nitrate interferences in fluorescence measurements of wastewater organic matter

Science of the Total Environment xxx (xxxx) xxx

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Inner filter effect, suspended solids and nitrite/nitrate interferences in fluorescence measurements of wastewater organic matter Massimiliano Sgroi, Erica Gagliano, Federico G.A. Vagliasindi, Paolo Roccaro ⇑ Department of Civil Engineering and Architecture, University of Catania, Viale A. Doria 6, 95125, Catania, Italy

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Inner filter effect (IFE) is negligible in

treated wastewater at excitation >340 nm.
IFE correction is always needed in primary wastewater effluents.
Error due to static/dynamic quenching of NO3–/NO2– is low at excitation >240 nm.
TSS produce fluorescence peaks in the tyrosine and tryptophan-like regions of EEM.
Fluorescence of tertiary wastewater after sand filtration is not affected by TSS.

a r t i c l e

i n f o

Article history: Received 21 June 2019 Received in revised form 24 September 2019 Accepted 24 September 2019 Available online xxxx Editor: Damia Barcelo Keywords: UV absorbance On-line monitoring Water quality Dissolved organic matter Particulate organic matter Wastewater treatments

a b s t r a c t In this study, it was assessed the effectiveness to correct for inner filter effect (IFE) the fluorescence spectra of several wastewaters (i.e., primary, secondary and tertiary wastewater effluents) and wastewaterimpacted surface waters using a common method based on UV absorbance measurements. In samples of secondary/tertiary wastewater effluents and surface waters, IFE was severe at excitation wavelengths <240 nm, and it was low (4–11%) at excitation wavelengths >340 nm. On the contrary, IFE has always been significant in primary wastewater effluents. After IFE correction, linear relationship was observed between fluorescence and absorbance in dilution series across the full excitation-emission matrix (EEM), although some distortions were still present. Particularly, experimental data showed the presence of static/dynamic quenching of fluorescence due to nitrite/nitrate, which cannot be corrected by IFE correction methods. Indeed, after addition of different nitrate/nitrite concentrations in wastewater (3– 40 mg/L as N), the estimated static/dynamic quenching error (QE) after IFE correction was often >20% for tyrosine and tryptophan-like fluorescence measured at excitation <240 nm. However, the QE was low (<5–10%) for fluorescence measured at excitation >240 nm. Overall, the QE increased with the increase of nitrite/nitrate concentration in wastewater. Total suspended solids (TSS) (i.e., particulate organic matter) in water produced intense fluorescence peaks in the tyrosine-like and tryptophan-like region of EEM, and TSS increased the absorbance values at all the excitation wavelengths of the UV–visible absorption spectra in unfiltered samples compared to 0.7 lm filtered samples. On the contrary, tertiary effluents employing full scale sand filtration (TSS < 2–4 mg/l) had similar UV absorbance and fluorescence spectra to 0.7 lm filtered samples. Finally, it was observed that uncorrected fluorescence intensities in the humic-like region of EEM were similar in both filtered and unfiltered samples, and it was independent of TSS concentration, dilution factor and water quality. Ó 2019 Elsevier B.V. All rights reserved.

⇑ Corresponding author. E-mail address: [email protected] (P. Roccaro). https://doi.org/10.1016/j.scitotenv.2019.134663 0048-9697/Ó 2019 Elsevier B.V. All rights reserved.

Please cite this article as: M. Sgroi, E. Gagliano, F. G. A. Vagliasindi et al., Inner filter effect, suspended solids and nitrite/nitrate interferences in fluorescence measurements of wastewater organic matter, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134663

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1. Introduction Fluorescence is a popular tool for studying and monitoring the concentration and nature of dissolved organic matter (DOM) in aquatic systems. In all natural waters, a fraction of the total dissolved organic matter pool that absorbs light also fluoresces in the UV and visible regions of the spectrum. This fluorescence is often measured across a range of excitation and emission wavelengths using excitation-emission matrix (EEM) spectroscopy, producing a 3-dimensional fluorescence intensity ‘map’ in which the presence of distinctive peaks can provide indications of sources, behavior, and biogeochemical cycling of DOM (Carstea et al., 2016; Coble, 1996). Spectroscopic surrogates based on fluorescence measurements have found large application in wastewater quality assessment as a tool for discharge detection in natural systems (Sgroi et al., 2017b) and for a continuous process control during wastewater treatments (Korshin et al., 2018). Spectroscopic surrogates have been proposed to monitor a wide range of water quality parameters during wastewater treatments, including chemical oxygen demand (COD) (Cohen et al., 2014; Sgroi et al., 2018b), biochemical oxygen demand (BOD5) (Hudson et al., 2008; Sgroi et al., 2018b), dissolved organic carbon (DOC) (Cohen et al., 2014; Shutova et al., 2014), pathogens (Baker et al., 2015; Gerrity et al., 2012), disinfection by-products (DBPs) (Li et al., 2017) and trace organic contaminants (TrOCs) (Chys et al., 2018b; Sgroi et al., 2018a). Fluorescence measurements in samples with high absorbance are common for wastewaters or wastewater-impacted surface waters and, thus, their accurate measurement is dependent on accounting for inner filter effect (IFE) (Lakowicz, 2006). As derived from the Lambert-Beer law, fluorescence intensity increases linearly with solute concentration/absorbance in water. However, this linearity is observable up to a threshold at which the fluorescence signal becomes suppressed by IFE due to the absorption of excitation (primary IFE) and emission (secondary IFE) light within the cuvette of a spectrofluorometer (Lakowicz, 2006). Hence, to obtain reliable fluorescence data, a series of methods for correcting the IFE have been developed (Kasparek and Smyk, 2018; Lakowicz, 2006; Luciani et al., 2009; MacDonald et al., 1997; Wang et al., 2017). The most commonly used method to correct DOM fluorescence spectra for IFE was proposed by Lakowicz (2006), whereby IFE is estimated from an absorbance measurement and removed algebraically (Murphy et al., 2010; Park and Snyder, 2018). Generally, proposed methods for IFE correction have been developed using selected fluorophores soluble in water and the efficacy of the correction method has been referred to the linearity between corrected fluorescence intensity and total absorbance (Atot) (i.e., sum of the absorbance of excitation and emission wavelengths) evaluated at different fluorophores concentrations (Kasparek and Smyk, 2018; Lakowicz, 2006; Luciani et al., 2009; Wang et al., 2017). However, in water samples containing DOM, several factors may cause deviation from the Lambert-Beer law, such as the presence of molecular aggregation, colloids, quenchers, enhancers or the formation of excimers (Lakowicz, 2006; Poulin et al., 2014; Wells et al., 2017). In addition, past research has demonstrated that DOM behaves as a complex mixture where charge transfer interactions play an important role (Aiken, 2014; Del Vecchio and Blough, 2004). Particularly, charge transfer interactions influence the optical properties of DOM molecules that are involved in ‘‘charge transfer complexes”, resulting in absorption and fluorescence spectra that cannot be expressed as the sum of the parts (Del Vecchio and Blough, 2004). Thus, a paramount research need is to evaluate the effectiveness of IFE correction methods in real surface water and wastewater samples. An interesting attempt in this sense has been conducted by Kothawala et al. (2013), who tested the effectiveness

