Absorbance and EEM fluorescence of wastewater: Effects of filters, storage conditions, and chlorination

Journal Pre-proof Absorbance and EEM fluorescence of wastewater: Effects of filters, storage conditions, and chlorination Massimiliano Sgroi, Erica Gagliano, Federico G.A. Vagliasindi, Paolo Roccaro PII:

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CHEM 125292

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Please cite this article as: Sgroi, M., Gagliano, E., Vagliasindi, F.G.A., Roccaro, P., Absorbance and EEM fluorescence of wastewater: Effects of filters, storage conditions, and chlorination, Chemosphere (2019), doi: https://doi.org/10.1016/j.chemosphere.2019.125292. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Title page information

Title: Absorbance and EEM fluorescence of wastewater: effects of filters, storage conditions, and chlorination Authors:

Massimiliano Sgroi Department of Civil Engineering and Architecture, University of Catania, Viale A. Doria 6, 95125, Catania, Italy. Email: [email protected]

Erica Gagliano Department of Civil Engineering and Architecture, University of Catania, Viale A. Doria 6, 95125, Catania, Italy. Email: [email protected]

Federico G.A. Vagliasindi Department of Civil Engineering and Architecture, University of Catania, Viale A. Doria 6, 95125, Catania, Italy. Email: [email protected]

Paolo Roccaro Department of Civil Engineering and Architecture, University of Catania, Viale A. Doria 6, 95125, Catania, Italy. Email: [email protected] Phone: +39 0957382716; fax: +390957382748

First author: Massimiliano Sgroi

Corresponding author: Paolo Roccaro

Declarations of interest: none

Fluorescence measurement interferences

Storage conditions

Effect of filters

Chlorination

H-O-Cl

Fluorescence (RU)

Fluorescence (RU)

Fluorescence (RU)

-O-Cl

1

Absorbance and EEM fluorescence of wastewater: effects of filters,

2

storage conditions, and chlorination

3

Massimiliano Sgroi, Erica Gagliano, Federico G.A. Vagliasindi, Paolo Roccaro*

4

Department of Civil Engineering and Architecture, University of Catania, Viale A. Doria 6,

5

95125, Catania, Italy

6

*

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[email protected]

8

ABSTRACT

9

Aim of this study was to delineate sample handling procedures for accurate fluorescence and UV ab-

10

sorbance measurements of wastewater organic matter. Investigations were performed using different

11

wastewater qualities, including primary, secondary and tertiary wastewater effluents, and a

12

wastewater-impacted surface water. Filtration by 0.7µm glass microfiber filter, 0.45µm polyvi-

13

nylidene fluoride (PVDF) membrane, 0.45µm cellulose nitrate membrane, and 0.45µm polyeth-

14

ersulfone (PES) syringe filter released manufacture impurities in water that affected fluorescence

15

measurements. However, pre-washing of filter by Milli-Q water was able to eliminate these interfer-

16

ences. Different storage conditions were tested, including storage of filtered and unfiltered samples

17

under different temperatures (25°C, 4°C, -20°C). According to the obtained results, the best practice

18

of wastewater samples preservation was sample filtration at 0.7/0.45 µm immediately after col-

19

lection followed by storage at 4°C. However, the time of storage that assured changes of these

20

spectroscopic measurements that do not exceed the 10% of the original value was dependent on

21

water quality and selected wavelengths (i.e., selected fluorescing organic matter component). As

Corresponding author. Phone: +39 0957382716; fax: +390957382748; e-mail address: procca-

22

a general rule, it is advisable to perform fluorescence and UV absorbance measurements as soon

23

as possible after collection avoiding storage times of filtered water longer than 2 days. Finally,

24

addition of chlorine doses typical for wastewater disinfection mainly affected tryptophan-like

25

components, where changes that exceed the 10% of the fluorescence intensity measured in the

26

unchlorinated sample were observed even at very low doses (≥ 1 mg/L). On the contrary, tyro-

27

sine-like and humic-like components showed changes < 10% at chlorine doses of 0.5–5 mg/L.

