Technical note—Performance evaluation of the PhytoFind, an in
Brett D. Johnston Jennifer L. Graham Guy M. Foster Bryan D. Downing
In 2019, the U.S. Geological Survey evaluated the performance of the Turner Designs, Inc. PhytoFind, an in-place phytoplankton classification tool. The sensor was tested with sample blanks, monoculture and mixed phytoplankton cultures, and turbidity challenges in a laboratory, and was tested on a 120-mile survey of the Caloosahatchee and St. Lucie Rivers in Florida, including Lake Okeechobee. Results include the following:The mixed phytoplankton group fluorescence channel (green excitation sensor) of the PhytoFind can be sensitive to interference.The PhytoFind generally overestimated chlorophyll concentration relative to laboratory-measured chlorophyll a concentrations.Turbidity interference may be less apparent in samples where green algae (chlorophytes) represent a high relative percentage of biovolume.The dissolved organic matter compensation feature was effective in the environmental waters sampled during this evaluation.The correlation between percent chlorophyll contribution per phytoplankton group measured by the PhytoFind and relative percent biovolume per phytoplankton group measured in the laboratory varied and was not explicitly determined...
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Ambient Water Temperature Test Methods

Ambient temperature is important because it can affect the function and response of complex electronic sensors. For fluorometers, this effect can be internal within the electrical components of the sensor or external from the water-temperature dependent fluorescence of target constituents. To quantify these potential effects, baseline, blank signal, and dye signal tests were performed over a range of ambient water temperatures: the baseline was assessed with covered photodetectors (the absence of light) in a dark container with DIW, the blank signal was assessed with uncovered photodetectors in a dark container with inorganic blank water (that is, quality-assured water, free of inorganic analytes), and the dye signal was assessed with uncovered photodetectors in a dark container with DIW colored with dyes. Subsequent tests were done with analytical-grade laboratory DIW; additional instrument blanks indicated that the laboratory DIW was not a source of interference in this study. Live cultures were excluded from temperature tests to avoid the introduction of additional photochemical variables affecting fluorescence intensity other than water temperature.

Methods of Baseline Signal Test

The baseline signal test measured the short-term variation of the instrument’s output when no input signal was present (that is, no light entered the photodetector; the photodetectors were covered). Variations of the output can be caused by electric fluctuations, changes in temperature, and other factors such as internal moisture. A temporally stable baseline is a crucial requisite for sensitive electronic instrumentation. To assess the effect of ambient water temperature on the stability of the dark signal baseline readings of chlorophyll concentrations from the PhytoFind, black tape was first placed over the photodetector lenses of the sensors to prevent detection of any light in the photodetector (fig. 4). The instrument was then submerged in DIW within the test vessel and gradually cooled to about 5 degrees Celsius (°C) by submersing the test vessel in a mixture of ice and rock salt. This method of cooling was used instead of a standard laboratory water bath because of the size of the instrument.

Data collection was initiated and maintained for 45 minutes while the water was heated gradually with an immersion heater to about 40 °C. During heating, the water temperature was stabilized briefly at 5 °C intervals. Water temperature was measured by the factory-installed water temperature probe of the PhytoFind. Prior to instrument testing, the factory-installed water temperature probe was verified by a National Institute of Standards and Technology (NIST)-traceable thermistor with a five-point check between 12 and 40 °C that showed the probe was accurate to within 0.2 °C.

Methods of Blank Signal Test

Instrument stability must also be evaluated in conditions with a null incident light intensity (that is, a zero-point check). To assess the effect of ambient water temperature on the chlorophyll concentrations from the PhytoFind, in the absence of fluoresced light with uncovered excitation lights and photodetectors, the instrument was submerged in inorganic blank water within the test vessel and gradually cooled to about 5 °C, as described for the baseline signal test. Data collection was initiated and maintained for 38 minutes while the water was heated gradually with an immersion heater to about 40 °C, allowing the water temperature to stabilize briefly at each 5 °C interval. Water temperature was measured by the factory-installed, NIST-verified water temperature probe on the PhytoFind.

Methods of Dye Signal Test

Water-temperature-induced changes in the fluorescence intensity of phytoplankton pigments may not be equivalent to changes in phytoplankton biomass (Hambrook Berkman and Canova, 2007). For this reason, corrections may be necessary and can be computed to compensate for this phenomenon when making optical fluorescence measurements (Downing and others, 2012). To assess the effect of water temperature on instrument-measured chlorophyll concentration, the test used DIW-diluted solutions of rhodamine water tracer (RWT) and basic blue 3 (BB3) dyes, based on the manufacturer’s recommendation. Both dyes trigger the green and brown phytoplankton group fluorescence channel (blue excitation); RWT also triggers the mixed group fluorescence channel (green excitation), and BB3 triggers the cyanobacteria group fluorescence channel (red excitation). Similar to phytoplankton pigment fluorescence, dyes exhibit water-temperature-induced changes in the fluorescence intensity, decreasing fluorescence with increasing water temperature (Foster and others, 2022). Target concentrations of dye were calculated, and dilutions were made using volumetric flasks. Light wavelength absorbances for the dye solutions were then measured on a Hach DR6000 benchtop spectrophotometer (Hach, 2020) for an independent verification of the dilution rates. The RWT was read at 556 nanometers (nm), and the BB3, at 654 nm; these wavelengths correlated with the peak absorbance of each dye.