of the IFE correction method proposed by Lakowicz (2006) in fluorescence measurements of dilution series of four different surface water samples. These researchers observed linearity between corrected fluorescence intensity and total absorbance across an EEM up to a value of Atot = 1.5. Nevertheless, a deeper investigation is still needed, particularly in the case of wastewater samples, for which the effectiveness of IFE correction methods has never been investigated. Indeed, wastewater organic matter has very distinct features from surface water (Hudson et al., 2007), which may be reason of significant deviation from the Lambert-Beer law. Particularly, the concentration of non-fluorescing quenchers, such as nitrite and nitrate, in wastewater is much more relevant then in surface water. Concerning this issue, it is noteworthy to highlight that methods for IFE correction have been developed to correct quenching that results from the presence of molecules that absorb light and it is not related to molecular interactions (Kasparek and Smyk, 2018; Lakowicz, 2006). On the contrary, fluorescence quenching related to the presence in the solution of chemical species, such as nitrate, nitrite or iron, results from interactions of the quenching species with the ground state of the fluorescing molecule (i.e., static quenching), and/or with the excited state of the fluorophore (.i.e., collisional/dynamic quenching) (Aiken, 2014; Poulin et al., 2014; Wells et al., 2017). Hence, errors in fluorescence measurements of DOM generated by different types of quenching need to be investigated. Generally, studies that have demonstrated the potential of fluorescence spectroscopy for water quality monitoring have been accomplished through off-line monitoring experiments, where surface water or wastewater samples have been filtered at 0.7/0.45 mm before analysis (Anumol et al., 2015; Gerrity et al., 2012; Sgroi et al., 2017a). Recently, researchers have started to investigate the use of fluorescence or UV absorbance portable devices for the in-situ and on-line monitoring of water quality at wastewater treatment plants (WWTPs) and water reuse systems (Carstea et al., 2018; Chys et al., 2018a; Mladenov et al., 2018). In this case, water sample filtration at 0.7/0.45 mm is hard to accomplish and studies addressed to evaluate the effect of suspended solids and particulate matter in these spectroscopic measurements are highly needed. Some previous studies have described fluorescence light attenuation by the presence of suspended sediments in river waters (Downing et al., 2012; Khamis et al., 2015). However, in these studies, the investigated sediments were mineral particles, which did not contain any fluorescing substances (Downing et al., 2012; Khamis et al., 2015). Thus, a paramount research need is to evaluate the contribution in fluorescence measurements of suspended solids in wastewater, which are essentially organic particulate matter not removed during wastewater treatments. To sum up, aims of the present study were: (i) to evaluate the extent of inner filter in fluorescence measurements of different wastewater qualities; (ii) to assess the effectiveness of the IFE correction method proposed by Lakowicz (2006), which is based on absorbance measurements, to correct fluorescence spectra of wastewater organic matter; (iii) to evaluate the error in fluorescence measurements related to the presence in wastewater of nitrites/nitrates, which produce static/dynamic quenching; (vi) to investigate the contribution of particulate organic matter of wastewater in fluorescence measurements.

2. Materials and methods 2.1. Tested waters and sample preparation To evaluate the extent of IFE correction using the method proposed by Lakowicz (2006), spectroscopic analyses were performed

Please cite this article as: M. Sgroi, E. Gagliano, F. G. A. Vagliasindi et al., Inner filter effect, suspended solids and nitrite/nitrate interferences in fluorescence measurements of wastewater organic matter, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134663

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on several samples collected in WWTPs (i.e., primary, secondary and tertiary wastewater effluents), and artificial lakes impacted by wastewater discharges located in Sicily (Italy). Description of the investigated aquatic systems is reported in the Data Article related to this manuscript (Sgroi et al., n.d.), whereas the main water quality parameters of the collected samples are indicated in Table 1. Furthermore, spectroscopic analyses were also performed on dilution series produced using samples collected from the primary wastewater effluent at Lentini WWTP (denoted as Lentini in Table 1), from the secondary effluents of Paternò WWTP (i.e., Paternò 1 and Paternò 2 in Table 1), from the tertiary effluent of Bronte WWTP (i.e., denoted as Bronte), and from Pozzillo Lake (i.e., Pozzillo in Table 1). All samples of wastewater effluents analyzed in this study were collected before final chlorination. All samples were stored at 4 °C with ice packs upon collection and were brought to the laboratory. Dilution series were prepared by diluting the original sample with Milli-Q water in a final volume of 100 mL. Starting from the undiluted sample, the volume of Milli-Q water added to the final volume of 100 mL was progressively increased by a step of 5/2.5 mL to obtain the dilution series. The number of dilutions of each series and the minimum volume of sample used for dilution were depended on water quality. Identical dilution series were prepared for filtered and unfiltered samples. Due to the high buffer capability of wastewater and wastewater-impacted surface water, no variation in pH across the dilutions was observed. Samples were filtered by 0.7 lm glass microfiber filters (Whatman, Clifton - NJ) pre-washed with Mill-Q water. Filtration and dilutions were immediately accomplished after samples collection and arrival in the laboratory. Analysis of water quality parameters and spectroscopic measurements were performed the same day of collection for unfiltered samples. Filtered samples were stored at 4 °C in the refrigerator and analyzed the day after.

interval of 1 nm. Excitation and emission slit widths were both set at 5 nm. Spectroscopic measurements were accomplished for filtered and unfiltered samples. The Raman scatter effect was minimized by subtracting EEMs of pure Milli-Q water from the sample EEMs; any negative intensity values produced by this subtraction were converted to zero values. Then, the emission intensity data were normalized to the Raman peak area of an emission wavelengths scan of Milli-Q water samples collected at the interval of 1 nm and related to an excitation wavelength of 350 nm to produce fluorescence intensities in Raman unit (RU). Non-trilinear data related to the Rayleigh scattering were eliminated (Murphy et al., 2010). Primary and secondary inner filter effect correction was accomplished according to the methodology proposed by Lakowicz (Lakowicz, 2006). This absorbance-based approach uses the measured absorbance at each pair of excitation (Akex) and emission wavelengths (Akem) across the EEM to convert the observed fluorescence intensity (Fobs) into the corrected fluorescence intensity (Fcorr). The sum of Akex and Akem is Atot. The formula used for inner filter correction is Eq. (1): Akex þAkem 2

F corr ¼ F obs  10

ð1Þ

Limit of reporting (LOR) for fluorescence measurements was calculated according to the methodology proposed in a previous study (Kothawala et al., 2013). Briefly, LOR was calculated for each pair of excitation-emission wavelengths from the average value (Fblank(kex,kem)) and the standard deviation (SD) of 10 individual blank EEMs, according to Eq. (2)

LORF kex;kem ¼ F blankðkex;kemÞ þ 10  SDðF blankðkex;kemÞ

ð2Þ

Analysis of ammonia, nitrite and nitrate were accomplished by colorimetric methods using the Hach Lange kits and a DR1900 spectrophotometer (Hach Lange). Analysis of other water quality parameters reported in Table 1 were accomplished according to standard methods (APHA, 2012).

2.2. Analytical methods 2.3. Assessing the effectiveness of IFE correction Ultraviolet light absorbance was analyzed using a double beam Shimadzu UV-1800 spectrophotometer (Kyoto, Japan). Absorbance spectra were measured from 200 to 800 nm at 1 nm intervals in a 1 cm quartz cuvette with Milli-Q water used as a blank. Fluorescence data were collected using a Shimadzu RF-5301PC fluorescence spectrophotometer (Kyoto, Japan) with the scanning range from excitation wavelength 220 nm–450 nm at an interval of 5 nm and emission wavelength from 250 nm to 580 nm at the

Five fluorescence peaks were selected across an EEM as representative indices of different DOM components by peak-picking method (Coble, 1996; Sgroi et al., 2017a). The excitation/emission wavelength positions of the selected fluorescence peaks are reported in Table 2. In the Data Article (Sgroi et al., n.d.), the locations of these pairs of excitation/emission wavelengths are shown within a typical EEM of wastewater samples. Furthermore, total

Table 1 Water quality parameters of tested waters. COD values for 0.7 lm filtered samples are shown in parentheses. Different samplings performed in same WWTP are denoted by different numbers.

