Two experiments were designed in the SLE for relating in situ chl determinations with satellite- and CASPER-derived fluorescence measurements. In the first experiment, water samples for chl were obtained from surface waters (0–20 m depth) of different sections of the Estuary as part of an oceanographic cruise on the Coriolis II vessel and organized by ISMER during 17–22 September 2011 (Figure 1). The chosen sampling depth interval is larger than the maximum depth range contributing to the fluorescence signal detected at the surface of SLE waters. However, due to the strong vertical mixing of SLE waters [14], magnitude of fluorescence or chl is quite homogeneous in the top 20 m of the water column. Thus, no fluorescence variations due to vertical layering of chl are anticipated in this study.

Discrete sample volumes (0.5 to 1 L) for chl measurements were collected at three depths (5, 10 and 20 m) using Niskin bottles attached to an oceanographic rosette. Vertical profiles were performed in 23 locations encompassing waters west of Rimouski city (48°26′N, 68°31′W) and east of Aux-Coudres Island (47°24′N, 70°22′W). Since some sampling stations were revisited on different days, a smaller number of sampling positions are shown in Figure 1. In the second experiment, we performed a time series of chl and CASPER fluorescence measurements in the lower estuary (hereafter ‘Frascati’ station, 48.483°N, 68.533°W) (Figure 1). This site was chosen near (∼5 km) the port of Rimouski city where a temporary lab was assembled to facilitate processing of samples. We used a small boat (7 m length) for collecting surface water samples (i.e., 3 m depth) during the morning (9:00 am and 11:00 am), noon (12:00 pm) and afternoon (13:30 pm and 14:45 pm) of 9 September 2011.

Discrete fluorometric determinations of chl were performed following the acidification technique [15]. Briefly, each volume of sample (100 and 500 mL) was concentrated using a fiber glass filter (GF/F, Whatmann, mean pore diameter = 0.7 microns). Then, organic-soluble pigments were extracted with 90% acetone during 24 h. The fluorescence before and after the addition of diluted acid (HCl 5%) was measured at room temperature (25 °C) and using a single excitation-emission wavelength (λ_exc = 443 nm, λ_emiss = 680 nm) fluorometer RD-10AU (Turner designs).

The fluorescence emission spectra of water samples were measured within the size range 0.2–30 microns using the laser-based fluorometer CASPER [16]. This instrument was originally designed to study desertification and includes the following innovations: (1) it is a portable optical system that can be operated by only one person, (2) it has two laser sources one in the UV (wavelength = λ = 266 nm, 2 mW) and one in the visible spectrum (λ = 405 nm, power = 25 mW), that make possible simultaneous measurements of multiple optical components (e.g., CDOM and chl), (3) it allows discrimination between dissolved and particulate phases due to two sets of filters, one to eliminate particulates above 30 μm (Millipore optiscale capsule, polycarbonate, pore size = 30 μm, diameter = 69 mm), and another one to separate fractions below and above 0.2 μm (Sartorius, Sartobran P, mixed cellulose esters, pore size = 0.2 μm, diameter = 47 mm), (4) it provides fast determinations since each sample (particulate + dissolved) can be analyzed in less than one minute, and (5) it has a miniature detector consisting of a fiber optic spectrometer (USB2000+, 2,048 pixels, 200–1,100 nm, Ocean Optics).

Each measurement requires five steps: (1) purge and fill up of system components (tygon tubing and quartz cuvettes) with water samples, (2) background light measurement for each excitation wavelength by alternatively turning off the lasers, (3) high spectral resolution (0.3 nm) fluorescence spectra measurements at UV excitation, (4) idem as (3) but using the visible wavelength, and (5) rinsing of system components with deionized water (Milli-Q). The minimum detection level of CASPER for measuring chlorophyll is 0.1 μg·L⁻¹ at an emission wavelength of 680 nm.

