Purpose and Scope

The purpose of this report is to provide information on the selection and use of field fluorescence sensors (also referred to as “field fluorometers”) by the USGS for in-place determination of the presence, relative abundance, and qualitative estimates of algal pigment concentrations in environmental waters. This report will help USGS personnel and others collect field data by using algal fluorometers in ways that are consistent and comparable across studies, sites, and instruments. For those who have selected fluorometric instrumentation for measurement of algal pigments in the field, this report provides guidance on instrument preparation and deployment, ongoing maintenance and field operations, quality-assurance procedures, and data reporting. This report supplements manufacturer user manuals for individual sensors. This report will not aid in quantifying or correcting for the various interferences discussed within. Quantifying and correcting for these interferences warrant their own investigations and these topics are beyond the scope of this report.

Measuring Algal Pigment Fluorescence

All algae contain chlorophyll a (chla), a water-insoluble, light-harvesting pigment involved in photosynthesis that emits approximately 3–5 percent of the absorbed light energy in the blue and red regions of the light spectrum as fluorescence (Falkowski and Kiefer, 1985). Chla is not simply contained within the cell, but rather, it is associated with biomolecules and packaged within cellular thylakoid membranes in ways that lead to variability in the peak absorption and emission bands as well as the relative fluorescence emissions (Roesler and Barnard, 2013). These variations in the chemical properties of chla also affect the intensity of measured fluorescence per unit of chla (commonly referred to in literature as “fluorescence yield”), which can vary several-fold based on the algal species and physiological status of cells (Proctor and Roesler, 2010; Roesler and Barnard, 2013).

Measurement of algal fluorescence using field-based sensors may be referred to as “in place” (within the original position of the sample, commonly called “in situ”) or “in body” (within the body or cells of a living organism, commonly called “in vivo”). “In-body fluorescence” is often used to refer to fluorescence measured from intact, living algae, which range in linear size from <0.2 micrometer (µm; picoplankton) to >2 millimeters (mm; macroplankton). To distinguish between laboratory-based approaches that may be used to measure fluorescence of live algae, field-based sensor measurements are referred to as “in place measurements” throughout this report. In contrast to in-body measurements, extracted (also called “in vitro”) measurements are laboratory-based fluorescence measurements typically made on samples of algae that are no longer living and have been collected on a filter and the pigments extracted with an organic solvent. Because extracted measurements are made on pigments dissociated from various proteins and membranes within living cells, the resulting fluorescence is typically higher than the observed in-body fluorescence (Hambrook Berkman and Canova, 2007). The relations between algal pigment-fluorescence measured in extracted samples of algae and by in-place sensors vary across hydrologic systems, algal species, sample depth, and even time of day; therefore, if the purpose of monitoring is to quantify pigment concentrations, each system should be evaluated independently.

In addition to chla, there are other fluorescent accessory pigments including chlorophylls b, c, and d, carotenoids, xanthophylls, and phycobilins (for example, phycocyanin and phycoerythrin) which are used by algae to harvest and shuttle photons from sunlight to make energy. Whereas all algae; including cyanobacteria, green algae, and diatoms contain chla; accessory pigments can be diagnostic of the different types of algae present (Wetzel, 2001). This is possible to measure because these pigments vary in their light absorption spectra, or how they absorb (and therefore use) or reflect light, at different wavelengths (fig. 3).

Together, the absorbed light and ensuing fluorescence produce characteristic excitation–emission maxima, or wavelength peaks, per different pigments. These wavelengths can be detected, quantified, and used to estimate pigment concentration using multiwavelength scans, or with single or dual wavelength sensors in a targeted manner. Chlorophyll a has a primary absorption peak in the blue (at about 430 nanometers [nm]) part of the visible light spectrum, and a smaller, secondary peak in the red part (at about 660 to 665 nm); plants are green because they reflect light in the green part of the spectrum (Wetzel, 2001). Cyanobacteria are of particular interest because of their potential to form harmful algal blooms (Graham and others, 2008). Important secondary pigments in cyanobacteria are the phycobilins PC and PE, which have peak absorption at fluorescence wavelengths near 595 nm and 670 nm, and 528 nm and 573 nm, respectively (Hambrook Berkman and Canova, 2007).