28

Keywords: UV absorbance; water quality; dissolved organic matter; chlorine; excitation-

29

emission matrix; standard protocol

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1. Introduction

31

Fluorescence is the release of energy in the form of light when molecules or moieties, named

32

fluorophores, are excited with a high-energy light source (Lakowicz, 2006). It has become a

33

popular tool for studying and monitoring the concentration and nature of dissolved organic mat-

34

ter (DOM) in aquatic systems (Aiken, 2014; Carstea et al., 2016; Korshin et al., 2018). Fluores-

35

cence is often measured across a range of excitation and emission wavelengths using excitation-

36

emission matrix (EEM) spectroscopy and producing a 3-dimensional fluorescence intensity

37

‘map’ in which the presence of distinctive peaks can provide indications of sources, behavior,

38

and biogeochemical cycling of DOM (Carstea et al., 2016; Coble, 1996). Fluorescence is an at-

39

tractive method because data collection is straightforward, fast, inexpensive, reagentless, highly

40

sensitive and non-invasive (Murphy et al., 2010; Park and Snyder, 2018). Particularly, fluores-

41

cence can be readily employed in laboratory studies to investigate DOM composition (Aiken,

42

2014; Tran et al., 2015), and it can be employed, in situ, allowing for the collection of environ-

43

mental data in real-time (Carstea et al., 2018; Mladenov et al., 2018). Due to the abovementioned

44

advantages, in the past decades, several studies have investigated the potential of fluorescence

45

spectroscopy as a monitoring tool of water quality in natural and engineered systems (Korshin et

46

al., 2018; Sgroi et al., 2017b; Yu et al., 2016). Particularly, spectroscopic surrogates have been

47

proposed to monitor a wide range of water quality parameters during water and wastewater

48

treatments, including chemical oxygen demand (COD) (Cohen et al., 2014; Sgroi et al., 2018b),

49

biochemical oxygen demand (BOD5) (Hudson et al., 2008; Sgroi et al., 2018b), dissolved organic

50

carbon (DOC) (Cohen et al., 2014; Shutova et al., 2014), pathogens (Baker et al., 2015; Gerrity

51

et al., 2012), disinfection by-products (DBPs) (Li et al., 2017; Roccaro et al., 2009; Roccaro and

52

Vagliasindi, 2012; Yang et al., 2015), trace organic contaminants (TrOCs) (Anumol et al., 2015;

53

Chys et al., 2018; Gerrity et al., 2012; Sgroi et al., 2018a, 2017a), and estrogenic activity (Huang

54

et al., 2019). Furthermore, fluorescence spectroscopy has been utilized to characterize changes of

55

DOM during the on-line chemical cleaning of membranes in membrane bio-reactors (Cai et al.,

56

2017, 2016; Sun et al., 2018).

57

However, the ease of data collection and the potentially powerful applications of fluorescence

58

spectroscopy belie the inherent complexity of the method. Indeed, fluorescence measurements of

59

DOM are influenced by artifacts (bias and error) from a range of sources, including the sample

60

constituents (concentration and matrix effects), sample acquisition, handling, and measurement

61

(Lakowicz, 2006; Murphy et al., 2010; Park and Snyder, 2018; Sgroi et al., 2019). Even though

62

use of fluorescence spectroscopy is emerging and increasingly draws attention for water quality

63

assessment and monitoring, to date only few studies have suggested standard methods and proto-

64

cols for samples handling and preservation before analysis, which may assure correctness of the

65

performed measurements (Schneider-Zapp et al., 2013; Spencer et al., 2007; Spencer and Coble,

66

2014; Wang et al., 2015). Particularly, these studies have investigated preservation of freshwater

67

and seawater samples, and have missed to describe the storage effect on wastewater organic mat-

68

ter. It was indicated that storage of 0.7/0.45 µm filtered samples at 4°C is a suitable procedure to

69

keep stable the fluorescence intensities of DOM for 7 days in surface waters (Spencer and Coble,

70

2014), whereas longer periods (i.e., from few months to 1 years) of storage are allowed for sea-

71

water samples (Schneider-Zapp et al., 2013; Spencer and Coble, 2014; Wang et al., 2015). Fur-

72

thermore, studies on sample handling do not recommend addition in water of antimicrobial pre-

73

servatives and oxidant quenching agents, acidification/poisoning, and freezing/thawing proce-

74

dures due to observed interference in fluorescence measurements (Park and Snyder, 2018;

75

Spencer et al., 2007; Spencer and Coble, 2014; Wang et al., 2015). Concerning fluorescence

76

measurements of wastewater organic matter, a recent study evaluated the effect of storage time

77

in total fluorescence (i.e., the summation of regionally integrated fluorescence intensities under

78

the whole EEM surface) measurements of a secondary wastewater effluent (Park and Snyder,

79

2018). Authors concluded that changes of total fluorescence values do not exceed 10% of the

80

original signal when samples are stored at 4°C over a period of 21 days. Nevertheless, a deeper

81

investigation is still needed to evaluate the effect of storage time and temperature in different

82

wastewater qualities (primary, secondary and tertiary wastewater effluents) at selected pairs of

83

excitation-emission wavelengths that are indicative of different DOM components, which are

84

characterized by very different biodegradability (Cohen et al., 2014; Sgroi et al., 2017a).