Chlorophyll-fluorescence is a major pathway for dissipating energy in excess going to photosystem II or PS II (i.e., the main protein complex of photosynthesis). The ratio fluorescence/chlorophyll is variable and depends on light harvesting pigments or antenna characteristics and rate of electron flow between PS II and PS I [17]. Fluorescence/chlorophyll increases with light as reaction centers become saturated and starts decreasing beyond saturating light intensities as absorbed photons are transformed into heat due to the xanthophyll cycle. In Canadian coastal waters, Neville and Gower [18] showed for the first time a positive correlation between chlorophyll a concentration and the magnitude of sun-stimulated fluorescence at 685 nm as estimated from airborne reflectance spectra. Based on these preliminary results, Borstad et al.[19] suggested the use of FLH for estimating chl even though there is a large variability of FLH/chl (0.01 to 0.08 mW·cm⁻²·sr⁻¹·μm⁻¹·mg·Chlorophyll⁻¹) between different water types. In simple terms, FLH is defined as follows: (1) FLH = L F − kL S − ( 1 − k ) L L (2) k = ( λ L − λ F ) ( λ L − λ S ) − 1where L_F, L_S and L_L are the radiance measurements coinciding with the chlorophyll fluorescence peak (L_F) and spectral limits (lower, L_S and higher, L_L) defining FLH, and λ_S, λ_F and λ_L are the corresponding wavelengths. The linear baseline between L_S and L_L is weakly influenced by atmospheric components such as oxygen and water vapor [10,20], and other optical components when waters are classified as case I (i.e., phytoplankton covaries with other optical components) [9,10]. Thus, most of the variability of FLH in oceanic waters can be attributed to phytoplankton abundance and physiology [9,10,20]. However, in case II waters, the use of FLH for estimating chl may be questionable or useless depending on the contribution of CDOM and detritus to the light attenuation in the fluorescence excitation and emission spectral ranges [9].

Response of FLH to different chl values was analyzed based on Raman-normalized (wavelength = 468 nm) CASPER spectra (hereafter FLH^CM). Raman normalization is intended to eliminate CASPER fluorescence variability due to changes in light source intensity or water temperature. FLH^CM was computed based on MERIS (MEdium Resolution Imaging Spectrometer) radiometric channels along with their respective bandwidths (L_S = 665 ± 10 nm, L_F = 681 ± 7.5 nm, and L_L = 709 ± 10 nm). Unlike MODIS (Moderate Resolution Imaging Spectroradiometer), spectral location of radiometric channels in MERIS is more suitable for chlorophyll fluorescence in terms of signal/noise ratio (1.4 fold higher) [20]. In addition, the use of FLH instead of single wavelength (e.g., centered at 680 nm) chl indices is justified by the fact that remotely sensed (airborne or spaceborne) FLH calculation minimizes elastic scattering contribution originated from water and atmospheric components [10,20].

Samples for FLH-chl experiments were obtained during a time series performed at the Frascati station (see Section 2.1), and from phytoplankton lab cultures. For the second comparison, we prepared four chl dilutions (8.2, 28.7, 64.8 and 309.3 mg·m⁻³) from the same culture of microalgae Nannochloropsis oculata. This Eustigmatophyceae species was cultivated using f/2 medium and samples were collected during the exponential phase of growth.

The analysis of spaceborne ocean color information allowed us to investigate three main questions: (1) How do satellite chl estimates based on CASPER-chl relationships compare with global ocean color products or local parameterizations developed using concurrent ship and satellite-derived fluorescence data?, (2) What is the influence of CDOM and detritus on chl estimates computed from FLH measurements?, and (3) How alike are regional spatial patterns of fluorescence- vs. L_w-based chl in the SLE?

The raw and processed fluorescence signal as measured by the CASPER spectrofluorometer and corresponding to samples obtained during the Frascati time series is shown in Figure 2(A,B). As expected, variability of the raw CASPER emission spectra (i.e., non-corrected by elastic contributions from the excitation source and inelastic water contributions from Raman) in the spectral range 380–760 was mainly attributed to changes in phytoplankton pigments when chl values varied between 1 and 2 mg·m⁻³ (Figure 2(A)). In fact, signal modifications due to the laser source performance (18.2% at λ = 405 nm) or water temperature variability (13.8% at λ = 468 nm) was small with respect to phytoplankton-related fluorescence (41.5% at λ = 680 nm). In addition, it is worthy to emphasize the spectral shift of the emission peak of chlorophyll (∼4 nm) to longer wavelengths when chl was doubled (Figure 2(B)). The relationship between chl and FLH^CM measurements based on in situ measurements or phytoplankton culture samples was linear for a chlorophyll concentration range between 1.1 and 309.3 mg·m⁻³ (Figure 2(C)). Regression slope coefficients were different from zero for both functionalities; however the magnitude of m for CASPER and Turner comparisons based on phytoplankton samples growing in controlled conditions was 2.7-fold greater (i.e., the fluorescence per unit of chl was 2.7-fold smaller) with respect to that computed using field samples (chl = FLH^CM (33.4 ± 9.3) + 0.13 ± 0.34, r² = 0.81, N = 5).