Fluorescence as a Proxy for Algal Biomass and Community Composition

Chla is a near-universal proxy for algal biomass (Wetzel, 2001; Hambrook Berkman and Canova, 2007). Fluorescence sensors do not directly measure chla or other algal pigments, but rather measure a relative fluorescence response of a whole water sample at a specific absorption wavelength. Measurement of pigments other than chla can give an indication of the presence and abundance of other algal groups of interest, such as cyanobacteria, or broader algal community composition. Fluorescence response is often (but not always) proportional to pigment concentration in water, and can be used, for example, to estimate chla or PC concentration. The fluorescence measurements described in this report are commonly compared to laboratory-measured concentrations of chla (most commonly, chla in micrograms per liter [µg/L]), to PC or PE (also in µg/L), or to the abundance or biovolume of total algae or specific algal groups (in cells per liter or biovolume per liter). This proxy-based relation between the in-place sensor measurement and the laboratory-measured quantity of interest is in contrast to sensors that make more direct measurements such as optical nitrate sensors; the magnitude of the absorbance at a peak wavelength (for example, 220 nm for nitrate) is directly proportional to the concentration of ions in solution (Pellerin and others, 2013). Users of algal fluorometers must be aware that algal fluorescence is a proxy (or indirect) measure and may not always be directly proportional to concentrations of chla or other pigments of interest.

Many factors influence the fluorescence response of living algae and can cause variability in the relation between in-place sensor measurements and extraction-based measurements in the laboratory (fig. 4). For example, not all taxa have the same amount of pigment and some taxa are more efficient at harvesting and using light, resulting in order-of-magnitude variability in chla fluorescence yields (Vincent, 1983; Proctor and Roesler, 2010; Roesler and Barnard, 2013; Bertone and others, 2018) that represent one of the largest sources of variability in the relation between field and laboratory-measured pigment concentrations (Roesler and Barnard, 2013; Proctor and Roesler, 2010). Another factor that causes variability in fluorescence yield is the general health of algae (Olaizola and others 1994), which affects the overall amount and type of pigments produced and hence their fluorescence. In addition, fluorescence may exhibit extreme diurnal variability caused by nonphotochemical quenching, resulting in apparent changes in pigment concentrations that are not indicative of changes in biomass concentration (Beutler and others, 2002; Roesler and Barnard, 2013; Bertone and others, 2018).

Although sensor-based measures of algal fluorescence may not always be directly related to concentrations of algal biomass or algal pigments, patterns in algal fluorescence contain inherently valuable information that can indicate the following:

Basic Sensor Design

In-place fluorometers generally work as follows: a light-emitting diode (LED) is used to generate a nearly monochromatic beam of light at a predetermined wavelength, and the light passes through a water sample of known dimensions and volume that is internal or external to the sensor body (fig. 5). A photodetector measures light returned from responsive fluorophores present in the sample at nearly the same time that it was emitted, as the process of fluorescence is nearly instantaneous (with a delay of only one hundred billionth to one hundred millionth of a second; Guilbault, 1990; Suggett and others, 2010). The specific wavelengths of fluoresced light measured by the sensor’s photodetector are determined by optical filters embedded within the sensor housing. After additional, instrument-specific filtering or processing of the light-derived electronic signal or both, a measurement is calculated in volts or counts and transmitted in analog or digital format (for example, Recommended Standard 232 [RS-232], serial-digital interface at 1200 baud [SDI-12], or Recommended Standard 485 [RS-485]) to a data-collection platform (DCP) for logging or transmission. The collected data can then be converted to other fluorescence or concentration units based on manufacturer or calibration-defined scale factors.

The physical components of field and laboratory fluorometers are essentially the same, and include a light source, optical filters, and a photodetector. Critical to the function of each component are the associated electronic circuits that include regulated power supplies, lamp drivers, instrumentation amplifiers, analog-to-digital converters, and a microcontroller programmed for the desired output format and data preprocessing. Some sensors are equipped with adjustable (as opposed to automatic) gain settings that can be used to maximize the linear range, which may be useful for specific applications at the low or high end of the dynamic range. However, a number of important physical modifications are necessary when laboratory fluorometers are miniaturized and adapted for field deployments. Modifications include the use of low-power LEDs as a light source instead of high-intensity xenon or mercury lamps, and elimination of the excitation monochrometer typically used in benchtop fluorometers for scanning across a range of excitation and emission wavelengths. These and other modifications, including ruggedized, waterproof housings and solid-state electronic components (such as integrated circuit boards and transistors which have no moving parts), efficient power and heat handling, and antifouling components, not only affect the ability to make accurate fluorescence measurements in place, but also affect the serviceability, longevity, stability, and cost of field fluorometers.