Initially, the PhytoFind was submerged in a solution of RWT with a measured absorbance of 0.129 absorbance unit (AU) at 556 nm. However, this resulted in a saturated measurement of the chlorophyll concentration equal to about 1,360 micrograms per liter (µg/L). Saturation means that there is no additional photocurrent output from the photodetector as the incident light increases (that is, the reading is at its maximum). Dilutions of RWT were made down to an absorbance of 0.003 AU at 556 nm, but these all resulted in saturated measurements, far above the chlorophyll concentration range maximum of 250 µg/L. Next, the PhytoFind was submerged in a solution of BB3, with a measured absorbance of 0.009 AU at 654 nm, which yielded a chlorophyll concentration of about 196 µg/L.

Because the PhytoFind uses multiple fluorescence signals in its calculation algorithms, a mixture of the dye solutions was used in testing. The initial mixture of RWT and BB3 (fig. 5) was diluted down to absorbances of 0.001 AU at 556 nm and 0.002 AU at 654 nm and used as the “high” concentration. The test was repeated for two additional dilutions, designated as the “middle” and “low” concentrations, to absorbances of 0.001 AU at 556 nm for RWT and 654 nm for BB3 for the middle concentration, and less than 0.001 AU at 556 nm for RWT and 654 nm for BB3 for the low concentration.

Data were recorded for each of the concentrations at water temperatures between 5 and 40 °C, allowing the temperature to stabilize briefly at each 5 °C interval. Water temperature was measured by the factory-installed, NIST-verified water temperature probe on the PhytoFind. The tests were run for an average of about 45 minutes, with the samples mechanically mixed using a magnetic stir plate to homogenize the sample volume used for the sensors.

Cultured Phytoplankton Test Methods

Laboratory tests were run using monoculture and mixed-population cultures of known freshwater phytoplankton species. Three cyanobacteria (Pseudanabaena, Microcystis aeruginosa, and Synechococcus) were cultured by the USGS (fig. 6), and a diatom (Navicula) and a chlorophyte (Chlorella vulgaris) were cultured by Algae Research Supply (Algae Research Supply, 2021). These taxa were chosen because they have the fluorescence characteristics of the target groups measured by the PhytoFind and are relatively common in freshwater systems. Because cyanobacteria are a particular group of concern, three species were selected to represent the group based on unique traits that affect the fluorescence characteristics of cyanobacteria. Pseudanabaena was chosen because of its filamentous morphology, as well as its phycoerythrin content; Microcystis aeruginosa, for its phycocyanin content and colonial growth form; and Synechococcus, for its phycocyanin content and unicellular growth form. Pigment content of Chlorella vulgaris and Navicula is primarily chlorophyll a, without the presence of either phycoerythrin or phycocyanin.

Outside the test vessel, all live cultures were stored at room temperature and at ambient light conditions with a photosynthetic photon flux density (a measure of received light) ranging from 7.23 to 7.83 micromoles per square meter per second, measured by a LI–COR, Inc. LI–193 spherical quantum sensor (LI–COR, Inc., 2021). These conditions were selected to minimize variations in the photochemical processes of the phytoplankton cells. Samples inside the test vessel were kept at room temperature, covered from ambient light, and mechanically mixed using a magnetic stir plate (fig. 7). These conditions were selected to maintain the phytoplankton cells, minimize interference from ambient light on fluorescence measurements, and homogenize the sample volume used in testing.

After a brief equilibration period (about 1 minute), sensor data were collected for about 5 minutes—except for the first run of Pseudanabaena, which was about 10 minutes—for each of the test conditions. The mean chlorophyll concentration and percent chlorophyll contributions per phytoplankton group were compared with discrete comparison samples collected immediately thereafter; the comparison samples were analyzed for laboratory-measured chlorophyll a concentration and phytoplankton identification and enumeration. Duplicate samples for chlorophyll a concentration analysis were collected in accordance with USGS protocols as described in Hambrook Berkman and Canova (2007) and analyzed at the USGS National Water Quality Laboratory in Denver, Colorado, in accordance with Arar and Collins (1997). Concentration of chlorophyll a, as opposed to chlorophylls b, c, d and e, is commonly used as a proxy for total chlorophyll concentration in fluorescence monitoring because it is present in all phytoplankton. As such, the total chlorophyll concentration may be greater than that of chlorophyll a.

