

Our laser fluorescence measurements at the NI-WB NERR also showed marked differences, exceeding one order of magnitude spatial variability in Chl-a and CDOM concentrations (see the red and blue bars in Figure 5), and high correlation between laser-stimulated Chl-a fluorescence/Raman ratio and Chl-a concentration (Rsq ~ 0.8). Laser assessments of physiological status, Fv/Fm, varied in the 0.3-0.5 range, along with 3-fold variability in the functional absorption cross-section, Sigma (presented by cyan and beige bars, respectively, in Figure 5). Remarkably, the lowest Fv/Fm magnitudes were observed in the area of the highest Chl-a biomass (sampling point DB-3), which likely indicates development of nutrient limitation at the late stage of an algal bloom. Spatial variability in FEX_5 patterns (see color 5-bar patterns in Figure_5) was less pronounced compared to the situation observed at the CBNERRVA site in June. The unusually high efficiency of green spectral area (532 nm, presented by green bars) in some FEX_5 signatures, somewhat contradicts relatively low phycoerythrin fluorescence observed in this area. Along with studying spatial variability, we conducted a short time series in selected NI-WB NERR locations to evaluate utility of ALF monitoring temporal variability at NERR sites. The results of these measurements are presented in Figure 6. Due to powerful tidal phenomena in the area, significant changes in bio-environmental variables were observed over 2-3 days of observations (see Figure 6). For example, a gradual, 5-fold rise in CDOM concentrations at station CB-B were apparent. These were accompanied by a drop in the physiological parameter, Fv/Fm. In addition, a significant change in the FEX_5 signature over the first night of observations was evident.

Tasks and activities for the next reporting period

Tasks for the next reporting period
The overall objective of the proposed research is to develop an advanced laser fluorescence (ALF) technology for environmental biomonitoring estuarine and coastal areas. During the next reporting period, we will focus our research effort on the following tasks:

Task # 5: To develop the prototype ALF system for bioenvironmental monitoring in estuarine and costal waters

• Finalize ALF technological solution based on research conducted in Tasks # 2-4
• Build a prototype of the ALF system capable of sample analysis in estuarine and coastal waters and real-time field measurements onboard a small vessel

Task # 6: Field deployments and tests of the ALF prototype on participating NERR sites

• Conduct extensive laboratory and field tests of the prototype ALF system on participating NERR sites

Work plan to accomplish tasks
The major research effort during the next reporting period will be focused on the Task # 5. The prototype ALF system will be designed, assembled, and tested in the laboratory conditions with phytoplankton cultures and field samples by Dr. Chekalyuk. At the end of the reporting period, we also plan to conduct field test of the ALF prototype in collaboration with Dr. Moore at the Chesapeake Bay NERR Site, VA (CBNERRVA) to initiate our work on Task # 6, which will be extended through the following reporting period to complete the research according to the project Work Plan.

Expenditures
The expenditures were in the range anticipated for the work accomplished to date.