FLUOROMETER PERFORMANCE EVALUATION REPORT 9/2002

Transcription

1 FLUOROMETER PERFORMANCE EVALUATION REPORT 9/22 ABSTRACT A series of performance evaluation tests were conducted at three tests sites to investigate the overall performance of the Turner Designs Submersible Fluorometer and the chlorophyll probe. Performance was determined through results designed to determine minimum detection limit of chlorophyll a, dynamic range, accuracy of readings to actual chlorophyll a, ambient light rejection, and response time. The results indicate that the fluorometer outperforms the sensor but the performance of both sensors is adequate for most environmental conditions. A performance limitation of the is the response time. Using a moving average calculation, it responds relatively slowly to changing concentrations and would not be effective for vertical profiling applications. In some extreme conditions, such as chlorophyll concentrations less than 1µg/L and high ambient light and/or high concentrations of interfering compounds, the may also not provide accurate measures of algal biomass. INTRODUCTION A series of performance evaluation tests were conducted to investigate the overall performance of the Turner Designs Submersible Fluorometer and the chlorophyll probe. Tests were conducted at three sites; Moss Landing Marine Laboratory, Turner Designs Testing Laboratory and the USGS Menlo Park facility. Performance was evaluated through designing tests to investigate the following areas; minimum detection limit, instrument range, response time, light rejection and accuracy of fluorometer reading to actual chlorophyll a concentration. Due to different specification standards, comparing instruments on published specifications is not always straightforward. Below is the published performance data for each instrument. Through our testing we have attempted to compare the instruments in a meaningful and informative way for the customer. We have compared instruments identically and have attempted to present the data in as an objective manner as possible. In most cases the raw fluorescence signal was recorded in order to eliminate scaling issues related to calibration. The raw fluorescence was recorded as %FS which indicates the % of a sample s signal in relation to 1% full scale of the particular instrument. Calibrated data is referred to as in vivo fluorescence or. Published Performance Specifications Range -4µg/L 4 orders of magnitude Minimum Detection Limit N/A.2 µg/l Resolution.1 µg/l,.1%fs 12 bit Max Sampling Rate NA 5Hz Depth -61m -6m Instrument Overview: While both instruments are chlorophyll fluorometers there are several key distinctions between the two units. Firstly, the physical size and appearance is significantly different. The fluorometer is designed to operate as a stand-alone unit and can also be interfaced to multi-parameter systems. Being a stand-alone unit, it incorporates many additional features that are not required in the probe. The following is a list of unique features; 6m depth capability, temperature compensation, internal data logging, autoranging, turbidity option, digital and analog output, configurable analog (-5V) limits, and calibration software. The presence of these features has resulted in a much different looking sensor. However, when interfaced to a multi-parameter system, the operates in a very similar manner to the probe. The has been designed to interface directly onto multi-parameter sondes. The probe is also now available on an optical sonde that only offers fluorescence, conductivity, 1 FLUOROMETER COMPARISON REPORT

2 temperature and depth. The probe provides an analog signal to the sonde which integrates the data from all of the sonde sensors into one digital data stream. The provides both digital and analog data continuously. MATERIALS AND METHODS All tests were conducted under controlled settings in a laboratory. In most cases, monocultures on algae were used as the test media (see table below for algal culture details). In some cases natural algal samples were used. Chlorophyll a extractions were conducted on all samples using the Non-acidification fluorometric method (Welschmeyer, 1997) and the Acidification technique (E.P.A. Method 445.) in the case of the pheophytin testing. Algal samples used: Genus Algal group 1 Accessory Pigment Dunaliella sp. chlorophyte chlorophyll b Thallassiosira sp. diatom chlorophyll c Phaeodactylum sp. diatom chlorophyll c San Francisco Bay Delta natural assemblage RESULTS MINIMUM DETECTION LIMIT The Minimum Detection Limit is the lowest concentration of algae in clean culture media than can is distinguishable from a blank culture media solution. Three detection limit tests were run by mixing a dilution series of algal cultures. The readings were allowed to stabilize and at least 1 datapoints were recorded per sample. Dunaliella Phaeodactylum Chl A Chl A Thallassiosira Chl A Table 1: Dunaliella and Phaeodactylum test data from Moss Landing Marine Laboratory (MLML). Data from Thallassiosira test from Turner Designs. The Chloro column is extracted chlorophyll a data. The and column is the in vivo fluorescence data. From the data shown it is clear the difference in detection limit between algal groups. The data supports the fact that the was unable to distinguish some algal cultures at concentrations of 1µg/L chlorophyll a or less while the was capable of distinguishing all samples. The lowest concentration tested was.34µg/l of Thallassiosira culture. Because each species of algae has varying in vivo fluorescence ():extracted chlorophyll, the limits of detection can vary between species. The demonstrated the ability to detect concentration as low as.79µg/l of Thallassiosira culture while not detecting other cultures that were above 1µg/L. 2 FLUOROMETER COMPARISON REPORT