Phytoplankton samples were collected in a 250-milliliter, amber, high-density polyethylene bottle, preserved with 25 percent glutaraldehyde, and shipped to PhycoTech, Inc. for analysis by imaging flow cytometry (IFC). IFC uses a combination of flow cytometry and video technology to capture high-resolution images of suspended particles; laser-induced fluorescence and light scattering from individual particles are measured and used to trigger targeted image acquisition. High-resolution images (about 3.4 pixels per micrometer) of suspended particles ranging from less than 8 to 150 micrometers (µm) are generated. These images are initially identified and counted using an automated classifier; the resulting data are used to provide estimates of phytoplankton concentration and biovolume (the volume of cells in a unit amount of water). Results are verified by an algal taxonomist.

Between 1,000 and 2,000 images per milliliter of sample were collected and classified. Images classified as “miscellaneous” or “unclassified” by IFC are denoted as “other” in the results of this report. A taxon classification of “miscellaneous” was defined as “all other groups, generally neutral. Includes small chlorophytes or cyanobacteria less than 9 µm in diameter”; and “unclassified,” as “images that the classifier cannot confidently identify. The “unclassified” group included small flagellates entrained in detritus, taxa not yet included in the classifier, partial images, and images with multiple taxa.” Although not specifically allocable to the target groups, fluorescence may be emitted by organisms within the “other” group. Thus, as relative biovolume of the “other” group increases, so does uncertainty in the characterization of sensor responses in those test conditions. In total, 25 discrete samples were collected and used for comparison analyses.

Phytoplankton Monoculture Dilution Series Test Methods

Dilution series for each of the monocultures were achieved by removing part of a sample from the test vessel and replacing it with ShopRite bottled spring water to achieve the targeted lower concentration (table 2). Spring water is a commonly used diluent for algal media, especially in short-term culturing, because of its content of minerals and salts and generally sterile condition. Optical properties (such as fluorescence, absorption, attenuation) of the culture media and spring water are considered minimal for these live phytoplankton tests. These dilution volumes were guided by the measured chlorophyll concentrations from the PhytoFind and the EXO Total Algae-PC sensor; the chlorophyll concentrations were used to estimate the relative concentration of the initial culture sample and thus subsequent dilutions. For example, if the initial culture was diluted five times, it would yield a total of six samples. As a result of this method, between three and six concentrations were run for each species to achieve a range of chlorophyll concentration within the 0 to 250 µg/L range specified by the manufacturer of the PhytoFind. The results are represented as ratios of the instrument-measured chlorophyll concentration estimations to the laboratory-measured chlorophyll a concentrations (chlorophyll-to-chlorophyll a concentration ratio), allowing for comparisons over the range of concentrations and between sensors to be normalized.

Table 2.

Summary of phytoplankton monoculture dilution series for evaluating the performance of the Turner Designs, Inc. PhytoFind in-place phytoplankton classification tool.

Dilution Sample volume removed (L) Buffer volume added (L) Laboratory-measured chlorophyll a concentration (µg/L) Incremental reduction from previous volume (%) Total reduction from initial volume (%) Chlorella vulgaris dilution series Initial 0.00 0.00 1,210 0.0 0.0 1 3.25 3.25 452 −62.6 −62.6 2 4.25 4.25 119 −73.7 −90.1 3 4.25 8.25 19.9 −83.8 −98.4 Navicula dilution series Initial 0.00 0.00 270 0.0 0.0 1 3.25 3.25 108 −60.0 −60.0 2 3.25 3.25 68.2 −36.9 −74.7 Microcystis aeruginosa dilution series Initial 0.00 0.00 354 0.0 0.0 1 2.25 1.25 231 −34.7 −34.7 2 5.25 5.25 66.8 −71.1 −81.1 Pseudanabaena dilution series Initial 0.00 0.00 919 0.0 0.0 1 1.25 1.25 732 −20.3 −20.3 2 1.25 1.25 554 −24.3 −39.7 3 1.25 1.25 393 −29.1 −57.2 4 1.25 1.25 310 −21.1 −66.3 5 4.00 3.25 130 −58.1 −85.9 Synechococcus dilution series Initial 0.00 0.00 698 0.0 0.0 1 4.00 4.00 138 −80.2 −80.2 2 4.00 4.00 25.6 −81.4 −96.3 Table 2. Summary of phytoplankton monoculture dilution series for evaluating the performance of the Turner Designs, Inc. PhytoFind in-place phytoplankton classification tool.

