CICEET Progress Report for the period 9/15/04 Through 3/15/05

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
Project Start Date: 9/15/03

Figures

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Tables

Table 1

The ALF technology seeks to improve measurements of pigment concentration, including Chl-a and phycobiliprotein (PBC) pigments, assess phytoplankton physiological/nutrient status, provide basic taxonomic phytoplankton characterization, as well as fluorescence assessment of chromophoric dissolved organic matter (CDOM), and measurements of water turbidity. These critical variables will provide valuable currently missing information, which can be utilized along with standard water quality data for improved bio-environmental characterization of estuarine and coastal environments.

Objective for the reported period
To determine and optimize ALF technical solutions with regard to specifics of coastal and estuarine aquatic environments

Tasks to meet objectives
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

Progress on Tasks
Project activities in the reported period included:

• Field measurements at the Chesapeake Bay NERR site in Virginia (CB-VA NERR)
• Data processing and analysis in order to finalize ALF instrumental configuration and operational protocols
• Specification of advanced algorithms for laser fluorescence analysis of coastal and estuarine aquatic environments
• Finalizing ALF design and acquiring the ALF instrument components
• Building the ALF instrument prototype and its initial laboratory tests

Accomplishments
ALF field measurements in the Chesapeake Bay and York River at Virginia NERR sites
The 3rd ALF field campaign was conducted in collaboration with the Virginia Institute of Marine Science (VIMS) in September 2004. Water samples were collected with a boat at 9 selected locations in the York River and Chesapeake Bay in the vicinity of the CB-VA NERR sites. The station locations are marked with empty dots in Figures 1-5. Two new sampling pointes (station #1 in the Chesapeake Bay and station # 5 in the York River) were selected in addition to the 7 points sampled during our June CB-VA NERR ALF field tests (see Progress Report); the Goodwin Island CB-VA NERR site is located close to station #2; the Catlett Island CB-VA NERR site is adjacent to station #6. The Data Flow YSI flow-through monitoring system was operated on a boat (Figure 6) to provide supporting underway measurements of chlorophyll-a (Chl-a, Figure 1) concentration, turbidity (NTU, Figure 2), water temperature (Figure 3), dissolved oxygen (Figure 4), and salinity (Figure 5).

Laser fluorescence measurements were conducted in the VIMS laboratory with time delay not exceeding 2-3 hours after sample collection. The house-built Laser Phytoplankton Analyzer was utilized for hyperspectral (420-750 nm) 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) fluorometer provided hyperspectral emission measurements at 8 excitation wavelengths in the blue-green spectral area (409, 440, 450, 460, 478, 495, 525, and 532 nm). The acquired water samples were filtered for the HPLC pigment analysis and fluorescence chlorophyll assessments. The filtrates were measured with the LPA and LEEM systems to assess CDOM pedestal contributions and phycocyanin/allophycocyanin fluorescence in the original sample spectra.

ALF Algorithm Development
The hyperspectral laser fluorescence signatures exhibited significant variability thus providing excellent data for refining the ALF analytical methods. The developed ALF spectral deconvolution algorithms allow accounting for spectral complexity and signal band overlap typical for coastal and estuarine waters. Examples of the spectral deconvolution for the ALF emission signatures measured at CB-VA NERR with laser excitation at 409 nm and 532 nm are presented in Figure 7 and Figure 8, respectively. The deconvolution procedure includes best fitting (blue line) of the measured spectra (red line) with spectral bands representing the major water constituents typical for coastal and estuarine environments, i.e. water Raman scattering, CDOM, Chl-a and phycobiliprotein (PBP) fluorescence (dashed lines). The constituent spectral bands have been retrieved from analysis of the natural samples and filtrates acquired at the participating NERR sites. The ALF hyperspectral emission measurements provide potential for improved characterization of bio-diversity in coastal and estuarine aquatic environments. In particular, we have identified relatively weak phycocyanin and allophycocyanin fluorescence (645 and 662 nm sub-peaks, respectively) in the PBP spectral band (gray dashed line in Figure 8), which have never before been resolved in natural seawater samples because of their overlap with more intense water Raman scattering (yellow dashed line in Figure 8).