3 Minimum Detection Limit Test #1 Minimum Detection Limit Test # R 2 =.9737 R 2 = chlorophyll a (ug/l) Graph 1 & 2: MDL test data from Moss Landing Marine Lab showing that the not differentiating between 2.5µg/L and 1.1µg/L Dunaliella samples. MDL Test#2 shows data from tests with Thallassiosira cultures indicate that the did not distinguish the blank and the first two culture samples. RANGE Both instruments provide an analog -5V output signal while the has a digital output as well. The -5V ranges are factory set for a given fluorescence range. The comes standard with a -1µg/L analog range while the comes standard with a -4µg/L range. As the analog range increases, the resolution of the data decreases and it is therefore advantageous to set the -5V limits to the environments in which you work. The digital outputs from the Sonde and the were used for all tests. Both instruments demonstrated good linearity for typical chlorophyll a ranges found in natural environments. The only difference of note for chlorophyll measurement is the ability to customize the -5V range on the. This abiltiy allows customers working in low chlorophyll environments to decrease the -5V range and therefore improve the resolution of the analog data R 2 =.9319 R 2 = chlorophyll a (ug/l) Linearity Test R 2 =.997 R 2 = chlorophyll a (ug/l) Graph 3: Data taken with calibrated values at MLML using Dunaliella cultures. 3 FLUOROMETER COMPARISON REPORT

4 ACCURACY In order to test the accuracy of measuring chlorophyll a fluorescence, the effects of various interfering compounds were investigated. In an ideal situation, an in situ fluorometer would measure chlorophyll a fluorescence only since chlorophyll a is the only photosynthetic pigment that all algae and photosynthetic bacteria have in common. By measuring chlorophyll a only you will obtain the best estimate of total, living algal biomass. Compounds that can interfere with fluorescent signals optimized to detect chlorophyll a include accessory pigments such as chlorophyll b and c, degraded chlorophyll a, pheophytin a, and dissolved organic matter. All of these interfering substances have fluorescence spectra that can overlap with chlorophyll a given the appropriate concentrations and fluorometer optics. When discussing an instrument s susceptibility to interfering compounds, the performance is determined by the optics. The optics in a fluorometer encompass three components; a light source, optical filters and a light detector. Both the and the instrument use solid-state components, LED light source and photodiode light detector, which makes them equivalent in this category except that the utilizes two LED lamps while the uses one. The difference between the optics lies in the optical filters, specifically the emission filters. The emission filter is placed in front of the light detector. It s purpose is to block all light except for the wavelengths of interest, in this case chlorophyll a fluorescence which peaks at 685nm. The more specific the filter is to the chlorophyll peak, the more accurate the fluorometer will be at measuring total algal biomass. The uses long-pass emission filters which allows in all light >6nm. This means that any material other than chlorophyll a that is excited by the blue LED light source (peak at 46nm) and fluorescing above 6nm will be detected. Likely candidates for this include pheophytin a, chlorophyll b, chlorophyll c and CDOM. The, on the other hand, utilizes a band-pass filter that allows in light from 66-7nm only, peaking at 685nm. Therefore, the fluorescence from compounds other than chlorophyll a are much less likely to be detected. The benefit of using a long-pass filter is the low material cost and higher transmission. The downside is greater susceptibility to interfering compounds. The tests were effective in demonstrating the effect of optical components in the pheophytin testing. One of the most likely interference s is pheophytin a. Pheophytin can accumulate in water column at density gradients, close to the bottom, and be increased due to active zooplankton feeding. A test was run to compare the fluorescence signal from a healthy algal culture and fluorescence from the same culture after exposed to a weak acid that converted much of the chlorophyll in the cells to pheophytin a. 1mL of 1N HCl was added to 35mL of Thallasisosira culture. The culture was stirred for 1 minute and then the fluorescence was read using both instruments. The results were as follows: Before After Before After FS% Ratio FLUOROMETER COMPARISON REPORT