*

Water Quality

Sample name

Conductivity (lS/cm)

pH

Ammonia (mg/L as N*)

Nitrite (mg/L as N)

Nitrate (mg/L as N)

COD (mg/L)

TSS (mg/L)

Primary effluent Primary effluent Primary effluent Primary effluent Primary effluent Primary effluent Secondary effluent Secondary effluent Tertiary effluent Secondary effluent Secondary effluent Secondary effluent Surface water Surface water Surface water

Lentini Bronte 1 Bronte 2 Adrano Taormina Letojanni Paternò1 Paternò2 Bronte Bronte 1 Bronte 2 Adrano Pozzillo Contrasto Ponte Barca

1074 – – – – – 870 1046 653 – – – 1144 549 974

7.58 7.8 7.9 7.48 7.57 7.38 7.78 7.81 7.42 7.7 7.8 7.29 7.95 8.29 8.5

31.8 21.5 23.5 44.6 5.8 11.9 2.1 <2.0 <2.0 2.8 3.2 1.9 <2.0 <2.0 <2.0

<0.6 0.4 0.5 <0.08 <0.1 <0.1 <0.6 <0.6 <0.6 0.2 0.2 0.5 <0.6 <0.6 <0.6

0.3 5.5 6.7 0.24 <0.1 <0.1 4.9 8.8 17.3 6.1 7.2 7.2 <0.2 1.56 4.73

201 (90) 387.0 756.0 378.0 128 213 23 (16) 23 (15) 18 (18) 68.3 89.5 64.0 18 (16) 10 19

80 – – – – – 94 4 2 – – – 21 – –

N = Nitrogen. Samples form secondary and tertiary wastewater effluents were collected before final chlorination

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Table 2 Fluorescence features and their coordinates selected by peak-picking method.

*

Fluorescence peak

Description

Excitation wavelength (nm)

Emission wavelength (nm)

I1 I2 I3 I4* I5

Aromatic protein, tyrosine-like fluorescence Aromatic proteins, tryptophan-like fluorescence Fulvic-like and humic-like fluorescence Microbial byproducts, proteins, biopolymers and tryptophan-like fluorescence fulvic-like and humic-like fluorescence

225 230 245 275 345

290 355 440 345 440

I4 was selected as excitation/emission 275/365 nm for Paternò2

absorbance values corresponding to the pairs of excitationemission wavelengths of fluorescence peaks I1, I2, I3, I4 and I5 were denoted as Abs1, Abs2, Abs3, Abs4 and Abs5, respectively. Using dilution series dataset, deviation from linearity of the relationship between fluorescence and absorbance of these five peaks was used to assess the effectiveness of IFE correction for different DOM components. The general relationship between fluorescence (F) and absorbance (i.e., Atot) can be described by Eq. (3) (Kothawala et al., 2013)

F ¼ q  Am tot þ b

ð3Þ

where the slope q depends on specific characteristics of fluorophores dissolved in water, b is the intercept, and m the exponent. Through a logarithmic transformation, it is possible to write Eq. (4)

b logðFÞ ¼ logðAm tot þ Þ þ logðqÞ q

ð4Þ 3. Results and discussion

In Eq. (4), the intercept b should be equal to zero after accounting for background fluorescence/absorbance via blank subtraction. Thus, it is possible to obtain Eq. (5):

logðFÞ ¼ m  logðAtot Þ þ logðqÞ

and absorbance across the EEMs of the investigated wastewaters and surface water. Further experiments to assess interferences due to static and collisional/dynamic quenching in fluorescence measurements of wastewater organic matter were performed by spiking different nitrate and nitrite concentrations in a further sample of secondary wastewater effluent collected at Paternò WWTP. Hence, defined amounts of sodium nitrate or sodium nitrite salts (ACS reagent grade, Sigma-Aldrich) were dosed in the selected wastewater. Spiked concentrations of nitrite and nitrate ranged from 3 to 40 mg/L as nitrogen (N) to investigate the fluorescence quenching effect of typical concentration of nitrate in municipal wastewater effluents. Fluorescence intensities corrected and uncorrected for IFE of samples with different addition of nitrites or nitrates were compared to a reference sample with no nitrite/nitrate spikes.

ð5Þ

In absence of IFE, the relationship between F and Atot measured at different fluorophores concentrations should be linear according to the Lambert-Beer law (Holland et al., 1977; Lakowicz, 2006). It derives that the exponent m in the Eq. (3), should be equal to 1.0. The parameter m is also the slope of the log-log relationship between fluorescence and absorbance obtained in Eq (5). Thus, when the relationship between F and Atot is truly linear, a plot of log(F) versus log(Atot) will have a slope equal to 1.0. On the contrary, if F trends downwards with increasing Atot (i.e., concave down) the slope is <1.0, whereas if it trends upwards with increasing Atot (i.e., concave up) the slope is >1.0. It is important to observe that in parts of scans where the ratio signal to noise (S/N) for fluorescence or absorbance measurements is low due to instrument limitations, background signals may cause the intercept b to be different than zero in Eq. (3). Hence, the slope of log(F) against log(Atot) may deviate from 1. Particularly, for DOM containing waters, it may happen at high excitation wavelengths where absorbance values are very low, and the sensitivity of UV absorbance measurements are highly reduced. It is important to take these considerations into account when evaluating the linearity of dilution series across a full EEM. Further details about this rationale can be found in the work of Kothawala et al. (2013). In linear and log transformed data set of EEMs of diluted series, regression coefficients were estimated by robust linear regressions with iteratively reweighted least squares fitting and a bi-square weighting function performed in Matlab (Kothawala et al., 2013). Robust linear regression differs from ordinary linear regression in that data points that deviate most from linearity receive lower weightings in the regression, making the fit more robust to the presence of random outliers (Holland and Welsch, 1977). In the present study, slopes in log-log plots were used to evaluate the deviation from linearity of the relationships between fluorescence

3.1. Extent of IFE in wastewater and wastewater-impacted surface water To evaluate the extent of IFE correction using the method proposed by Lakowicz (2006) on fluorescence measurements of the investigated wastewaters and surface waters, the relative percent difference was calculated between corrected and uncorrected fluorescence intensities for the five peaks reported in Table 2, which are indicative of different DOM components. For these calculations, only filtered and undiluted samples were considered. Fluorescence suppression was highly severe for peaks I1 and I2, which correspond to fluorescence intensities measured at low excitation wavelengths, in all tested waters (Table 3). Particularly, for these fluorescence peaks the suppression was the highest in samples collected from the final wastewater effluents, probably, due to the presence of high nitrate concentrations (Table 1). On the contrary, IFE was very low for peak I5 in secondary/tertiary wastewater effluents and surface waters. In these cases, the relative percent difference was often < 5% and always lower than 10 – 11%. It is noteworthy to observe that previous studies have indicated the need for IFE correction in natural waters when the total absorbance at a determined wavelengths pair across an EEM exceeds 0.042 (IFE > 5%) (Kothawala et al., 2013; Lakowicz, 2006; Miller, 1981). In the investigated surface waters and secondary/tertiary wastewater effluents, absorbance values of Abs5 have always been found lower than or comparable with this threshold. Nevertheless, IFE suppression for index I5 was significant and not negligible (8– 25%) in samples of primary wastewater effluents. For peaks I3 and I4, fluorescence suppression was low/moderate (7–23%) in wastewater-impacted surface waters and secondary/tertiary wastewater effluents, whereas it was high in primary wastewater effluents (24–52%). 3.2. Effectiveness of IFE correction The linearity between fluorescence intensities and total absorbance values for the five peaks selected in Table 2 was evaluated