Since our intention here is to propose a fluorescence-based index and not an absolute metrics of chl that can be applied to optical satellite measurements over the SLE, and given the limited range of pigment values used when comparing field samples, we decided to use the chl-FLH^CM function obtained from phytoplankton cultures for modeling spaceborne chl based on fluorescence line height estimates.

The direct relationship between in situ chl and satellite fluorescence was supported by shipboard surveys performed during 21 September 2011 (Figure 3). Given the range of pigment concentrations measured during the cruise (0.2 to 5.8 mg·m⁻³), a non-linear variation should be expected between averaged surface (0–20 m depth) chl determinations and MODIS-Aqua nFLH values. However, this effect could not be detected since only 3 of 23 cruise stations coincided in time and space with satellite measurements. The resulting linear regression function was developed with samples obtained across the lower estuary, had a regression slope greater than 1 (P < 0.05), and was characterized by a regression intercept not different from zero (P > 0.05). Despite the small time difference between MODIS-Aqua (17:10 GMT) and MERIS (16:53 GMT) overpasses during 28 September 2011, the linear covariation between chl^SM and chl^CASPER was relatively weak (r² = 0.335) (Figure 4(A)). Nevertheless, the co-dependency between chl^SM and chl^CASPER values was greater with respect to that seen between CASPER and L_w-based chl indices (r² up to 0.255, Figure 4(C)). Notice that the number of valid data points differ between regression curves due to geographic differences in algorithm performance. In general and based on the regression slope, chl^CASPER was underestimated and overestimated with respect to chl^SM (m > 1, P < 0.05, Figure 4(A)) and chl^NN (m < 1, P < 0.05, Figure 4(D)), respectively. The regression intercept was in all cases greater than one but for chl^CASPER − chl^SM and chl^CASPER − chl^oc3 where I was not different from zero (P < 0.05). The hyperbolic regression model only converged when chl^CASPER was related with chl^GSM and chl^NN (Figure 4(C,D); Table 2), and presented values of r² that reflected a slight improvement on explaining residual variability (up to 7%) with respect to the linear regression functions.

The spatial distribution chl^CASPER values throughout the SLE and during noon of September 28, 2011, was very irregular with values changing up to one order of magnitude (2.27–21.5 mg·m⁻³) (Figure 5(A)). Highest pigment concentrations (>20 mg·m⁻³) were found north of the MTZ and near the mouth of the most important north shore rivers draining in the lower part of the St Lawrence Estuary (Saguenay, Manicougan and Outardes). Intermediate to high values (1–10 mg·m⁻³) were observed south of the Outardes Delta and over the main channel, and around the archipelago extending along the south shore of the lower estuary (Figure 1). These spatial patterns were also observed in satellite images collected 15 days before or after September 28 (data not shown). In general and consistent with Figure 4, the ratio between chl^CASPER and chl^oc3, chl^GSM and chl^NN over the whole SLE varied 134, 48 and 860-fold, respectively (Figure 5(B–D)). Lower chl^oc3 values with respect to CASPER estimates were mainly computed in the upper estuary (ratio < 0.5) (Figure 5(B)). Conversely, oc3 pigment predictions overestimated CASPER (ratio > 20) over waters near the coast of the north and south shores of the lower estuary. In general, chl^GSM and chl^CASPER estimates followed comparable spatial patterns even though large differences were observed in specific locations of the south shore (ratios < 0.2) and waters north-east of Rimouski city (ratios > 5) (Figure 5(C)). Lastly, the spatial distribution of chl^NN showed relatively low values (ratio up to 0.005) with respect to chl^CASPER in a large portion of the Estuary including upper and lower sections, and mostly coinciding with deeper waters along the Laurentian Channel (Figure 5(D)).