Mixed-Population Culture Test Methods

Formulations for mixed-population tests (table 3) were guided by chlorophyll concentration readings of the initial culture samples as described in the “Monocultures” part of this section. The computed volumes of monoculture samples and spring water were added to the test vessel to achieve six unique mixed conditions within the 0 to 250 µg/L range specified for chlorophyll concentration by the manufacturer of the PhytoFind. The mixed-population conditions (table 3) were Chlorella vulgaris, Microcystis aeruginosa, and Navicula in about equal concentrations (CVL–MIC–NAV #1); Chlorella vulgaris dominant in the presence of Microcystis aeruginosa and Navicula (CVL-MIC-NAV #2); Microcystis aeruginosa dominant in the presence of Chlorella vulgaris and Navicula (MIC–CVL–NAV); Navicula dominant in the presence of Chlorella vulgaris and Microcystis aeruginosa (NAV–CVL–MIC); Pseudanabaena dominant in the presence of Chlorella vulgaris, Microcystis aeruginosa and Navicula (PAB–CVL–MIC–NAV); and Microcystis aeruginosa, Pseudanabaena, and Synechococcus in about equal concentrations (MIC–PAB–SYN).

Table 3.

Species composition of mixed-population phytoplankton cultures for evaluating the performance of the Turner Design, Inc. PhytoFind in-place phytoplankton classification tool.

Test condition Chlorella vulgaris Microcystis aeruginosa Navicula Pseudanabaena Synechococcus CVL–MIC–NAV #1 X X X — — CVL–MIC–NAV #2 X1 X X — — MIC–CVL–NAV X X1 X — — NAV–CVL–MIC X X X1 — — PAB–CVL–MIC–NAV X X X X1 — MIC–PAB–SYN — X — X X Table 3. Species composition of mixed-population phytoplankton cultures for evaluating the performance of the Turner Design, Inc. PhytoFind in-place phytoplankton classification tool.

Turbid Mixed-Population Culture Test Methods

To assess the effects of suspended particles on the optical sensors, multipoint add-in turbidity was tested for five of the six mixed-population conditions; CVL–MIC–NAV #1, CVL–MIC–NAV #2, MIC–CVL–NAV, NAV–CVL–MIC, PAB–CVL–MIC–NAV. Elliott silt loam soil (U.S. Department of Agriculture, 2015) obtained from the International Humic Substances Society (https://humic-substances.org/) was added incrementally to each mixed-population sample, using continuously measured turbidity sensor data from the EXO2 multiparameter sonde as a guide to achieve a desired range of 0 to 1,000 nephelometric turbidity units (NTU; fig. 8). Samples for turbidity were collected before the first soil addition and then at three to five additional points of increasing turbidity through the test duration.

The turbidity test samples were analyzed on a calibrated Hach 2100AN laboratory turbidimeter (Hach, 2015) to verify the sensor readings. Select samples were used to define a rating equation to correct the EXO turbidity sensor readings (measured in formazin nephelometric units) based on comparison values obtained by the turbidimeter (measured in nephelometric turbidity units). The selected samples were from the test condition with Chlorella vulgaris, Microcystis aeruginosa, and Navicula in about equal concentrations (CVL–MIC–NAV #1). Samples from this condition were assumed to be the best representation of the mean response from the EXO turbidity sensor based on variations in light scattering or attenuation by differences in cell morphology. Turbidity sensor measurements are detailed in Foster and others (2021). The rating equation was derived by a regression analysis between the turbidimeter values and those made by the EXO turbidity sensor at the time of sample collection. The equation defining the relation is:

NTU

= 0.0009(

FNU

)

2

+ 1.0936(

FNU

) + 2.1982,

(1)

where NTU is turbidity in nephelometric turbidity units and FNU is turbidity in formazin nephelometric units. The coefficient of determination (R2) for this relation is 1, and the number of samples is 4. Samples from the other turbid mixed-population culture test conditions were used to validate the equation. The correction was done to facilitate a high-definition comparison between additive turbidity levels and the corresponding changes in the continuous measurements from the PhytoFind.

Results of Monoculture Dilution Series

Chlorella vulgaris Dilution Results

In a four-point dilution series of Chlorella vulgaris (table 2), the PhytoFind overestimated chlorophyll concentration relative to laboratory-measured chlorophyll a concentration within the operating range and underestimated chlorophyll concentration above the operating range of 0 to 250 µg/L chlorophyll. Chlorophyll concentration was overestimated by 350 percent at 19.9 µg/L chlorophyll a and about 75 percent at 119 µg/L chlorophyll a. When the operating range was exceeded, the PhytoFind underestimated chlorophyll concentration by about 50 percent at 452 µg/L chlorophyll a and about 80 percent at 1,210 µg/L chlorophyll a. Overall, the chlorophyll-to-chlorophyll a concentration ratio for the PhytoFind decreased exponentially with increasing chlorophyll a concentration. Reported contribution percentages from the green and brown phytoplankton group decreased from 87 to 0 percent with increasing chlorophyll a concentrations (fig. 14).