Please cite this article as: M. Sgroi, E. Gagliano, F. G. A. Vagliasindi et al., Inner filter effect, suspended solids and nitrite/nitrate interferences in fluorescence measurements of wastewater organic matter, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134663

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M. Sgroi et al. / Science of the Total Environment xxx (xxxx) xxx Table 3 Relative percent difference (%) between peak fluorescence intensities of samples (0.7 lm filtered) corrected and uncorrected for inner filter effect. Water Quality

Sample name

Primary effluent Primary effluent Primary effluent Primary effluent Primary effluent Primary effluent Secondary effluent Secondary effluent Tertiary effluent Secondary effluent Secondary effluent Secondary effluent Surface water Surface water Surface water

Lentini Bronte 1 Bronte 2 Adrano Taormina Letojanni Paternò1 Paternò2 Bronte Bronte 1 Bronte 2 Adrano Pozzillo Contrasto Ponte Barca

Fluorescence peak I1

I2

I3

I4

I5

91 108 108 121 98 108 66 97 145 126 125 94 25 27 65

68 82 79 90 73 82 35 50 85 71 73 50 18 16 35

32 51 48 52 41 52 12 12 23 18 22 13 14 8 12

24 47 45 51 35 52 11 10 17 16 20 12 11 7 11

8 25 12 20 12 21 4 4 11 4 7 3 4 3 4

by plotting in linear scale fluorescence values (corrected and uncorrected for IFE) versus absorbance values measured in samples of dilution series of wastewater or surface water filtered at 0.7 mm. Produced plots are reported in Figs. 1-2 for Lentini and Paternò 2 waters, respectively, and in the Data Article (Sgroi et al., n.d.) for Paternò 1, Bronte and Pozzillo waters. Deviations from the theoret-

ical 1.0 slope of regression analysis of transformed dataset in log-log scale are reported in Table 4. Investigation of IFE for peaks I3, I4, I5 in filtered Bronte and Paternò1 wastewaters was not accomplished due to significant scatter of points and high x-intercept values observed in plots of fluorescence intensity versus absorbance, and probably caused by interferences with background signal.

Fig. 1. Relationship between fluorescence intensities of peaks I1, I2, I3, I4, I5 and total absorbance values for filtered samples corrected and uncorrected for inner filter effect of Lentini wastewater.

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Fig. 2. Relationship between fluorescence intensities of peaks I1, I2, I3, I4, I5 and total absorbance values for filtered samples corrected and uncorrected for inner filter effect of Paternò2 wastewater.

Table 4 Coefficients (slope m, intercept z, coefficient of determination R2, number of data points n) in robust linear regressions between the logarithm of total absorbance and logarithm of fluorescence intensity of peaks I1, I2, I3, I4, I5 for the investigated water samples corrected and uncorrected for inner filter effect. Sample

Peak

Corrected for inner-filter

Uncorrected for inner-filter

m

z

R2

n

m

b

R2

n

Lentini Paternò1 Paternò2 Bronte Pozzillo

I1 I1 I1 I1 I1

1.0476 1.0420 1.1792 1.2210 0.5515

0.9413 0.2153 0.3797 0.5195 0.7055

0.9869 0.9658 0.9430 0.9678 0.8804

20 18 24 19 12

0.8569 0.6485 0.5956 0.1729 0.4014

0.6422 0.5451 0.7543 0.9289 0.9015

0.9731 0.8894 0.7144 0.5426 0.8421

20 18 24 19 12

Lentini Paternò1 Paternò2 Bronte Pozzillo

I2 I2 I2 I2 I2

0.9432 0.9835 0.9988 1.2983 0.8312

0.7273 0.2300 0.3888 0.0612 0.1986

0.9791 0.9399 0.9308 0.9693 0.9433

20 18 24 19 12

0.8290 0.7732 0.8744 0.6936 0.6407

0.5142 0.0049 0.2151 0.3246 0.0283

0.9647 0.8924 0.9057 0.9137 0.982

20 18 24 19 12

Lentini Paternò2 Pozzillo

I3 I3 I3

0.9933 1.1844 1.1125

0.8258 1.0792 0.8295

0.9739 0.9593 0.9936

20 24 12

0.9016 1.1207 1.0213

0.6634 0.9710 0.6897

0.9073 0.9526 0.9940

20 24 12

Lentini Paternò2 Pozzillo

I4 I4 I4

1.1648 1.0632 1.0501

1.2199 0.8301 0.4163

0.9936 0.9593 0.9719

20 24 12

1.1055 1.0087 1.0034

1.0905 0.7328 0.3299

0.9951 0.9538 0.9772

20 24 12

Lentini Paternò2 Pozzillo

I5 I5 I5

1.8968 1.1988 1.6101

2.4134 1.6789 1.9371

0.8132 0.9537 0.9898

20 24 12

1.8274 1.1767 1.5778

2.3005 1.6307 1.8728

0.9101 0.9514 0.9918

20 24 12

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The highest suppression of fluorescence intensities was observed for peaks I1 and I2, where concave down curve for uncorrected fluorescence intensities was evident for all the waters (Figs. 1–2, and data in (Sgroi et al., n.d.)). In this case, the IFE correction method was able to substantially recover the linearity between fluorescence and absorbance as can be observed in plots, and by the calculation of the coefficient of determination (R2) of the performed linear regression analysis. Particularly, when IFE correction was performed, slopes of robust linear regression analysis for log-log transformed data related to peak I1 and I2 for Lentini and Paternò 1 waters, and peak I2 for Paternò 2 wastewater were found very close to the theoretical value of 1.0. Small deviations from the theoretical value of 1.0 were observed for slopes related to peak I1 (m = 1.179) in Paternò 2 wastewater and for peaks I1 (m = 1.221) and I2 (m = 1.298) in Bronte wastewater (Table 4). It is important to highlight that Bronte and Paternò 2 wastewaters were the waters with the highest nitrates concentration, which are quenching molecules that highly absorb in the range of wavelengths 220–240 nm (Buck et al., 1954), and that may have produced the observed deviations. In Pozzillo water, slopes for corrected fluorescence intensities of peak I1 and I2 remained low and equal to 0.55 and 0.83, respectively (Table 4). In this case, the obtained results can be explained by the typical low concentration of tyrosine and tryptophan-like fluorescing substances in surface water (Baker, 2001; Hudson et al., 2007), and, thus, by probable interferences with the background signal (Kothawala et al., 2013). For peaks I3, I4, I5 both corrected and uncorrected fluorescence intensities seemed to show a linear trend with the increase of the absorbance values, as shown in Figs. 1–2 and in the Data Article (Sgroi et al., n.d.). In addition, slopes of the linear regression of the log-log transformed data were close to 1.0 for both corrected and uncorrected datasets of indexes I3 and I4 for all the five investigated waters, and peak I5 for Paternò2 wastewater. Furthermore, the calculated slopes for the three indexes had very similar values,