Additional significant improvement in accuracy of the quantitative assessments of water constituents is achieved by normalization of fluorescence intensities to the intensity of synchronously measured water Raman scattering. This method, which utilizes the Raman intensity as ‘internal standard’ for calibration of other signals, has been successfully utilized in laser remote sensing for the past two decades. Commercially available fluorometers do not possess the narrow-band laser excitation required for Raman measurement and the hyperspectral signal detection critical for correct assessment of the signal intensities in complex coastal waters. Therefore, they require frequent calibration in optically variable coastal environments. The ALF technology, which utilizes the hyperspectral deconvolution algorithm, provides for further improvement in the accuracy of the Raman calibration and eliminates the need of frequent system calibrations. To illustrate the efficiency of the combined spectral deconvolution and Raman normalization algorithms (SD/RN), Figure 9 presents correlation between independent HPLC pigment measurements (vertical axis) and the laser-stimulated Chl-a fluorescence measured over a range of bio-environmental and optical conditions in the Delaware River, Delaware Bay and in ‘blue’ waters of the Middle Atlantic Bight with (left panel) and without (right panel) Raman normalization (Rsq = 0.97 and 0.51, respectively).

Preliminary Data
As evident from the data displayed in Figures 1-5 and in Table 1, the surveyed area represented a range of bio-environmental conditions. In particular, ratios of maximal to minimal magnitudes for water depth, salinity, dissolved oxygen, and turbidity at the sampling stations were respectively 9.7, 6.6, 2.6, and 7.7. Water temperature and pH varied a few percent; Chl-a concentration also showed relatively small, 40% variability compared to the 10-fold range observed in June in this area (see Progress Report).

The laser fluorescence parameters calculated with the SD/RN algorithms showed high correlation with independent supporting measurements of comparable characteristics. For example, Figure 10 displays the results of regression analysis between laser fluorescence assessments, FC/R_409, and Data Flow measurements of Chl-a concentrations (Rsq = 0.89, combined June-September data set is presented to extend the dynamic range). The correlation analysis has revealed that laser measurements can provide additional useful information. In particular, the intensity of laser elastic scattering, Sctr_532, showed high correlation with water turbidity measured by the Data Flow system (Rsq = 0.79 for combined data measured in June and September, see Figure 11; Rsq as high as 0.99 (!) was observed for the September data set). The ALF prototype instrument (see below) was designed to provide the turbidity measurements in addition to the originally planned parameters.

A representative data set of hyperspectral signatures with laser fluorescence excitation at 11 wavelengths was acquired. As discussed in our previous progress report, blue (405 nm) and green (532 nm) laser excitation wavelengths were selected for the ALF prototype instrument, which is currently under development (see below). An extended set of parameters that can be retrieved from the hyperspectral measurements was evaluated for utilization in the ALF biomonitoring in the coastal and estuarine environments. For example, Figure 12 displays relative spatial variability in the 9 parameters, retrieved from our Sept. 2004 CBNERRVA data measured with the blue (409 nm) and green (532 nm) laser excitation. While the magnitudes of SD/RN Chl-a fluorescence (violet and green bars in Figure 12) showed relatively low variability confirmed by the YSI Chl-a measurements (see Table 1), the SD/RN fluorescence of phycoerythrin (PE, presented by orange bars in Figure 12), a pigment indicator of PBC-containing algal groups (cyanobacteria, cryptophytes, and rhodophytes), exhibited significant, 10-fold variability, reaching its maximum and minimum magnitudes at the CB_VA NERR site (station #2) and station #9 in the upper York River, respectively. The same trend was observed in the PE/Chl-a fluorescence ratio measured with the green excitation (light green bars in Figure 12), which is another useful indicator of the relative abundance of the PBC-containing algal groups in the phytoplankton community. The ratio of green/blue efficiencies of Chl-a fluorescence excitation (red bars in Figure 12), which reflects variability in accessory photosynthetic carotenoids, was relatively conservative throughout the surveyed region except its significant drop in the Queen Creek, stations 7 and 8.