In the same four-point dilution series of Chlorella vulgaris, the EXO Total Algae-PC overestimated chlorophyll concentration relative to chlorophyll a concentration within its operating range, overestimated chlorophyll concentration just above the operating range, and underestimated chlorophyll concentration well above the operating range of 0 to 400 µg/L chlorophyll. Chlorophyll concentration was overestimated by about 300 percent at 19.9 µg/L chlorophyll a and about 150 percent at 119 µg/L chlorophyll a. When the operating range of 0 to 400 µg/L chlorophyll was exceeded, the EXO Total Algae-PC overestimated chlorophyll concentration relative to chlorophyll a by 58 percent at a concentration of 452 µg/L chlorophyll a and underestimated chlorophyll concentration by about 40 percent at 1,210 µg/L chlorophyll a. Overall, the chlorophyll-to-chlorophyll a concentration ratio for the EXO Total Algae-PC declined exponentially with increasing chlorophyll a concentrations (fig. 14).

Navicula Dilution Results

Phytoplankton analysis indicated that about 75 percent of the Navicula cells present were compromised; however, because of the potential for a sample with dead or damaged cells, the results of the tests are discussed in this section. In a three-point dilution series of Navicula (table 2), the PhytoFind underestimated chlorophyll concentration relative to laboratory-measured chlorophyll a concentration within and above the operating range of 0 to 250 µg/L chlorophyll. The PhytoFind underestimated chlorophyll concentration by about 50 percent at 68.2, 108, and 270 µg/L chlorophyll a. Overall, the chlorophyll to chlorophyll a concentration ratio for the PhytoFind did not change with increasing chlorophyll a concentration. Reported contribution percentages from all three phytoplankton groups were consistently about 30 percent, even when chlorophyll a concentration increased (fig. 15).

In the same three-point dilution series of Navicula, the EXO Total Algae-PC underestimated chlorophyll concentration within the operating range of 0 to 400 µg/L chlorophyll. The operating range of the EXO Total Algae-PC was not exceeded. The EXO Total Algae-PC underestimated chlorophyll concentration by about 70 percent at 68.2, 108, and 270 µg/L chlorophyll a. Overall, the chlorophyll to chlorophyll a concentration ratio for the EXO Total Algae-PC did not change with increasing chlorophyll a concentration (fig. 15).

Microcystis aeruginosa Dilution Results

In a three-point dilution series of Microcystis aeruginosa (table 2), the PhytoFind overestimated chlorophyll concentration relative to laboratory-measured chlorophyll a concentration at a lower chlorophyll a concentration within the operating range of 0 to 250 µg/L chlorophyll, measured accurately at a higher chlorophyll a concentration within the operating range, and underestimated the chlorophyll concentration above the operating range. Chlorophyll concentration was overestimated by about 50 percent at 66.8 µg/L chlorophyll a, measured within 1 percent at 231 µg/L, and underestimated by about 30 percent at 354 µg/L chlorophyll a. Overall, the chlorophyll to chlorophyll a concentration ratio for the PhytoFind decreased proportionally when chlorophyll a concentration increased. Reported contribution percentages from the cyanobacteria group decreased from 85 to 71 percent when chlorophyll a concentration increased (fig. 16).

In the same three-point dilution series of Microcystis aeruginosa, the EXO Total Algae PC underestimated chlorophyll concentration relative to laboratory-measured chlorophyll a concentration consistently within the operating range of 0 to 400 µg/L chlorophyll. The operating range of the EXO Total Algae-PC was not exceeded. Chlorophyll concentration was underestimated by about 75 percent at 66.8, 231, and 354 µg/L chlorophyll a. Overall, the chlorophyll-to-chlorophyll a concentration ratio for the EXO Total Algae-PC was consistent when chlorophyll a concentration increased (fig. 16).

Pseudanabaena Dilution Results

In a five-point dilution series of Pseudanabaena (table 2), the PhytoFind overestimated chlorophyll concentration relative to laboratory-measured chlorophyll a concentration consistently within and above the operating range of 0 to 250 µg/L chlorophyll. Chlorophyll concentration was most overestimated, by about 60 percent, at 130, 310 and 393 µg/L chlorophyll a. Chlorophyll concentration was least overestimated, by about 10 percent, at 919 µg/L chlorophyll a, the highest concentration tested. Overall, the chlorophyll-to-chlorophyll a concentration ratio for the PhytoFind decreased when chlorophyll a concentration increased. Reported contribution percentages from the cyanobacteria group (phycocyanin fluorescence channel) decreased from 34 to 21 percent with increasing chlorophyll a concentrations. Additionally, the contribution percentage from the mixed phytoplankton group (phycoerythrin fluorescence channel) ranged from 66 to 79 percent when chlorophyll a concentration increased. This response from the PhytoFind can be attributed, in part, to the content of phycoerythrin in Pseudanabaena (fig. 17).