These remarkable differences in chl indices may lead to ambiguous spatial patterns of phytoplankton along and across the Estuary. In fact, latitudinal and longitudinal changes in chlorophyll during the September 2011 cruise showed no increase toward the southwest, a trend clearly defined by MODIS-Aqua or MERIS fluorescence measurements (Figure 6(A–D)). Notice that these discrepancies were also present even after analyzing satellite scenes corresponding to other days during September. Differences in large scale spatial patterns were also striking between satellite fluorescence and global products of chl. In general, latitudinal and longitudinal distribution of chl^oc3 magnitude was quite constant compared to those associated with satellite fluorescence (Figure 6(C–F)). Geographic variability of chl^GSM and satellite fluorescence was comparable even though this correspondence tended to disappear over the westernmost locations of the upper estuary (Figure 6(E)). Conversely, the chl^NN showed an inverse spatial variation with respect to nFLH, and consequently was a very misleading index for mapping phytoplankton distributions in the SLE (Figure 6(F)).

Relationships between satellite-derived nFLH, CDM, and TSM values during 28 September 2001 are shown in Figure 7. In general, CDM and TSM presented higher values over areas influenced by river discharge and turbidity plumes (e.g., NE of the MTZ in the upper estuary and along the north shore in the lower estuary) (Figure 7(A–B)). The highest concentrations of CDM (up to 5.7 m⁻¹, north shore) and TSM (up to 20 g·m⁻³, MTZ) were found in the same sub-regions where maximum values of chl^CASPER (>20 mg·m⁻³) and nFLH (up to 0.20 mW·cm²·μm⁻¹·sr⁻¹) were obtained (Figure 5(A) and Figure 7(C,D)). Likewise, the lowest CDM (<0.1 m⁻¹) and TSM (<1 g·m⁻³) values were detected in those locations characterized by the lowest chl^CASPER (<3 mg·m⁻³) and nFLH (<0.005 mW·cm²·μm⁻¹·sr⁻¹) values (e.g., central portion of lower estuary) (Figures 5(A) and 7(A,B)). In general, the magnitude of nFLH varied independently from CDOM variability when CDM magnitude was less than 0.3 m⁻¹ (e.g., all sub-regions). Also, nFLH appeared to be positively associated with CDOM at CDM values greater than 0.5 m⁻¹ (e.g., north shore and MTZ) even though the slope of this trend was not different from zero (P > 0.05) (Figure 7(C)). Conversely, nFLH was positively related with TSM when TSM concentrations were relatively low (<2 g·m⁻³) (all sub-regions, r² = 0.20, nFLH = 0.028 TSM (8·10⁻⁴, 1 standard error) + 0.006 (9·10⁻⁴), N = 4719, Figure 7(D)). Likewise, this correspondence was weaker at greater TSM values (2–10 g·m⁻³), and disappeared at TSM greater than 10 g·m⁻³ when nFLH reached a relatively constant value around 0.1 mW·cm²·μm⁻¹·sr⁻¹.

Lab measurements using CASPER demonstrated that fluorescence emission at a wavelength of 680 nm varied less with respect to chlorophyll changes. Indeed at chl >0.5 mg·m⁻³, a variation in chlorophyll of 100% only corresponded with a 42% relative variation of normalized fluorescence line height. At higher pigment concentrations (i.e., chl > 0.5 mg·m⁻³), light excitation is less efficient due to partial absorption of emitted fluorescence and a smaller phytoplankton optical cross section (i.e., a_ph/chl smaller) associated with a greater ‘packaging effect’ [20]. Another interesting feature of the CASPER fluorescence spectra was the spectral shift of the phytoplankton fluorescence peak at higher chl values. This phenomenon was previously reported based on field measurements [9,34] and is related with a spectral shift to longer wavelengths on L_w and driven by a greater contribution of elastic scattering with respect to chlorophyll fluorescence [9]. Certainly, this spectral shift can not be detected using conventional fluorometers based on a single-wavelength excitation. Thus, we suggest that CASPER and other comparable spectrofluotrometers are a better choice to calibrate in-water and satellite fluorescence-based models of chl.