7

and slopes of corrected samples were only slightly higher than those of uncorrected data. Slopes were generally higher than 1.0, excluding data for peak I3 of Lentini wastewater (Table 4). These observations may be interpreted as an overcorrection of the method and absence of IFE for these DOM components. However, absorbance values at wavelengths higher than 250 nm are low in wastewater and surface water, and this fact is exacerbate in diluted samples. Hence, interference with background signal may have produced positive deviation from 1.0 of the slope of log-log data and caused this misinterpretation. This fact was particularly evident for index I5. It is noteworthy to highlight that fluorescence measurements used for regression analysis were always above the calculated LOR in all the dilution series. Between these five selected waters, absorbance values >1.5, which is the threshold set by Kothawala et al. (2013) to consider IFE correction accurate, were observed only for Abs 1 in Bronte wastewater. According to Kothawala et al. (2013), absorbance values >1.5 can produce deviation from the linearity (i.e., concave up curve) resulting in an overcorrection of the method. Nevertheless, in this study, no link or thresholds between linearity of the relationship and maxima of absorbance values were observed. Slopes of log-log relationship between fluorescence and absorbance of samples corrected and uncorrected for IFE for all the pairs of excitation-emission wavelengths across an EEM are shown in Fig. 3 for three of the investigated waters. Results from Fig. 3 confirm the observations reported in Table 4, and that IFE correction was effective to substantially recover the linearity between fluorescence and absorbance for spectroscopic measurements conducted at excitation wavelengths lower than ~250 nm. At higher excitation wavelength, slopes of both corrected and uncorrected dataset were close to or higher than 1.0. Generally, slope values increased at higher excitation wavelengths due to reduced absorbance of water samples and diminished measurement sensitivity, which caused evident interferences with background signals and positively biased the regression analysis. Further reason of

Fig. 3. Slopes of log-log relationships between fluorescence and total absorbance for dilution series of filtered (0.7 lm) samples of three investigated waters: a) fluorescence intensities uncorrected for inner filter effect; b) fluorescence intensities corrected for inner filter effect. Value of slopes lower than 0.8 are colored as 0.8 value. EEM regions with few data to perform robust regression have missing slopes.

Please cite this article as: M. Sgroi, E. Gagliano, F. G. A. Vagliasindi et al., Inner filter effect, suspended solids and nitrite/nitrate interferences in fluorescence measurements of wastewater organic matter, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134663

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deviation from linearity for all the fluorescence components across an EEM may be attributed to the fact that DOM is a complex mixture, where charge transfer interactions play an important role, and where the presence of molecular aggregation, colloids, quenchers and enhancers may cause deviation from the LambertBeer law (Del Vecchio and Blough, 2004; Lakowicz, 2006). To sum up, analysis of the deviation from linearity of the relationship between fluorescence and absorbance in wastewater and wastewater-impacted surface water gives useful information about the effectiveness of the utilized IFE correction method to reinstate the linearity of this relationship in agreement with the Lambert-Beer law. This fact was particularly evident for fluorescence intensities measured at excitation wavelengths lower than ~250 nm, which correspond to regions of EEMs highly affected by IFE. However, due to biases caused by interference with background signals, and possible deviation from the Lambert-Beer law caused by the complex mixture of DOM in wastewater or wastewater-impacted surface water, this approach cannot give precise information about possible overcorrection or under correction of the utilized method. 3.3. IFE correction and nitrite/nitrate interferences As discussed above, one of the reasons of deviation from the Lambert-Beer law during spectroscopic measurements in wastewaters may be the presence of non-fluorescing quenchers (Lakowicz, 2006). Nitrite and nitrate are light absorbing molecules, which produce static and collisional quenching of fluorescence (Aiken, 2014). Nitrates and nitrites can be present in biologically treated wastewater effluents or industrial wastewaters at very high concentrations (Fernández-Nava et al., 2010; Sgroi et al., 2018b). Furthermore, the similarity between the absorbance spectra of the investigated wastewater effluents and typical absorbance spectra of nitrite and nitrate (Sgroi et al., n.d.) clearly highlight the important contribution of these molecules to fluorescence quenching in wastewaters. Nitrates have an intense absorbance band with maxima around 200 nm, and a smaller absorbance peak at 302 nm. On the contrary, nitrites show absorbance peaks at 208 nm and 354 nm (Buck et al., 1954). To assess interferences due to static and dynamic quenching produced by nitrites/nitrates in fluorescence measurements of wastewater organic matter, an additional sampling was performed at Paternò WWTP, and the collected wastewater effluent was filtered at 0.7 lm. Spikes of different concentrations of nitrate

(NO–3) and nitrite (NO–2) were accomplished in this wastewater. Then, UV absorbance and fluorescence spectra were acquired for each of the obtained samples. The wastewater sample with no spikes was considered as the reference sample. Results of the experiments are reported in Table 5 and Table 6 for the five fluorescence peaks selected in Table 2. Quenching of fluorescing signal was evaluated in spiked samples by calculation of the relative variation (%) of fluorescence intensities compared to the reference sample (Table 5–6). Fluorescence suppression was considered relevant when higher than 5%, which is the error regarded as significant for IFE correction in natural waters (Kothawala et al., 2013; Lakowicz, 2006). Since wastewaters are very complex water matrices, in this study a further threshold of 10% was established to consider acceptable the error of fluorescence measurements affected by nitrites or nitrates (i.e., quenching error % of fluorescence intensities). When considering uncorrected samples, additions of NO–3 in wastewater significantly suppressed the fluorescence of peaks I1 and I2, whereas fluorescence intensities of peaks I3, I4 and I5 were substantially unaffected (Table 5). Nevertheless, peak I4 showed 8% reduction of fluorescence intensity at the highest nitrate addition (35 mg/L as N). Spikes of NO–2 highly suppressed fluorescence peaks I1 and I2, moderately suppressed fluorescence intensities of peaks I3, I4, whereas fluorescence peak I5 was slightly suppressed (Table 6). For fluorescence peaks I1 and I2, fluorescence quenching was relevant (often >50%) even after the addition of small amounts of nitrites or nitrates. Furthermore, uncorrected fluorescing intensities of these two peaks have often been reduced below the calculated LOR. IFE correction methods have been elaborated to correct quenching of molecules that absorb light without being involved in direct interactions with chemical species that result in changes of the fluorophore energetics (Lakowicz, 2006). Hence, IFE correction methods are not intended to correct for other types of quenching, such as static and collisional quenching. These types of quenching can result from interaction of nitrates/nitrites with the ground state and excited state of organic fluorophores (Aiken, 2014). In agreement with this assumption, data in Table 5–6 clearly show that the used IFE correction method was not able to correct the quenching effect of nitrate/nitrite, and the IFE corrected fluorescence intensities affected by nitrate or nitrite spikes have always been lower than fluorescence intensities measured in the reference sample. Particularly, after IFE correction the error associated with peaks I1 and I2 has always been very high and often higher than

Table 5 Fluorescence intensities of peaks I1, I2, I3, I4, I5 and corresponding total absorbance values of wastewater samples spiked with different nitrate (NO–3) concentration. Quenching error (QE) for fluorescence intensities are calculated as relative variation (%) compared to the reference sample for samples uncorrected and corrected for inner filter effect. Relative variations 5% compared to the reference sample are featured in a bold type, whereas relative variations 10% are featured in italics. Calculated values for the limit of fluorescence reporting (LOR) are provided in the bottom. N-NO-3 spike (mg/L)

Abs1

I1 (RU)

QE (%)

Abs2

I2 (RU)

QE (%)

Abs3

I3 (RU)