In the same five-point dilution series of Pseudanabaena, the EXO Total Algae-PC underestimated chlorophyll concentration relative to laboratory-measured chlorophyll a concentration consistently by about 90 percent within and above its operating range of 0 to 400 µg/L chlorophyll. Chlorophyll concentration was tested at the concentration range of 130 to 919 µg/L chlorophyll a. Overall, the chlorophyll-to-chlorophyll a concentration ratio for the EXO Total Algae-PC was consistent when chlorophyll a concentration increased (fig. 17).

Synechococcus Dilution Results

In a three-point dilution series of Synechococcus (table 2), the PhytoFind overestimated chlorophyll concentration relative to laboratory-measured chlorophyll a concentration within the operating range and underestimated chlorophyll above the operating range of 0 to 250 µg/L chlorophyll. Chlorophyll concentration was overestimated by about 150 percent at 25.6 µg/L chlorophyll a and about 40 percent at 138 µg/L chlorophyll a. When the operating range was exceeded, the PhytoFind underestimated chlorophyll concentration by about 65 percent at 698 µg/L chlorophyll a. Overall, the chlorophyll-to-chlorophyll a concentration ratio for the PhytoFind decreased exponentially when chlorophyll a concentration increased. Reported contribution percentages from the cyanobacteria group decreased from 100 to 76 percent with increasing chlorophyll a concentration (fig. 18).

In these same test conditions, the EXO Total Algae-PC underestimated chlorophyll concentration relative to laboratory-measured chlorophyll a concentration consistently by about 90 percent through the concentration range of 25.6 to 698 µg/L chlorophyll a within and above the operating range of 0 to 400 µg/L chlorophyll. Overall, the chlorophyll to chlorophyll a concentration ratio for the EXO Total Algae-PC was consistent when chlorophyll a concentration increased (fig. 18).

Results of Mixed-Population Culture Testing

Mixed-Population Cultures Chlorophyll Results

In mixed-population cultures, the PhytoFind consistently overestimated chlorophyll concentration relative to laboratory-measured chlorophyll a concentration (table 6). The overestimation was most exaggerated, by 400 percent, in the mixture dominated by the chlorophyte Chlorella vulgaris. The least-overestimated chlorophyll concentration, by about 63 percent, was in the mixture dominated by the phycoerythrin-containing cyanobacterium Pseudanabaena (fig. 19; table 6).

Table 6.

Summary of chlorophyll concentrations and laboratory-measured chlorophyll a concentrations in mixed-population phytoplankton cultures.

Test condition Laboratory-measured chlorophyll a concentration (µg/L) PhytoFind chlorophyll concentration (µg/L) EXO Total Algae-PC chlorophyll concentration (µg/L) CVL–MIC–NAV #1 53.6 132 120 CVL–MIC–NAV #2 20.0 98.2 94.2 MIC–CVL–NAV 15.3 43.4 17.3 NAV–CVL–MIC 15.2 33.5 18.8 PAB–CVL–MIC–NAV 37.5 61.0 20.2 MIC–PAB to SYN 36.6 76.3 5.47 Table 6. Summary of chlorophyll concentrations and laboratory-measured chlorophyll a concentrations in mixed-population phytoplankton cultures.

The EXO Total Algae-PC overestimated chlorophyll concentration relative to laboratory-measured chlorophyll a concentration in mixtures where the chlorophyte relative biovolume was greater than 15 percent and underestimated chlorophyll concentration in mixtures dominated by cyanobacteria with chlorophyte relative biovolume of less than 15 percent (table 6). The EXO Total Algae-PC overestimated chlorophyll by about 400 percent in the mixture dominated by the Chlorella vulgaris. The most severe underestimation was about 85 percent in the mixture containing only the cyanobacteria species. Of all the test conditions, the EXO Total Algae-PC measured the closest chlorophyll-to-chlorophyll a concentration, with a variability of less than 10 percent, in the mixture dominated by the cyanobacterium Microcystis aeruginosa (fig. 19; table 6).

Mixed-Population Culture Community Composition Results

Percent chlorophyll contributions per phytoplankton group of the total chlorophyll measured by the PhytoFind were compared with laboratory-measured relative percent biovolume as determined by IFC analyses (table 7). The comparison of community composition measured by the PhytoFind with relative percent biovolume is not direct; however, community composition measured by the PhytoFind and relative percent biovolume were assumed to be comparable (fig. 20). Of the four test conditions containing the chlorophyte Chlorella vulgaris with the diatom Navicula or the cyanobacterium Microcystis aeruginosa or all three species, the green and brown phytoplankton group was overestimated by the PhytoFind by about 40 percent in comparison to relative percent biovolume when Chlorella vulgaris was less than 40 percent relative percent biovolume. This result coincided with monoculture tests observations, indicating an increasing overestimation of chlorophyll concentration from Chlorella vulgaris as the chlorophyll a concentration decreases. Thus, the lower the Chlorella vulgaris concentration is in a mixture, the more likely its contribution to total chlorophyll concentration may be overestimated and underestimated in reverse.

Table 7.

Phytoplankton group proportions measured in mixed-population phytoplankton cultures.