The light source in the current (2022) generation of field fluorometers is typically composed of one or more LEDs, which reduces the cost, power consumption, longevity, and size of instruments relative to other types of lamps used in field spectrophotometers (such as deuterium and xenon flash lamps). When excited by light emitted by LEDs, the stability, lifetime, and quantum yield (ratio of photons emitted to photons absorbed) of fluorophores varies by wavelength; but measurements of chla and PC excitation by LEDs indicate little drift even over thousands of hours of operation. Photodetectors are the true sensing elements of the instrument and, typically, are photodiodes or diode arrays characterized by low cost, low power consumption, and small size. The angle at which the excitation light source and the emission detector are placed relative to each other (typically from 90 to 140 degrees) determines the sample volume; the volume is geometrically defined by where the excitation light beam intersects with the cone of reception of the detector (fig. 5). As the acceptance angle or detection cone is increased, the length of the sample volume and the amount of light attenuated along the sample path is decreased. Attenuation of received light can be caused by particles scattering the light along the sample path, and by photons reabsorbing the fluoresced light within the sampling volume. Optical filters are made of glass or other materials that filter out unwanted wavelengths emitted from the light source, and from fluoresced light returning to the detector. Bandpass filters used in most field fluorometers typically allow for a broad spectral distribution. The common specifications of bandpass filters are the wavelength with peak transmittance, the peak center wavelength (the average of two half-height wavelengths in a spectrum) and the peak wavelength full width at half maximum (the wavelength at half of the maximum transmittance; fig. 2).

All field fluorometers are equipped with an electronic signal processor that converts the raw current from a photodetector to a useful measurement output. Instruments can use a system of analog and digital electronic circuits, or a specialized microcontroller known as a digital signal processor to detect, convert, and emit signals at very high internal sample frequencies, which are then integrated to output high-frequency data (from 1 to 20 hertz [Hz]) by an analog or digital (RS-232) signal output. A microcontroller also controls the modulation of the LED so that the system can detect and differentiate between fluorophores and background noise (that is, photodetector dark current). Some sensors may employ dynamic gain to increase sensitivity and prevent saturation over a broader range of conditions. Most field fluorometers are designed to be integrated with external DCPs that provide power to the sensors and a datalogger controller to record the output of the fluorometer. Field fluorometers equipped with digital output in the form of serial communication (for example, RS-232, RS-485, USB or SDI-12) are common and desirable compared to analog output fluorometers for most continuous-monitoring deployments. Although there are many other protocols for serial communication, the majority of DCPs currently in use are designed to directly interface using RS-232, SDI-12 and RS-485 serial protocols.

Sensor Specifications

Field fluorometers are typically characterized by the three following data-quality specifications: (1) detection limit, (2) dynamic range (typically within the linear range of the sensor), and (3) resolution. Currently (2022), there are no accepted national or international standards to which these measurements can be compared, such as those available for other water quality measurements published by the National Institute of Standards and Technology (NIST) or the International Organization for Standardization (ISO). In addition, the accuracy of field fluorometers is the degree of agreement between the sensor-measured and true-fluorescence intensity, which is primarily affected by two sources of uncertainty: (1) instrument noise and (2) interferences. Although instrument noise; which is caused by the electronic components, the light source, and the detector resolution; is often random (Skoog and others, 2007), a systematic error can result in good precision but poor accuracy (that is, bias). Therefore, instruments with low electronic noise and a greater tolerance (that is, lower uncertainty because of matrix interferences) for interferences from suspended particles or dissolved organic matter (DOM) have greater accuracy. However, it is important to recognize that these specifications relate to the fluorescence measurements themselves; they do not reflect the correlation of the measurement to laboratory-measured pigment concentrations, algal abundance, or algal community composition.