QE (%)

Abs4

I4 (RU)

QE (%)

Abs5

I5 (RU)

QE (%)

49.4 61.1 75.3 90.3 95.2

0.758 1.061 1.317 1.575 2.157 2.584

0.416 0.298 0.238 0.216 0.135 0.092

– 28.4 42.8 48.1 67.6 77.9

0.096 0.109 0.119 0.128 0.153 0.175

0.762 0.788 0.758 0.759 0.764 0.747

– 3.4 0.6 0.4 0.2 2.0

0.066 0.069 0.070 0.069 0.075 0.075

0.457 0.446 0.445 0.463 0.441 0.419

– 2.6 2.7 1.2 3.7 8.4

0.012 0.012 0.012 0.011 0.013 0.012

0.918 0.920 0.920 0.931 0.930 0.920

– 0.3 0.2 1.4 1.3 0.2

22.8 21.6 47.8 66.9 73.4

0.758 1.061 1.317 1.575 2.157 2.584

0.758 0.671 0.640 0.683 0.594 0.466

– 11.5 15.5 9.9 21.6 38.6

0.096 0.109 0.119 0.128 0.153 0.175

0.841 0.878 0.851 0.858 0.879 0.873

– 4.5 1.2 2.0 4.6 3.8

0.066 0.069 0.070 0.069 0.075 0.075

0.492 0.481 0.481 0.500 0.479 0.456

– 2.3 2.3 1.6 2.6 7.3

0.012 0.012 0.012 0.011 0.013 0.012

0.930 0.932 0.932 0.943 0.943 0.932

– 0.2 0.2 1.4 1.4 0.2

Samples uncorrected for inner filter effect Reference 5 10 15 25 35

1.624 2.276 2.674 2.885 3.006 3.031

0.147 0.075 0.057 0.036 0.014 0.007

Samples corrected for inner filter effect Reference 5 10 15 25 35

1.624 2.276 2.674 2.885 3.006 3.031

LOR

0.036

0.548 0.422 0.429 0.286 0.181 0.146

0.102

0.059

0.034

0.009

Please cite this article as: M. Sgroi, E. Gagliano, F. G. A. Vagliasindi et al., Inner filter effect, suspended solids and nitrite/nitrate interferences in fluorescence measurements of wastewater organic matter, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134663

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Table 6 Fluorescence intensities of peaks I1, I2, I3, I4, I5 and corresponding total absorbance values of wastewater samples spiked with different nitrite (NO–2) concentration. Quenching error (QE) for fluorescence intensities are calculated as relative variation (%) compared to the reference sample for samples uncorrected and corrected for inner filter effect. Relative variations 5% compared to the reference sample are featured in a bold type, whereas relative variations 10% are featured in italics. Calculated values for the limit of fluorescence reporting (LOR) are provided in the bottom. N-NO-2 spike (mg/L)

Abs1

I1 (RU)

QE (%)

Abs2

I2 (RU)

QE (%)

Abs3

I3 (RU)

QE (%)

Abs4

I4 (RU)

QE (%)

Abs5

I5 (RU)

QE (%)

50.3 86.4 93.2 98.7 99.9 100.0

0.758 1.181 1.999 2.407 2.928 3.070 3.119

0.416 0.264 0.123 0.084 0.038 0.021 0.006

36.5 70.3 79.8 90.9 95.0 98.5

0.096 0.132 0.189 0.222 0.292 0.348 0.428

0.762 0.756 0.714 0.687 0.644 0.597 0.555

0.9 6.4 9.9 15.6 21.7 27.2

0.066 0.077 0.093 0.101 0.123 0.137 0.157

0.457 0.429 0.411 0.406 0.394 0.385 0.360

6.3 10.2 11.3 14.0 15.9 21.2

0.012 0.021 0.032 0.038 0.053 0.063 0.079

0.918 0.904 0.896 0.890 0.871 0.845 0.805

1.5 2.4 3.0 5.1 8.0 12.4

0.758 1.181 1.999 2.407 2.928 3.070 3.119 0.102

0.758 0.697 0.585 0.510 0.287 0.244 0.125

8.0 22.8 32.8 62.2 67.8 83.6

0.096 0.132 0.189 0.222 0.292 0.348 0.428 0.059

0.841 0.856 0.842 0.829 0.818 0.787 0.773

1.8 0.2 1.3 2.7 6.4 8.1

0.066 0.077 0.093 0.101 0.123 0.137 0.157 0.034

0.492 0.467 0.456 0.455 0.453 0.450 0.432

5.1 7.3 7.6 8.0 8.6 12.3

0.012 0.021 0.032 0.038 0.053 0.063 0.079 0.009

0.930 0.926 0.930 0.931 0.927 0.910 0.883

0.5 0.0 0.1 0.3 2.2 5.0

Samples uncorrected for inner filter effect Reference 3 10 15 20 30 40

1.624 2.124 2.924 3.022 3.082 3.086 3.107

0.147 0.073 0.020 0.010 0.002 0.000 0.000

Samples corrected for inner filter effect Reference 3 10 15 20 30 40 LOR

1.624 2.124 2.924 3.022 3.082 3.086 3.107 0.036

0.548 0.440 0.225 0.163 0.057 0.006 0.000

19.6 59.0 70.2 89.7 98.8 100.0

20%. Only exception was the error estimated for peak I2 (around 10%), when the smallest concentration of nitrite and nitrate (3– 5 mg/L as N) was added in the wastewater. On the contrary, error estimated for peak I3, I4, and I5 was generally <10% at all the investigated concentrations of nitrates/nitrites. Particularly, the error due to quenching effect was found 5% for peak I5 at all the investigated nitrite concentrations, and it was 5% for peak I3 up to a concentration of nitrites in water of 20 mg/L as N. Quenching of fluorescence intensity I4 was <5% only for a nitrite concentration in water of 3 mg/L as N. In the case of nitrate addition, a quenching error of 8% for peak I4 was reduced to 7% after correction, when it was added a nitrate concentration of 35 mg/L as N. At lower nitrate concentrations, I4 was not affected by the presence of these lightabsorbing molecules in water. Overall, it was observed that static/dynamic quenching of fluorescence due to nitrite/nitrate in wastewater cannot be eliminated by IFE correction. Particularly, the observed quenching error chan-

ged significantly with the selected pair of excitation-emission wavelengths, the dose of nitrite and of nitrate. For peaks I1 and I2, where the fluorescence suppression was very high, the observed quenching error was often >20%. In the case of moderate/slight quenching of fluorescence by nitrites/nitrates, such as the fluorescence suppression that was observed for peaks I3, I4, and I5, IFE correction resulted in a quenching error often <5%, which can be considered negligible. In the Data Article (Sgroi et al., n.d.) are reported EEM fluorescence spectra of wastewater samples with different NO–3 spikes, corrected and uncorrected for IFE. 3.4. Effect of suspended solids in fluorescence measurements Absorbance spectra of filtered and unfiltered samples for the five investigated waters during this set of experiments are shown in the Data Article (Sgroi et al., n.d.). Fluorescence spectra of filtered and unfiltered samples are reported in Fig. 4 and in

Fig. 4. EEMs non corrected for inner filter effect of a) unfiltered and b) filtered (0.7 lm) samples of Lentini and Paternò waters with indication of TSS concentration.