Test condition Green and brown phytoplankton Mixed phytoplankton Cyanobacteria Other phytoplankton PhytoFind (% chl) IFC

(% BV) PhytoFind (% chl) IFC

(% BV) PhytoFind (% chl) IFC

(% BV) PhytoFind (% chl) IFC

(% BV) CVL–MIC–NAV #1 53.62 42.50 2.49 NA 43.90 35.78 NA 21.72 CVL–MIC–NAV #2 76.53 78.08 0.41 NA 23.06 11.04 NA 10.88 MIC–CVL–NAV 55.28 18.12 3.03 NA 41.70 63.82 NA 18.06 NAV–CVL–MIC 69.80 25.65 9.19 NA 21.02 21.58 NA 52.77 PAB–CVL–MIC–NAV 11.39 8.59 40.87 NA 47.74 82.29 NA 9.12 MIC–PAB–SYN 0.00 NA 12.40 NA 87.60 88.29 NA 11.71 Table 7. Phytoplankton group proportions measured in mixed-population phytoplankton cultures.

In the test condition with these species plus the dominant addition of the cyanobacterium Pseudanabaena, the PhytoFind overestimated the mixed phytoplankton group and underestimated the cyanobacteria group compared with relative percent biovolume. Although IFC analysis indicated that the relative percent biovolume of the cyanobacteria group was about 82 percent, the PhytoFind reported about 48 percent from the cyanobacteria group and about 41 percent from the mixed phytoplankton group. As with the monoculture tests, the mixed phytoplankton group (that is, phycoerytherin-containing phytoplankton) contribution was overestimated because this fluorescence channel detected the phycoerythrin present in Pseudanabaena. When the PhytoFind was tested in a mixture of only the cyanobacteria species—Pseudanabaena, Microcystis aeruginosa, and Synechococcus—the reported percent chlorophyll contribution and relative percent biovolume for the cyanobacteria group were 87.6 and 88.3 percent, respectively.

Turbid Mixed-Population Cultures Results

Multipoint additions of Elliott silt loam soil (U.S. Department of Agriculture, 2015) were applied to five of the six mixed-population culture mixtures—CVL–MIC–NAV #1, CVL–MIC–NAV #2, MIC–CVL–NAV, NAV–CVL–MIC, PAB–CVL–MIC–NAV—addressing effects of turbidity on chlorophyll concentration measurements (fig. 21). In conditions where the relative percent biovolume of Chlorella vulgaris was greater than 40 percent, chlorophyll concentration measured by the PhytoFind varied by approximately ±10 percent around the mean in turbidity ranges from 0 to 1,000 NTU. For those conditions where relative percent biovolume of Chlorella vulgaris values were lower than 40 percent, chlorophyll concentration from the PhytoFind showed a proportional response to increasing turbidity levels, increasing by about 100 percent through the range of 0 to 1,000 NTU. As the turbidity increased, the percent chlorophyll contribution of the mixed phytoplankton group measured with the PhytoFind increased and the percent chlorophyll contribution of the green and brown phytoplankton group concurrently decreased.

In conditions where the relative percent biovolume of Chlorella vulgaris was greater than 40 percent, the EXO Total Algae-PC had an inversely proportional relation to increasing turbidity levels, particularly for concentrations greater than 100 NTU, decreasing by about 60 percent through the range of 0 to 1,000 NTU. For those conditions where relative percent biovolume of Chlorella vulgaris values were lower than 40 percent, the EXO Total Algae-PC chlorophyll concentration measurements were consistent overall and had a slightly inversely proportional relation, decreasing by only about 5 µg/L, or about 25 percent, through the range of 0 to 1,000 NTU (fig. 21).

Chlorophyll Field Test Results

The PhytoFind overestimated chlorophyll concentration relative to laboratory-measured chlorophyll a concentration at 17 of 20 sample locations. Based on IFC analyses, the phytoplankton communities were dominated by organisms classified in either the green and brown phytoplankton group or the cyanobacteria group. The highest overestimations were observed in samples dominated by the cyanobacteria group, by about 200 percent. In the three sample locations where chlorophyll concentration was underestimated (varying by about 10 to 30 percent less than the laboratory-measured chlorophyll a concentration), a chlorophyte was dominant (fig. 22). Overall, the magnitude of overestimation by the PhytoFind in the range of chlorophyll a concentrations sampled was similar to results of the laboratory tests using phytoplankton cultures, whereas the magnitude of underestimations was not. IFC analyses indicated phytoplankton community compositions with more than 10 different species or classifications at each location and, thus, may have led to sensor responses not observed during laboratory tests.