In addition, manufacturer specifications are often determined under controlled operating conditions, averaged for a limited number of sensors, and by using primary reference materials (for example, an algal culture of a single species) in solutions that typically result in greater accuracy, greater precision, and lower detection limits than would be observed if measured in environmental waters where interferences are a factor. The results are then sometimes scaled across a range using an algorithm and secondary reference materials (for example, rhodamine WT, see Smith, 2020). Individual sensors can vary in performance because of even small variations in the manufacturing process. Prior to deploying an instrument in the field, it is important to independently verify instrument performance in a controlled environment, such as a laboratory, with appropriate standards and interferences (such as suspended particles and DOM). The baseline performance data collected prior to deployment serves as a basis for evaluating instrument performance after deployments and during instrument servicing.

The detection limit for fluorometers can be defined as the lowest value measurable by a given sensor that can be distinguished from a blank value with some level of statistical confidence (usually 99 percent; 40 CFR §136 app. B). Detection limits for in-place fluorometers are a function of both the detector sensitivity and the instrument electronic noise. In general, the detection limits of the current (2022) generation of in-place fluorometers are very low in environmental waters and may be important for waters with low levels of algal pigments.

The dynamic range is defined as the difference between the lowest and highest concentrations that can be measured, although the range over which the instrument response is linear (linear dynamic range) is often of greater interest. Linearity is typically reported by the manufacturer as the coefficient of determination, r², or can be determined by users from a calibration curve across the dynamic range. The maximum detectable value is particularly important for fluorometers, especially when used in systems with excessive algal biomass or algal blooms, and is determined by several factors including the chemical composition of the target constituent and the instrument’s optical path length and components. Some sensors are equipped with adjustable (as opposed to automatic) gain settings that can be used to maximize the linear range, which may be useful for specific applications at the low or high end of the dynamic range.

Instrument resolution is defined as the minimum difference between measured values reliably detected by the sensor. The current (2022) generation of field fluorometers can typically measure at a resolution of two decimal places.

Processes Affecting Fluorescence Response

Several environmental and chemical variables affect fluorescence response because they affect the capacity of the fluorescing molecule to dissipate energy, which thereby reduces detectable excitation. These effects are referred to as photochemical quenching. Nonphotochemical quenching, a form of dissipation of absorbed light employed by plants and algae to protect themselves from high light intensity, is a commonly observed effect specific to fluorometric measurement of algal pigments.

Interferences Affecting In-Place Measurements of Algal Pigment Fluorescence

Fluorescence measurements of all types are susceptible to interference from substances that affect how the emitted (excitation) light propagates through the solution, as well as how the fluoresced (emission) light propagates back through the solution to the photodetector. In addition to particle concentration, physical properties such as size and type of particles can affect both absorption and scattering of photons, whereas chemical properties such as DOM and other dissolved constituents primarily affect absorption (fig. 7). Both scattering and absorption, collectively known as “matrix effects” (Pellerin and others, 2013), attenuate light because they result from properties of the matrix in which the fluorescence measurement is being made. Strategies for reducing matrix effects could be necessary in challenging environments. One such strategy that has been applied to other measurement types (nitrate, DOM, and so on) is physically filtering out particles prior to sensor measurements, but filtering cannot be applied to algal fluorescence, because the algal material would be removed by most filters.

Instrument Selection

While several commercially available in-place fluorometers may meet the necessary specifications (for example, detection limit, operating range, and resolution) for most freshwater and coastal applications, several additional factors are worth considering when selecting a sensor for algal fluorescence measurements. Practical considerations for sensor selection such as the desired configuration (open face or flow through) and the data-collection platform may be particularly important. For example, users of a multiparameter instrument may want to consider a sensor that integrates with that platform for consistency and ease of use and consider possible interferences (see section “Factors Influencing Observed Fluorescence”) and other potentially important informational data (see section “Ancillary Data”).

Sensor cost is also an important consideration, as the current (2022) generation of commercially available field fluorometers range from about $2,000 to $30,000 based on several factors. For example, integrated wipers, internal dataloggers, and controllers are included with some but not all sensors. Similarly, the choices of housing materials, microprocessors, and the number of LEDs—which may be important considerations in some environments, particularly for long-term deployments in coastal waters—affect the cost of the instruments.