The magnitudes of variable fluorescence, Fv/Fm (yellow bars in Figure 12), indicated generally good phytoplankton physiological status and photosynthetic efficiency. The Fv/Fm magnitudes were ~15% higher in the York River mouth and adjacent portion of the Chesapeake Bay (Stations 1, 2, 3, and 4) compared to the up-the-river area (stations 5, 6, 7, 8, 9) and the Queen Creek (Station 8). Another physiological parameter, “Sigma”, which characterizes the efficiency of phytoplankton photosynthetic light absorption, also exhibited a 30% gradual increase down the York River towards the Chesapeake Bay. The increased phytoplankton photosynthetic capacity in the lower York River might likely be caused by the nutrient enrichment (should be later confirmed by data on nutrients). The CDOM concentration (light blue bars in Figure 12) showed a gradual 2-fold decline from station 9 towards the York River mouth with its lowest magnitude at station 1 in the Chesapeake Bay; the maximal CDOM concentration at station 8 in the Queen Creek exceeded 3 times the minimal one observed in the Chesapeake Bay at station 1. Similar trends were observed in spatial distribution of the green laser scattering (dark blue bars in Figure 12), an indicator of water turbidity (see above), which showed high (Rsq = 0.83) correlation with CDOM concentration.

Overall, the ALF measurements allow quite comprehensive characterizations of the bio-environmental conditions in the surveyed area. We anticipate that pending results of microscopic taxonomic and HPLC pigment analyses will provide additional useful data for detailed interpretation of the laser fluorescence measurements. For example, significant spatial variability observed in the ALF fluorescence excitation bar patterns (Figure 13) may provide additional information about accessory pigments and phytoplankton taxonomic composition. Meanwhile, the presented preliminary data can be used to illustrate the potential of the ALF technology for improved characterization of biodiversity in coastal and estuarine environments. Presently, only one biological parameter, Chl-a concentration, is incorporated in the NERR System-Wide Monitoring Program to assess the environmental bio-variability. The observed low variability in Chl-a biomass indicated by the YSI system (see Table 1), might lead to the conclusion about generally low algal bio-variability in surveyed area. The multivariable ALF data set indicates that, despite the low Chl-a variability, there were significant spatial changes in the composition of the phytoplankton community in the surveyed area; pronounced variations in phytoplankton physiological status, pigment composition, CDOM content and turbidity were also indicated.

The ALF Prototype System Development
The observed spatial and temporal variability in the LPA and LEEM spectral patterns have been analyzed to choose the optimal design decisions. As discussed above, the key ALF technological solutions have been identified as:
(1) Utilization of two, blue and green, compact lasers as excitation sources for various ALF measurements
(2) Hyperspectral emission detection with a high-resolution CCD spectrograph
(3) Utilization of the spectral deconvolution/Raman normalization algorithms

A compact, laptop-footprint ALF prototype system, which implements the above technological requirements and solutions, is currently under development (see Figure 14 and Figure 15). The ALF configuration includes 2 miniature lasers, a compact hyperspectral CCD spectrometer with a fiber light collector, a Pump-During-Probe (PDP) sensor with a PMT detector and a waveform digitizer, a flow-through sample unit with a miniature pump, a GPS module for shipboard operation, and a laptop PC with a USB system controller.