The EXO Total Algae-PC underestimated chlorophyll concentration relative to laboratory-measured chlorophyll a concentration at 19 of 20 sample locations; the greatest underestimations, by about 60 percent, were in samples dominated by cyanobacteria. In the one sample location where chlorophyll concentration was overestimated, by about 15 percent relative to laboratory-measured chlorophyll a concentration, a cyanobacterium was dominant (fig. 22). Overall, the magnitude of underestimations by the EXO Total Algae-PC in the range of chlorophyll a concentration sampled—especially when cyanobacteria were dominant—was similar to results of the laboratory tests (fig. 22). Overestimation of chlorophyll concentration relative to laboratory-measured chlorophyll a concentration when cyanobacteria were dominant in the sample corresponds to the laboratory-testing results of the mixed population test condition MIC–CVL–NAV (where the cyanobacterium Microcystis aeruginosa was dominant in the presence of Chlorella vulgaris and Navicula) where chlorophyll concentration was also overestimated.

Community Composition Field Test Results

Community compositions were diverse during the July 15–18, 2019, moving-boat survey in southern Florida. IFC analyses indicated about 10 to 20 different phytoplankton taxa for each of the 20 locations. Of these taxa, six were dominant in one or more samples, including chlorophytes, cyanobacteria, and diatoms. Relative percent biovolume measured by IFC analyses shows that the green and brown phytoplankton group ranged from about 8 to 80 percent; the mixed phytoplankton group, from about 0 to 1 percent; and the cyanobacteria group, from about 8 to 70 percent (table 8). For algae not specifically classified by IFC as belonging to one of the PhytoFind target phytoplankton groups, the relative percent biovolume was accounted for in a group labeled “other” and ranged from about 8 to 60 percent.

Table 8.

Summary of phytoplankton group proportions at locations sampled during a moving-boat survey from July 15–18, 2019, in Florida.

Sample location Green/brown algae Mixed algae Cyanobacteria Other algae PF

(% chl) IFC

(% BV) PF

(% chl) IFC

(% BV) PF

(% chl) IFC

(% BV) PF

(% chl) IFC

(% BV) 1 0.00 7.61 40.02 0.31 59.98 30.88 NA 61.20 2 3.25 8.01 54.43 0.00 42.32 68.65 NA 23.34 3 0.00 18.47 65.34 0.00 34.66 66.75 NA 14.77 4 0.00 34.08 87.20 0.04 12.80 31.86 NA 34.02 5 0.00 38.78 83.98 0.00 16.02 35.25 NA 25.96 6 22.43 61.67 54.06 0.00 23.52 17.30 NA 21.02 7 72.01 71.18 10.55 0.47 17.44 13.09 NA 15.26 8 69.76 59.03 4.88 0.00 25.36 32.92 NA 8.05 9 71.62 35.32 4.61 0.08 23.77 49.67 NA 14.92 10 37.12 28.71 44.51 0.13 18.37 37.77 NA 33.39 11 78.38 61.44 0.63 0.34 20.99 17.16 NA 21.06 12 41.47 61.75 35.23 0.00 23.30 13.13 NA 25.11 13 62.35 67.01 6.81 0.82 30.85 16.49 NA 15.68 14 51.93 47.78 14.48 0.20 33.59 34.65 NA 17.37 15 73.36 41.13 0.00 0.23 26.64 27.16 NA 31.47 16 63.74 54.21 6.82 0.05 29.43 27.26 NA 18.48 17 63.58 34.16 12.59 0.12 23.83 39.29 NA 26.42 18 75.55 51.57 0.00 1.03 24.45 23.66 NA 23.74 19 86.31 83.54 0.00 0.00 13.69 8.40 NA 8.06 20 69.19 56.17 4.84 0.14 25.97 17.83 NA 25.86 Table 8. Summary of phytoplankton group proportions at locations sampled during a moving-boat survey from July 15–18, 2019, in Florida.

No consistent correlation was found in the relations between percent chlorophyll contribution per phytoplankton group from the PhytoFind and relative percent biovolume per phytoplankton group from IFC analysis. The relationship was not determined to be controlled by dominant taxa, specific population ratio, or environmental condition. Most commonly, the PhytoFind overestimated the mixed phytoplankton group, which happened for 17 of the 20 locations sampled, and the PhytoFind overestimations of the mixed phytoplankton group chlorophyll contributions were the largest in magnitude out of all the target phytoplankton groups. The IFC reported mixed phytoplankton biovolume at each location as about 0 to 1 percent, whereas the PhytoFind reported several chlorophyll contribution percentages as high as 87 percent (fig. 24; table 8). These data indicate potential sensitivity of the mixed phytoplankton group fluorescence channel to interference from internal or external sources beyond the intended target phycoerythrin.

Because the algorithm of the PhytoFind factors in signals from all fluorescence channels (phycoerythrin, phycocyanin, chlorophyll and DOM) to determine estimates for chlorophyll concentration and percent chlorophyll contributions, it is possible that interference in one fluorescence channel could affect the estimates of all values reported. Additional testing across a range of water-quality and phytoplankton community types may better elucidate potential interferences.