There are numerous single, dual, and multichannel fluorometers available. The instrument selected depends on several considerations, including the following:

Many of these considerations can be addressed with historical water-quality data, site documentation, and data-quality objectives determined for the study. The user can then evaluate instrument needs relative to manufacturer-stated or user-defined specifications, as well as relevant publications. It is important to note that in-place algal fluorescence measurements might be impractical in some systems during times of extreme conditions.

Data Comparability

Not all fluorometers will yield comparable results, even among the same make and model (Roesler and others, 2017). Fluorometric instruments of different designs commonly do not yield identical or equivalent results in the same sampled water or for different algal species and assemblages. Even sensors (of differing make and model) that report measurements in the same units (µg/L, RFU, and so on) and calibrated with the same standards may not yield identical or equivalent results in environmental waters. For example, in a sensor comparison exercise in the San Francisco Estuary, chlorophyll fluorescence response varied considerably among identical probes during a 2-week period (fig. 9; Stumpner and others, 2022).

This variation is because of engineering specifications and the many interferences (chemical, physical, and biological) that are unique to each combination of sensor type and location. Fluorometric sensor measurements are dependent on the optical configuration and the excitation and emission wavelengths of the sensors. This means that the wavelength of light used and the angle and number of detectors used to measure emission response from intracellular pigment molecules all play a role in sensor response. These factors must be well understood when comparing data across sensor networks. When operating field fluorometers, information on sensor type, calibration practices, and so on must all be well documented and available to data users.

Laboratory Calibration and Calibration Checks

There is no “truth” when it comes to field fluorometers, at least when “truth” is defined as the real measurement of an extracted pigment concentration or cellular abundance, because fluorescence is a proxy measure based on the fluorescent properties of algal pigments. Even comparison to an extracted pigment sample is only accurate relative to past and future readings if phytoplankton communities and ambient conditions are similar. However, achieving relative comparability in sensors across a network—or in a single sensor over time—is critical for fluorometric datasets. This comparability is achieved through consistent and rigorous calibration practices.

There are many factors that contribute to the frequency and number of calibration checks needed. In general, current (2022) optical sensors like fluorometers should not need to be recalibrated often. However, calibration checks must be made routinely to verify sensor function and verify there is no calibration drift. For continuous deployments, sensors should never go unverified for more than 3 months. Calibration guidance can be found in Anderson (2005) and Hambrook Berkman and Canova (2007) and may be defined in quality assurance plans. For more general information on calibration checks, users should refer to the National Field Manual for the Collection of Water-Quality Data book 9 (U.S. Geological Survey, variously dated), Wagner and others (2006), and the user manual for the instrument.

Calibration checks generally require a two-point check in solutions of known concentration. Deionized water (DIW) is typically used to verify the measurement at zero concentration as the first point, with a secondary standard (rhodamine WT, for example) used to verify another point at a high concentration measurement. A third point could be checked if additional confirmation of linear instrument response is needed. Checks that are within the calibration criteria (table 1) indicate that the instrument is performing as expected. For instruments that fall outside of the calibration criteria, users should ensure that the optical parts are clean and free from staining or scratches, and the measurement is not affected by bubbles, temperature variability, or direct sunlight prior to repeating the calibration check.

In some cases, it may be appropriate to repeat with a separate set of standards, ideally from a previously unopened (or freshly made) bottle, to confirm the readings prior to recalibrating the instrument. Although many dyes are stable for at least a year at room temperature when stored in a dark bottle, it is important to recognize that they will degrade over time (diluted dyes can last only hours to days depending on storage conditions) and will need to be replaced. Users should follow expiration dates and conduct routine laboratory checks if degradation is suspected.

Other factors to consider during the calibration of field fluorometers are the following:

Recalibration of a sensor should only be done when all potentially interfering factors, including those listed above, are confidently eliminated, thereby ensuring that the sensor is in fact out of calibration specifications (table 1). Otherwise, increased possibility of human-induced error, particularly in preparing the secondary reference standard, can be introduced through the erroneous recalibration of instruments. Recalibrating a sensor should not be needed routinely; sensors found out of calibration specifications are more likely indicative of calibration error or sensor failures than calibration drift.