The ALF instrument is designed to provide discrete laboratory sample analysis and continuous flow-trough underway measurements onboard a small vessel or motorboat (Figure 6). In the latter case, combined with the Data Flow system that yields the standard water quality data set, it provides for detailed bio-environmental characterization in coastal and estuarine aquatic areas. A compact high-capacity ALF rechargeable battery provides up to 5 hours of autonomous system operation without an external power supply. The ALF instrument is assembled in a compact aluminum case, which can be enclosed in a waterproof Pelican case for system transportation and operation onboard a small vessel (Figure 16). Two dedicated ALF software packages were developed during the reported period that provide semi-automatic laboratory measurements of water samples acquired in the surveyed areas and automatic flow-through monitoring on-board a small vessel. For example, Figure 17 displays a screen capture taken during laboratory measurements of the coastal water sample acquired in the Chincoteague Bay. The prototype system is currently at its final stage of instrument development and laboratory testing. An ALF upgrade with a water temperature sensor is considered to further improve its capacity for biomonitoring onboard a small vessel.

Project Objectives for Next Reporting Period

Objectives
During the next reporting period, our research effort will be focused on the 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

The ALF prototype system will be tested on participating NERR sites during 3 field campaigns planned in spring and summer 2005. The ALF spring field tests and deployments have been scheduled in March and April at the CB-VA and South Slough (OR) NERRs, respectively. The ALF instrument configuration, measurement protocols, data processing and analytical algorithms will be refined based on the initial instrument field tests. The upgraded system will be extensively tested in the laboratory conditions with natural water samples. The 3rd ALF field test campaign is planned during summer 2005 with a special emphasis on the flow-through underway monitoring onboard a small vessel. A final project report will describe project activities, analyze research results, and provide recommendations how the Advanced Laser Fluorescence technology can be operationally utilized for bio-monitoring estuarine and coastal areas. This will complete the initial stage of ALF development funded by CICEET in 2003.

Anticipated Success in Meeting Project Objectives
We do not anticipate significant problems with meeting the project objectives in the scheduled project period. The project team may consider requesting a no-cost project extension for several months after September 1, 2005 to finalize the ALF analytical protocols, publication preparation and completing additional dissemination activities. As suggested by Dr. Nieder, a President of the NERR Association, it is planned to present our final project report and demonstrate the operational ALF prototype to the NERR network at the 2005 NERRA fall meeting at Rookery Bay NERR in FL. The ALF research results will be also presented at the 18th Biennial Conference of the Estuarine Research Federation (October 16-21, 2005, Norfolk, VA) as an invited talk during the Chesapeake Bay Colloquium and as an interactive poster at the SYM-04 Symposium “Observing the Coastal and Ocean Environment: Developments in Sensor Technology and the Use of Long-Term Data Sets for Operational Ecology”.

Anticipated Difficulties
(1) Comparison of data acquired in the Chesapeake Bay VA (CB-VA) and North Inlet ­ Winyah Bay (NI-WB) NERRs has shown significantly different patterns in pigment abundance, phytoplankton composition, and photo-physiological status. The entire NERR network of 26 sites represents very diverse aquatic environments. Therefore, more extensive ALF field tests in a broad range of bio-environmental conditions are needed to adjust measurement protocols and analytical algorithms with regard to diverse regional, local and seasonal conditions.

(2) The ALF technology development is eventually targeted for operational utilization within SWMP biomonitoring by the NERR personnel, who may or may not have relevant background in laser spectroscopy and optical measurements. Though the ALF prototype instrument and software are designed to be user-friendly and do not require special professional skills for their operation, the ALF technology per se is relatively new and may need an ‘educated end user’ for its future flawless incorporation in the SWMP biomonitoring program.

We have submitted the follow-on CICEET proposal “Advanced Laser Fluorescence (ALF) Biomonitoring in the Middle-Atlantic NERR Sector and Great Lakes” that addresses the above issues. The proposed research is considered as the next important step towards successful ALF operational utilization. At the following stage of the ALF development, we propose to conduct a series of seasonal and annual tests and evaluations of the ALF technology in the Middle-Atlantic NERR Sector. Field deployments will be also conducted in the Old Woman Creek (OWC, Lake Erie, OH) NERR for ALF evaluation in the Great Lakes-type freshwater aquatic environment.

Overall Project Timeline Update
No major updates except the potential request for no-cost extension (see above)

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