The relationships found between CASPER-based nFLH and chl samples (field and derived from cultures) are not expected to be substantially influenced by the fluorometric method (i.e., with acidification) or type of fluorometer (excitation wavelength = 436 nm) used in this study. This statement is supported by previous experiments in the SLE during spring 2000 and 2001, and comparing chl measurements based on non-acidification method (chl^WEL) [35] or High-Pressure liquid Chromatography (chl^HPLC) with fluorometric determinations (chl^acid = (0.74 ± 0.05, 1 standard error) chl^HPLC +(0.09 ± 0.25), r² = 0.80, N = 70; chl^acid = 0.88 chl^WEL ± 0.03+(0.06 ± 0.15), r² = 0.93 N = 70) performed using the same technique and optical instrument (chl^acid) [36].

Although a direct relationship was always observed between FLH^CM and chl, the fluorescence yield per unit chlorophyll was lower when experiments were performed with phytoplankton cultures. The discrepancy was likely attributed to the greater light levels used in the incubated samples with respect to those characteristic of field measurements. PAR (i.e., photosynthetic available radiation, wavelength = 400–700 nm) above a threshold of 1.0·10⁻⁴ mol·quanta·m⁻²·s⁻¹ can be photo-inhibitory and tends to lower fluorescence/chl [27]. Averaged PAR of incubated samples was 3.5·10⁻⁴ mol·quanta·m⁻²·s⁻¹ or more than 3-fold greater than theoretical in situ estimates computed as <PAR> = PAR(0+)/(H_mix K_PAR) [37], where PAR(0+) is the PAR at noon and incident at the sea surface during 28 September 2011 (1.04·10⁻³ mol·quanta·m⁻²·s⁻¹), H_mix is the averaged mixed layer depth (10 m), and K_PAR is the lowest vertical light attenuation coefficient for PAR measured in the SLE (1.1 m⁻¹). Therefore, the above calculations suggest potential modifications on fluorescence/chl in phytoplankton cultures due to excessive light.

Variations in fluorescence between experiments due to changes in micronutrients concentration, water temperature, taxonomic differences, and CDOM are anticipated to be secondary due to the following facts. As specified by Turner designs, the variation of acetone-extracted chlorophyll fluorescence due to temperature is 0.3%/°C. This effect may introduce a negligible error on FLH calculation (up to 3%) if we consider a maximum temperature shift of 10 °C between field and lab measurements.

Based on published reports, the phytoplankton culture used in this study (N. oculata) has a fluorescence emission spectrum that is comparable to those associated with bacillariophytes (i.e., diatoms) [38]. Thus, since diatoms dominate over the lower St Lawrence Estuary (i.e., where field samples were collected for CASPER comparisons) [39], uncertainties on CASPER fluorescence curves due to phytoplankton composition are likely minor. In general, phytoplankton growth is not nutrient-limited in the SLE [40], thus nutrients should not have a major influence on phytoplankton fluorescence in our experiments.

The linear behavior between chl and FLH^CM variations seemed to contradict empirical and theoretical parameterizations reported by other studies [9,20,33]. However, we attribute this deviation to the lack of ‘packaging effect’ and re-absorption of photons as a function of chl in the phytoplankton culture experiments. As chl increases, the a_ph* tends to decrease and re-absorption of photons tends to increase creating a saturation plateau on fluorescence values [20, 27]. This phenomenon did not occur in our phytoplankton cultures since we worked with diluted samples (i.e., same abundance per volume) and phytoplankton cells were exposed to similar light conditions during the incubation.