Resetting the calibration of a sensor to the factory default may be needed when calibration error is apparent, and in other rare circumstances. For instance, when the calibration history of the sensor is in question, a factory reset (or “uncal”) can be performed on some sensors according to the guidance in the user manual. Care should be taken when considering whether to reset a sensor, as it may be difficult or impossible to formulate a mathematical correction to apply to data in the current time series collected prior to the factory reset. After resetting the sensor's calibration, verify and recalibrate if necessary.

Secondary Standards

As stated previously, unlike many other types of sensors, there are no true primary standards for algal field fluorometers. The measurement being collected by field fluorometers is the fluorescence response of algal pigments, and not the concentration of algal pigments. A secondary standard can take many forms; however, rhodamine WT dye has been recommended by several manufacturers because of its high solubility in water, known optical properties, and stability over periods of months to years when stored in the dark at room temperature. Rhodamine WT solutions at various concentrations are available through several manufacturers. A two-point calibration conducted with a zero solution (preferably DIW) and a temperature-indexed value from a rhodamine WT secondary standard allows for standardization across sensors.

If users choose to prepare diluted standards from a concentrated stock solution, it should be done using class A volumetric glassware (volumetric flasks, graduated cylinders, or pipettes) under carefully controlled laboratory conditions as described by Wilson and others (1986), and the concentration of the diluted standard should be verified using an independent benchtop or portable instrument. Care is essential when transferring rhodamine WT using pipettes because the viscosity of the solution may result in inaccuracies in volumes mixed. In addition, diluted rhodamine WT mixtures are not as stable and should be used within 5 days of preparation.

Absorption of rhodamine dyes typically ranges between about 440 and 680 nm (Gofman, 1972; Licha and Resch-Genger, 2014; Sugiarto and others, 2017). Measuring absorbance at the peak wavelength (for example, about 556 nm for rhodamine WT) with a spectrophotometer is one way to evaluate rhodamine WT standards to determine the expected absorbance at a given concentration, and thus verify the accuracy of a diluted standard. This is especially true if making rhodamine WT dilutions from concentrate, because error can come about from the operator or the equipment. The ability to verify rhodamine WT standard will depend on the availability of benchtop photospectrometers or fluorometers. Correct benchtop readings of properly diluted rhodamine WT must be determined empirically for any given benchtop instrument. It is important to ensure that benchtop readings are consistent for every batch of rhodamine WT standard, thereby eliminating the standard as a source of calibration error. While this approach to ensuring standard consistency over time is focused on rhodamine WT, the same general approach (that is, determining absorption maxima and measuring every batch of standard made) would apply to any standard created or used.

Alternate Standards

Instrument manufacturers are developing alternative standards, and some now offer options available to users. Solid standards incorporate a piece of solid material that will produce a known fluorescence response. However, the method and material used for alternative standard calibration may not produce equivalent results to those of typical methods and materials. It is up to the end user to follow the manufacturer’ instructions, evaluate new standards, their effects on data quality, and data comparability across sites and data collection networks.

Reference Sensors

Maintaining a field fluorometer for dedicated laboratory use may be a better option for validating the accuracy of diluted standards relative to the sensor output (Alliance for Coastal Technologies, 2017). Such a sensor (sometimes referred to as a reference sensor) is one that has been validated, either by the manufacturer or through internal procedures, and is maintained in the servicing office for no other purpose than for calibrated reference measurements. A well-documented quality assurance plan, like that used to track NIST traceable thermometers, should be maintained to ensure proper functionality of the reference sensor, and provide a traceable calibration history. A reference sensor should be used under the following guidelines:

Reference sensors are not necessary if you have a benchtop instrument that can verify the accuracy of standards. However, reference sensors are useful to ensure that the standard solutions are consistent so that intercalibration among different sensors in a network can be achieved and validated. For example, standard values outside of ±5 percent of the expected range based on the reference sensor would need to be remade.

Manufacturers’ Instructions

Considering the wide variety of fluorometers on the market, and the inherent differences caused by different manufacturing techniques, components, and styles, it is unlikely that a universal approach to calibrations would be appropriate. Each instrument’s operating manual should be the authoritative reference for calibrating any given fluorometer model. These procedures should be weighed against the requirements of other overarching organizational standard protocols.