Please cite this article as: M. Sgroi, E. Gagliano, F. G. A. Vagliasindi et al., Inner filter effect, suspended solids and nitrite/nitrate interferences in fluorescence measurements of wastewater organic matter, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134663

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(Sgroi et al., n.d.), where plotted EEMs were not corrected for IFE. Indeed, methods for IFE correction have been developed using fluorophores soluble in water, and under conditions of validity of the Lambert-Beer law. High organic matter concentration and presence of particulate in unfiltered samples can be reason of light scattering, and significant deviation from the Lambert-Beer law, with much higher distortions than in filtered samples (Lakowicz, 2006). Hence, developed methods for IFE correction may be not appropriate for unfiltered samples. In this investigation, for a consistent comparison, both filtered and unfiltered samples were not corrected for IFE. It is interesting to observe that fluorescence EEMs of samples with a significant concentration of total suspended solids (TSS) (i.e., Lentini, Paternò1, Pozzillo) showed intense fluorescence peaks in the region of tyrosine and tryptophan-like substances in unfiltered samples, whereas fluorescence intensities in the region of humic-like compounds were similar for both filtered and unfiltered samples. On the contrary, fluorescence spectra of filtered and unfiltered samples with low TSS concentration (i.e., Paternò2, Bronte), showed similar fluorescence intensities across the whole EEM. Similar conclusions can be inferred when comparing absorbance spectra. Indeed, only waters with very low TSS concentration showed overlapped UV absorbance spectra for filtered and unfiltered samples. On the contrary, unfiltered samples with high TSS concentration had increased absorbance values (Sgroi et al., n.d.). These observations suggest that the use of fluorescence for monitoring tertiary wastewater effluents, where TSS are generally removed, does not need a previous filtration at 0.7 lm. Dilution series of filtered and unfiltered samples were produced to evaluate the effect in fluorescence measurements of particulate matter, when it is present in water at different concentrations. Particularly, IFE and light scattering effect due to TSS presence is expected to be lower in diluted samples. For this purpose, fluorescence intensities and total absorbance values of the pairs of excitation-emission wavelengths selected in Table 2 were compared for the obtained dilution series using scatter plots (Figs. 5– 6 and data in (Sgroi et al., n.d.)). Results show that fluorescence intensities and total absorbance values of both filtered and unfiltered dilution series are linearly related. The linearity of the relationship between the spectroscopic measurements in filtered and unfiltered samples is highlighted by the high R2 values of the performed linear regressions, which were often >0.9, and only in two cases lower than 0.7 (i.e., peak I2 in Paternò1 and Paternò2 waters). Generally, humic-like fluorescence peaks (i.e., I3 and I5) had higher R2 values than fluorescence intensities typical of tyrosine and tryptophan-like substances (i.e., I1, I2, I4). These latter fluorescence peaks are representative of the regions of EEM that in this study were found to be influenced by fluorescing particulate matter (Fig. 4 and data in (Sgroi et al., n.d.)). When considering dilution series in terms of total absorbance, the R2 values were often >0.9, and always >0.95 for Abs1 and Abs2. Particularly, the R2 decreased for absorbance values measured at higher excitation wavelengths. Indeed, the lowest R2 values were observed for Abs5 in all waters. At this pair of excitationemission wavelengths, organic matter absorbance in water is very low, the sensitivity of the spectroscopic measurement is reduced, and interferences with the background signal may affect the measurements at high dilution rates. Figs. 5–6 and scatter plots in the Data Article (Sgroi et al., n.d.) show that for Bronte and Paternò2 wastewaters, which are the waters with very low TSS concentration, data points of dilution series for all the investigated fluorescence peaks were very well aligned along the first bisector when comparing filtered and unfiltered samples (excluding some scattered points for I2 in Paternò2 water). Similar results are observable when comparing total absorbance values (except small deviations for Abs3, Abs4 and Abs5 val-

ues in Paternò2 water). Thus, it seems that when TSS are absent or present at very low concentration in water, 0.7 lm filtered samples and unfiltered samples have very similar fluorescence and UV absorbance spectra. On the contrary, for Lentini, Paternò1 and Pozzillo waters, where TSS concentration was not negligible, data points of dilution series for absorbance values have never been found aligned along the first bisector. Thus, a significant presence of particulate matter in water produced a moderate/important increase of absorbance values at all the excitation wavelengths of the UV–visible absorption spectra. Furthermore, the presence of TSS in these waters caused deviation from the first bisector of the regression line for fluorescence peaks I1, I2 and I4. Particularly, the deviation for peaks I2 and I4 was higher than for I1. These fluorescence peaks are typical of tyrosine and tryptophan-like substances, and correspond to regions of EEMs shown in Fig. 4 and in the Data Article (Sgroi et al., n.d.), where intense fluorescence peaks were observed in samples with high TSS content. Fluorescence intensities and absorbance values have always been higher in unfiltered samples compared to filtered samples. In addition, the slope of the linear regressions in Figs. 5–6 and in the scatter plots reported in the Data Article (Sgroi et al., n.d.), and its deviation from the value 1.0 can be considered an indicator of the impact of TSS on the UV absorbance and fluorescence measurements of water samples. Finally, it is very interesting to observe that data points of dilution series of fluorescence peaks I3 and I5 have always been aligned with the first bisector for all the investigated waters, and it was independent of TSS concentration. Although, TSS presence produced an important increase of total absorbance measurements at these pairs of excitation-emission wavelengths in unfiltered samples compared to filtered samples in Lentini, Paternò1 and Pozzillo waters, fluorescence emissions in the region of humic-like substances have always resulted unaffected by TSS. This fact was particularly surprising in the primary wastewater effluent of Lentini WWTP, where very high absorbance values can be observed. Particularly, IFE is expected to increase due to higher absorbance in unfiltered samples compared to filtered samples with following suppression of fluorescence signal if assuming that TSS do not fluoresce in the humic-like region of EEM. Otherwise, particulate matter should fluoresce the same light energy that it is absorbed in this region of EEM. In any case, for peaks I3 and I5, measured fluorescence intensities were similar for filtered and unfiltered samples in all the waters (i.e., in waters with different organic matter content and composition) and at all the dilution rates. This is the first study to compare fluorescence spectra of filtered and unfiltered wastewater samples, and an interesting behaviour of fluorescence emission in water samples containing particulate matter and suspended solids has been highlighted. To better explain the observed phenomenon, authors encourage further research. 3.5. Implications for on-line monitoring Results of this study have important implications for on-line monitoring of water quality during wastewater treatment processes by spectroscopic measurements. On-line monitoring requires the use of fluorescence sensors to be installed in situ. The use of a coupled on-line UV sensor is advisable to perform IFE correction. In this study, fluorescence peak I5 was found to be very slightly affected by IFE, when fluorescence measurements were accomplished in secondary/tertiary wastewater effluents or surface waters (Table 3). Furthermore, fluorescence measurements of peak I5 were negligibly affected by TSS concentration in water. Thus, reliable data may be obtained by fluorescence portable devices submerged in water, when measuring fluorescence at this pair of excitation-emission wavelengths. No IFE correction or removal of TSS seems to be needed when measuring this fluorescence intensity in treated wastewater.

Please cite this article as: M. Sgroi, E. Gagliano, F. G. A. Vagliasindi et al., Inner filter effect, suspended solids and nitrite/nitrate interferences in fluorescence measurements of wastewater organic matter, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134663

M. Sgroi et al. / Science of the Total Environment xxx (xxxx) xxx

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Fig. 5. Comparison between fluorescence intensities (uncorrected for inner filter) and total absorbance values in filtered and unfiltered diluted samples of Lentini wastewater (TSS = 80 mg/L). Dashed line indicates the first bisector.