5 The Difference in the ratios indicates a difference in the fluorometers ability to detect pheophytin fluorescence. The ratio of 2.76 indicates that it is less sensitive to pheophytin fluorescence than the sensor that had a ratio of Regression Comprison R TD USGS-Thal TD-Thal TD-Thal2 MLML-Pheo MLML-Pheo MLML-Dun MLML-Dun MLML-Dun TD Fresh USGS-Delta Graph 4: The bar graph displays the correlations (r 2 ) obtained from the minimum detection and linearity tests conducted at all three testing sites. In most cases the data correlated with actual chlorophyll a more closely. All tests returned an r 2 of greater than.85 or better. The samples market TD Fresh and USGS-Delta are natural water samples with an assemblage of algal species. All other samples were moncultures of algae. AMBIENT LIGHT REJECTION Ambient light rejection refers to the ability of the fluorometer to be unaffected by sunlight or other ambient light sources. Light rejection is typically performed by flashing the LED light source. When the light source is on, the instrument detects the fluorescence and any ambient light, when the light source is off, the instrument detects the ambient light. The instrument compares the two to determine the fluorescence. Ambient light rejection test results indicate that there is a difference in how the two instruments correct ambient light. The displayed no significant difference in fluorescence signal while the showed a decrease in signal as ambient light increased and became more variable Ambient Light Rejection Test Scufa no light light at 9 degrees light at 18 degrees Position of Light Source % Full Scale Ambient Light Rejection Test #2 Lit Lab Dark Lab Light 12" Direct Light 12" Variable Graph 5 & 6: Graph 5 displays data from Moss Landing Marine Lab. The instruments were placed in a solution of rhodamine WT dye. A halogen light source was used to test ambient light. Graph 6 displays data from tests run at Turner Designs with a halogen light source. The units were not placed in a solution. 5 FLUOROMETER COMPARISON REPORT

6 RESPONSE TIME The graph below shows the response time of the two instruments as they were alternately placed into samples of natural water containing algae and blank deionized water. The readings were taken at one-second intervals. The difference in the graphs displays a significant difference between the two instruments. The system uses a moving average as opposed to the realtime sampling of the. The moving average allows the system to smooth the data as well as enables it to improve the limit of detection. The, designed for high-speed vertical profiling applications, samples at a rate of 1Hz and outputs data at 1Hz or 5Hz. The spikes at the beginning of the readings of the algal cultures is due to removing the instrument from the blank water and then placing it into an algal culture. The change from air to liquid creates some bubbles that cause spikes in the readings. The sensor is less sensitive to these changes due to the slow moving average (~1sec) of the data which smoothes the data but does not supply instantaneous data. RESPONSE TIME TEST IN VIVO FLUORESCENCE :55:55 13:56:38 13:57:22 13:58:5 13:58:48 13:59:31 14::14 14::58 14:1:41 TIME Graph 7 CONCLUSIONS The results of the performance tests indicate that both instruments are effective at estimating chlorophyll a concentrations in typical environmental conditions. Although accuracy tests favored the fluorometer, most correlations between the in vivo fluorescence and the actual chlorophyll concentration exceeded.9 for both instruments. It is in the presence of interfering compounds where the data from the two units may differ significantly. Results from testing with degraded chlorophyll, pheophytin, support this conclusion, with the not differentiating between chlorophyll and pheophytin as well as the. Other extreme environmental conditions could limit the effectiveness of the probe. Conditions such as high ambient light,from shallow waters with reflective sediment, and low productivity waters could be problematic. Also, due to the slow response time of the probe, vertical profiling would be a challenge. On the other hand, the probe seems well suited for deployed use in coastal or freshwater environments where chlorophyll levels exceed 1µg/L and ambient light and interfering compounds are not a significant issue. The performed well in all categories. It is a more versatile instrument because of ability to be used in self-contained mode. 6 FLUOROMETER COMPARISON REPORT

7 REFERENCES: Arar, E.J. and G.B. Collins In vitro determination of chlorophyll a and pheophytin a in marine and freshwater algae by fluorescence. E.P.A. Method 445., Revision 1.2 Welschmeyer, N.A Fluorometeric analysis of chlorophyll a in the presence of chlorophyll b and pheopigments. Limnol. Oceanogr. 39: TESTING FACILITIES: MOSS LANDING MARINE LABORATORY, MOSS LANDING, CA. Tests run under the supervision of Dr. Nick Welschmeyer. TURNER DESIGNS TESTING FACILITY, SUNNYVALE, CA. UNITED STATES GEOLOGICIAL SURVEY, MENLO PARK, CA Report written by Robert Ellison, Turner Designs, Inc., 22 7 FLUOROMETER COMPARISON REPORT