Laboratory Use

In some instances, instruments designed to be used as field fluorometers are maintained in the laboratory, and environmental samples are collected and brought to the laboratory for fluorescence measurements of algal pigments. This approach can reduce environmental influences and interferences on fluorescence measurements, but long holding times between sample collection and measurement could affect the health and viability of algae, thereby affecting fluorescence response. Using field fluorometers in the laboratory for discrete sample measurements should be approached with a full understanding of the effects that discrete sample collection and transport may have on resultant measurements and data interpretation. Data collected using this approach should not be stored under the same parameter code as in-place field measurements, because the approaches are not directly comparable.

Field Use

There are numerous ways algal fluorometers can be used in the field, including continuous fixed site or moored deployments, as a field meter during operation and maintenance of a in-place sensor, for point sample measurements during sample collection, and spatial surveys. Each of these approaches are discussed in more detail below. Additional guidance for field use of algal fluorometers can be found in Hambrook Berkman and Canova (2007) and U.S. Geological Survey (2018c).

Fouling and Drift Corrections

Corrections for continuous water-quality sensors should be applied when a specified threshold for the sum of the absolute values of the computed fouling and drift corrections are exceeded (table 1; Wagner and others, 2006). Whenever the thresholds are not exceeded (table 1), corrections do not need to be applied. However, users may wish to evaluate instrument accuracy in the laboratory to determine if more stringent, project-specific criteria are justified to meet data quality objectives. If less stringent corrections criteria are applied to a dataset, it should be released in an acceptable digital repository such as ScienceBase (https://www.sciencebase.gov/catalog/) rather than NWIS.

Reporting Parameter and Method Codes

There is not one universal NWIS parameter code for algal fluorescence, because there are several different types of algal pigments that can be measured, and measurement units that can be used. The recommended NWIS parameter codes for fluorescence sensor output of the algal pigments most commonly measured by the USGS are described in table 2.

The “f” included with all abbreviations designates that data are the result of fluorescence-based measurements. If new parameter codes for algal fluorescence data need to be requested, the “f” designation should be used. Parameter codes for other less-commonly measured algal pigments also are available for use in NWIS.

The “estimated from reference material” description included with parameter units other than RFU (RFU is a sensor-specific unit) means that reported concentrations are based on a manufacturer supplied conversion table between a secondary standard (such as rhodamine WT dye) and a primary standard commonly based on a single algal reference culture. If new parameter codes need to be requested for reporting units other than RFU that are based on a manufacturer-supplied conversion, the “estimated from reference material” description should be included.

Method codes are used to identify the equipment used to carry out measurements. NWIS method codes for algal fluorometers most commonly used by the USGS are listed in table 3.

Method codes for other algal fluorometers also are available in NWIS and can be looked up at https://help.waterdata.usgs.gov/code/method_cd_query?fmt=html. New method codes can be requested for sensors or configurations not currently on this list. Method code requests for new makes and models of algal fluorometers should include information about the following: manufacturer; model; excitation, emission and bandpass wavelengths; and reference organisms used to develop the conversion table between a secondary standard and pigment concentration.

Field Fluorescence to Estimate Quantitative Algal Data

Some users may want to use field fluorescence data as a surrogate for algal pigment concentrations (for example, chla or phycocyanin), algal biomass, or other measures of an algal community. In these cases, empirical or statistical modeling can potentially be used to quantify the relation between algal fluorescence-sensor data and the desired endpoint, typically using results from discrete water-quality sample analyses. Relations must be based on site-specific data. The ability to develop a good relation depends on a variety of factors, such as those previously discussed in the “Factors Influencing Observed Fluorescence” and “Ancillary Data” sections. Flexibility in model development may be required to account for seasonal or long-term changes in the relation between field fluorescence and laboratory measurements because of changes in species composition or other factors influencing algal fluorescence response. There are several methods (regression analysis, for example) that can potentially be used to achieve model-derived computations of pigment concentrations or algal abundance in the waterbody (Roesler and others, 2017), however the techniques, methods, and reporting policy for such efforts are beyond the scope of this report. Similarly, any published time-series regression or other model that relies on discrete samples should maintain some level of sampling effort if publication of the computed result is to continue.

Computed pigment concentration or algal abundance based on fluorescence may be stored in NWIS but must be entered under a parameter code that clearly indicates the concentrations are indirect estimates. In addition, the model used to compute concentrations must be appropriately documented and published. Available NWIS parameter codes for estimated concentrations of algal pigments are listed in table 4.