Fluorescence peak I5 was shown to be a very useful fluorescence based index for tracking wastewater discharge in rivers and streams, and for monitoring the behavior of some emerging contaminants in

surface water (Sgroi et al., 2017b). Furthermore, this humic-like component was correlated with the removal of emerging contaminants during filtration of wastewater in granular activated carbon

Please cite this article as: M. Sgroi, E. Gagliano, F. G. A. Vagliasindi et al., Inner filter effect, suspended solids and nitrite/nitrate interferences in fluorescence measurements of wastewater organic matter, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134663

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Fig. 6. Comparison between fluorescence intensities (uncorrected for inner filter) and total absorbance values in filtered and unfiltered diluted samples of Bronte wastewater (TSS = 2 mg/L). Dashed line indicates the first bisector.

filters, and it was the fluorescing component that showed the slowest breakthrough compared to other fluorescing components of wastewater organic matter and to UV absorbance at 254 nm (Sgroi

et al., 2018a). Fluorescence peak I3 was unaffected by TSS presence, but affected by IFE (Table 3). Due to influence of TSS on UV absorbance measurements, filtration is needed for IFE correction.

Please cite this article as: M. Sgroi, E. Gagliano, F. G. A. Vagliasindi et al., Inner filter effect, suspended solids and nitrite/nitrate interferences in fluorescence measurements of wastewater organic matter, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134663

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Fluorescence peaks I1, I2 and I4 were affected by both IFE and TSS. Hence, a pre-filtration before measurement able to remove TSS is advisable to get reliable data, to avoid TSS interferences, and to correct for IFE. The use of portable devices based on spectroscopic measurements seems particularly suitable for the on-line monitoring of water quality during tertiary and advanced wastewater treatments (e.g., wastewater treatments at indirect potable reuse systems), where TSS have generally been removed by previous treatments. Indeed, when TSS are present at very low concentration (<2–4 mg/ L), absorption and fluorescence spectra of unfiltered and 0.7 lm filtered samples are similar. Finally, nitrate/nitrite presence in wastewater at concentration 3–5 mg/L as N is reason of high fluorescence suppression for measurements accomplished at excitation wavelength <240 nm, where the produced error is often >20%. Thus, tyrosine and tryptophan-like fluorescence indexes with excitation wavelength <240 nm cannot be used for on-line monitoring of wastewater containing nitrate/nitrite. Although the present work provides useful information to perform correct on-line monitoring of water quality by fluorescence portable devices, the current level of the development and implementation of spectroscopic methods for online/real time water quality monitoring is far from its real potential. Particularly, authors encourages further studies to investigate the effect of environmentally relevant components or parameters of wastewater, which may produce significant interference in fluorescence measurements. For example, metals such as iron, manganese and aluminum can influence DOM fluorescence and investigations to quantify and correct the produced errors are paramount. Furthermore, consistent approaches to design and operate an inherently robust sensor network for any wastewater treatment plant or reuse system needs to be pursued, together with the development of novel sensitive and stable sensors that provide multiwavelength absorbance and fluorescence, and other relevant data. Hence, all correlations observed in laboratory studies between fluorescence based surrogates and water quality parameters need to be validates by real-time and in situ measurements.

4. Conclusions In this study, several experiments were performed to assess the effectiveness of the IFE correction method proposed by Lakowicz (2006) in fluorescence measurements of wastewater and wastewater-impacted surface water samples. In this method, IFE is estimated from an absorbance measurement and removed algebraically. The effectiveness of IFE correction was also evaluated in the presence of non-fluorescing quenchers, such as nitrite and nitrate. Further objective of the study was to evaluate the effect of suspended organic solids in fluorescence measurements. Based on the results of the study, the following conclusions can be obtained:
In secondary/tertiary wastewater effluents, and in wastewater impacted surface waters, IFE was very severe for fluorescence measurements performed at excitation wavelengths <240 nm, whereas fluorescence measurements performed at high excitation wavelengths (>340 nm) were slightly affected by IFE (<5– 11%). On the contrary, IFE has always been significant for all the DOM components detected in the EEM of primary wastewater effluents;
The investigated method for IFE correction was effective to reinstate the linearity of the relationship between fluorescence and absorbance in dilution series for all the investigated DOM components, although small deviations were still observed;
Addition of nitrate/nitrite (3–40 mg/L as N) in wastewater produced static and dynamic quenching that resulted in reduced

13

fluorescence intensity, which cannot be corrected by IFE correction methods. Particularly, the observed quenching error was very high (often >20%) for tyrosine and tryptophan-like fluorescence measured at excitation wavelength <240 nm, and it was low (<5–10%) for fluorescence intensities measured at excitation >240 nm;
Presence of TSS (i.e., particulate organic matter) in water produced intense fluorescence peaks in the tyrosine-like and tryptophan-like region of EEM. Furthermore, a significant presence of TSS in water increased the absorbance values at all the excitation wavelengths of the UV–visible absorption spectra. On the contrary, water samples with low TSS concentration (2– 4 mg/L) had similar UV absorbance and fluorescence spectra to 0.7 lm filtered samples;
Uncorrected fluorescence intensities in the humic-like region of EEM were similar in unfiltered and 0.7 lm filtered samples, and it was independent of TSS concentration, dilution factor and water quality. Results of this study have important implications for on-line monitoring of water quality by fluorescence measurements. Indeed, the humic-like fluorescence index (i.e., index I5 - ex/em 345/440 nm) does not need correction for IFE, when measured in treated wastewater or surface water, and it is negligibly affected by the presence of both organic solids and nitrite/nitrate in water. Hence, under these circumstances, fluorescence portable devices can be directly submerged in water for measurement of this fluorescence intensity. To measure other fluorescence intensities across an EEM, filtration is needed to perform IFE correction or avoid TSS influence. However, on-line monitoring by spectroscopic measurements seems particularly suitable during advanced wastewater treatments, where TSS have been removed by previous treatments, and fluorescence and absorbance spectra are not affected by particulate matter. Thus, the use of a coupled on-line UV sensor is advisable to perform IFE correction. Finally, fluorescence intensities measured at excitation wavelength <240 nm may not be useful for on-line monitoring in wastewater with high nitrate/nitrite concentrations (>3–5 mg/L as N) due to severe quenching effect. Declaration of Competing Interest The authors declare that there is no conflict of interest regarding the publication of this article. Acknowledgements This study was partially funded by the University of Catania within the ‘‘Piano della Ricerca Dipartimentale 2016-2018” of the Department of Civil Engineering and Architecture, Project ‘‘Advanced treatment processes for the removal of emerging contaminants from water (PACEm)”. Authors are thankful to Davide Gionfriddo, master student at the University of Catania, for his support during sampling, analysis of water quality parameters and spectroscopic measurements. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.scitotenv.2019.134663. References Aiken, G.R., 2014. Fluorescence and dissolved organic matter: a chemist’s perspective. In: Coble, P.G., Lead, J., Baker, A., Reynolds, D.M., Spencer, R.G.M. (Eds.), Aquatic Organic Matter Fluorescence. Cambridge University Press, New York, pp. 35–74.

Please cite this article as: M. Sgroi, E. Gagliano, F. G. A. Vagliasindi et al., Inner filter effect, suspended solids and nitrite/nitrate interferences in fluorescence measurements of wastewater organic matter, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134663

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Please cite this article as: M. Sgroi, E. Gagliano, F. G. A. Vagliasindi et al., Inner filter effect, suspended solids and nitrite/nitrate interferences in fluorescence measurements of wastewater organic matter, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134663