Although chl^CASPER and chl^SM were both based on fluorescence line height changes, the empirical model developed with MODIS-Aqua and shipboard measurements only corresponded with CASPER estimates in 34% of the cases (Figure 4(A)). In addition, both variables did not follow a 1:1 relationship. This is not surprising given three main reasons: (1) the increase in fluorescence variability in field samples with respect to laboratory measurements, due mainly to changes on a_ph* (Equation (2) in [20]), (2) differences between spectral bands used in MODIS-Aqua and CASPER-MODIS models to compute FLH (Figure 1 in [20]), and (3) bias attributed to spectral changes in excitation light source (i.e., laser in CASPER vs. sun-induced in MODIS or MERIS) and receiver’s differences (i.e., detector response of CASPER vs. satellite sensors). As highlighted in Figure 4(C), a variation in chl^CASPER was followed by a comparable rate of change of chl^GSM even though the regression intercept suggested a ‘residual’ pigment as computed by the GSM model when chl^CASPER was 0 mg·m⁻³. This pigment offset was attributed to a greater CDOM interference on chl^GSM with respect to chl^CASPER. CDOM is a dominant optical component that is responsible for absorbing most of the blue light in the SLE [7]. Thus, in CDOM-rich waters, the total light absorption at 443 nm (i.e., maximum absorption peak for chlorophyll) increases, resulting in an overestimation of chl when this pigment is measured using remote sensing reflectance ratios. To illustrate this effect we have that chl is inversely proportional to R1 = R_rs(443)/R_rs(550) = (a(550) b_b(443))/(a(443) b_b(550)), where a and b_b are total absorption (i.e., water + particulates + CDOM) and backscattering coefficients at the corresponding wavelengths. Therefore, if chl and b_b(443)/b_b(550) are constant, and magnitude of a_CDOM(443) is larger, 1/R1 becomes smaller and chl is higher.

Why did chl^oc3 and chl^NN not have a good correspondence with chl^CASPER? Unlike the other global products of chl, the performance of chl^oc3 is highly influenced by CDOM interference since it uses less spectral information than the other models to tease out the optical contribution of phytoplankton from that associated with CDOM. The lack of covariability between chl^NN and chl^CASPER in the SLE is not surprising since chl^NN was originally developed with specific neural network coefficients obtained from European coastal waters. In fact, this neural network is initialized with TSM measurements made over the North Sea and the Baltic Sea. Thus, a large uncertainty on chl^NN can be expected between the SLE and European coastal environments due to changes on SPM optical properties between different water bodies and associated to variations on organic content and size distribution of particulates. As shown in Table 2, a hyperbolic function was a more accurate regression model for relating chl^CASPER with global chl products. These results support the non-linear behavior between chl and fluorescence, and corroborate semi-empirical relationships suggested by Gower’s and Gilerson’s studies [9,20].

The use of discrete shipboard measurements for characterizing large scale patterns of phytoplankton in surface waters of the Saint Lawrence Estuary may be misleading if oceanographic surveys are inadequate in terms of spatial coverage or density of sampling locations. Likewise, satellite measurements based on L_w may not be useful for mapping horizontal variations of chlorophyll in this environment if global ocean color products (e.g., chl^NN) are not locally validated. In that regard, we demonstrated the superior ability of spaceborne FLH measurements for deriving more realistic distributions of chl in SLE waters.

Consistent with ‘in situ’ historical fluorescence measurements [11], chl^CASPER presented maximum values (i.e., >10 mg·m⁻³) in three areas of the Estuary: north of Aux-Coudres Island in the upper Estuary (47.7°N, 69.9°W, I), nearby Forestville city (48.55°N, 69.145°W, II), and the Manicougan Peninsula (49.07°N, 68.19°W, III) in the north shore of the lower Estuary. These high chl spots are likely created by downstream advection and resuspension of benthic diatoms (I) [41], divergence fronts (II) [42], and river plumes (III) [43]. Unlike other studies, the analysis of chl^CASPER fields during September 28 also suggested relatively high pigment concentrations in the proximity of the Saguenay Fjord entrance and along the opposite coast extending to 48.49°N, 68.55°W. These sites correspond with major tidal flats environments (e.g., Bay Sainte-Anne, Berte Island, Figure 1) and an active transport of sediments including attached microalgae [41].

Although chl^CASPER and chl^SM are more suitable metrics for describing variations of chlorophyll in the SLE with respect to global ocean color products, the interpretation of geographic variability of chlorophyll based on satellite fluorescence may be compromised if one or more of the following factors play an important role altering FLH: (1) inaccurate atmospheric correction, (2) modified phytoplankton physiological parameters affecting quantum yield of fluorescence (ΦF) and/or a_ph*, and (3) influence of high concentration of detritus and/or CDOM on fluorescence excitation/emission spectra. Regarding the first factor, we compared MERIS top-of-the atmosphere (TOA) reflectance measurements over the SLE with simulated TOA values computed using a radiative transfer model (6SV, [44]) and local meteorological information obtained from AERONET and airport weather stations located at Mont Joli and Quebec City. During 28 September 2011, measured TOA radiance due to aerosols accounted for 20 and 40% of total radiance at λ_S and λ_L, respectively. Satellite-derived optical thickness of aerosols at 869 nm, the most important uncertainty in atmospheric models, was relatively small and invariable (0.03 to 0.08) suggesting relatively low concentration of aerosols over the study area. Preliminary results show a maximum bias of 25% (29.9% at λ_S, 20.8% at λ_L) between simulated and measured TOA reflectance. Thus, we conclude that atmospheric correction of ocean color products used in this study was satisfactory and its uncertainty is relatively small with respect to the nFLH variation observed in the SLE (several orders of magnitude, see Figures 6(C,D) and 7(C,D)). However, satellite measurements of nFLH under turbid atmospheres and high solar zenith angles are expected to be more inaccurate by increasing the detection limit of chl retrievals up to 2.6-fold [10].

With reference to the second factor, nFLH is expected to change in a direct way with respect ΦF and a_ph* (Equation (2) in [27]). Previous investigations have shown that the magnitude of a_ph* during April–May 2000 and 2001 cruises in the SLE varied between 2.8 and 6.4-fold, respectively [45]. Therefore, in our study area, the impact of this variability for satellite chlorophyll fluorescence should be secondary. The influence of ΦF on nFLH is more difficult to assess over the SLE since there are no direct in situ measurements of ΦF (e.g., based on fast rate pulse fluorometers). However, no major changes on ΦF are expected in our study area given the lack of nutrient-limiting conditions for phytoplankton growth [43]. Based on radiative transfer simulations, Gilerson et al.[9] concluded that ΦF is relatively constant and equal to 1% in coastal waters having chl values ranging from 1 to 50 mg·m⁻³.

Reduction in nFLH due to non-photochemical quenching may occur if phytoplankton cells are exposed to damaging solar radiation levels [27]. However, mid-day fluorescence depression due to photo-inhibition is unlikely in our satellite-derived nFLH values (see Section 4.1) unless waters are relatively shallow (bottom depth <10 m) and clear (K_PAR < 1 m⁻¹). These environments are spatially constrained as they only represent less than 10% of the SLE surface area [46]. The third factor affecting nFLH mainly relates to the interference exerted by dominant optical water constituents other than pigmented particulates. The analysis of relationships between the CDM and nFLH for different regions of the SLE did not support a hypothetical suppression of phytoplankton fluorescence due to CDOM-mediated spectral overlap of chlorophyll excitation wavelengths as proposed by other studies [9]. This apparent discrepancy was probably attributed to the larger effect of detritus-derived elastic scattering with respect to CDOM light attenuation on nFLH variability.

In general, nFLH and TSM were directly related over different waters of the SLE (Figure 7(D)). However, this correspondence varied for different TSM intervals (m = 0.028 for TSM = 0.5 and 2 g·m⁻³ and m = 0 for TSM = 2.5 to 20 g·m⁻³). TSM includes a variable contribution of algal particulates, thus we suggest that nFLH variability is dominated by microalgae at lower TSM values (e.g., south shore and upwelling regions). Conversely, the leveling off of nFLH at higher TSM values could be interpreted as an increase in detritus mass with respect to microalgae (e.g., MTZ and north shore). The aforementioned impact of detritus on chlorophyll fluorescence peak and FLH has been investigated by Gilerson et al.[9,33] who showed that chl explains one-third of FLH variability when concentrations of non-algal particulates (NAP) are relatively low (<2 g·m⁻³). However, FLH may not be an accurate proxy for chl when NAP concentrations are above 2 g·m⁻³ and becomes an useless index at NAP concentrations above 10 g·m⁻³ due to the overwhelming contribution of elastic scattering (>95%) with respect to fluorescence. In this study, we estimate a NAP range between 0.5 (e.g., upwelling and south shore) and 20 g·m⁻³ (e.g., MTZ and north shore) [7], thus our chl^CASPER index may be used in the SLE only in those locations characterized by relatively low to moderate turbidity and having concentrations of total suspended particulate matter below 13 g·m⁻³.