85

Furthermore, DOM characterization by fluorescence spectroscopy needs sample filtration at

86

0.7/0.45 µm before analysis. Filters of different materials, including polyethersulfone (PES)

87

membrane, polyvinylidene fluoride (PVDF) membrane, cellulose nitrate membrane or glass mi-

88

crofiber, are available in the market. However, investigations of possible interference in fluorescence

89

measurements due to presence of leaching substances from filters have never been accomplished.

90

Further reason of interference may be the presence of chlorine in disinfected wastewater effluents.

91

Hence, there is the need to evaluate differences in the EEM spectra between chlorinated and unchlo-

92

rinated wastewater secondary effluents to provide indication about correct sampling procedures at

93

full-scale wastewater treatment plants (WWTPs) before spectroscopic analyses. Therefore, this study

94

aims to delineate sample handling procedures for accurate fluorescence measurements in wastewater

95

samples. Specific objectives are: (i) to evaluate the effect of filtration, time and temperature of stor-

96

age in fluorescence analysis of different wastewater qualities, including primary, secondary and ter-

97

tiary wastewater effluents and of a wastewater impacted surface water; (ii) to evaluate the inter-

98

ference of leaching substances from filters of different material in fluorescence spectroscopy;

99

(iii) to assess the effect of chlorine doses typical for disinfection in fluorescence measurements

100

of wastewater organic matter.

101

2. Materials and methods

102

2.1. Tested waters

103

Experiments in this study were performed using samples of four aquatic systems located in Sicily

104

(Italy). Samples were collected from the primary wastewater effluent at Lentini WWTP, from the

105

final effluents of Paternò WWTP and Bronte WWTP, and from Pozzillo Lake, which is a

106

wastewater-impacted surface water. Comparing the two final wastewater effluents investigated

107

in this study, it is noteworthy to highlight that the wastewater collected at Bronte WWTP was a

108

tertiary effluent treated by sand filtration, whereas wastewater samples at Paternò WWTP were

109

collected after the secondary settling. Descriptions of the investigated aquatic systems are report-

110

ed in the Data Article related to this manuscript (Sgroi et al., n.d.), whereas the main water quali-

111

ty parameters are indicated in Table 1. Samples of the final effluent were collected before final

112

chlorination at the investigated WWTPs.

113

2.2. Analytical methods

114

Ultraviolet light absorbance was analyzed using a Shimadzu UV-1800 spectrophotometer (Kyo-

115

to, Japan). Absorbance spectra were measured from 200 to 800 nm at 1 nm intervals in a 1 cm

116

quartz cuvette with Milli-Q water used as a blank.

117

Fluorescence data were collected using a Shimadzu RF-5301PC fluorescence spectrophotometer

118

(Kyoto, Japan) with the scanning range from excitation wavelength 220 nm to 450 nm at an in-

119

terval of 5 nm and emission wavelength from 250 nm to 580 nm at the interval of 1 nm. Excita-

120

tion and emission slit widths were both set at 5 nm. The Raman scatter effect was minimized by

121

subtracting EEMs of pure Milli-Q water from the sample EEMs; any negative intensity values

122

produced by this subtraction were converted to zero values. Then, the emission intensity data

123

were normalized to the Raman peak area of an emission wavelengths scan of Milli-Q water sam-

124

ples collected at the interval of 1 nm and related to an excitation wavelength of 350 nm to pro-

125

duce fluorescence intensities in Raman unit (RU). Non-trilinear data related to the Rayleigh scat-

126

tering were eliminated. Inner filter effect correction was accomplished according to the method-

127

ology proposed by Lakowicz (2006).

128

Limit of reporting (LOR) for fluorescence measurements was calculated according to the meth-

129

odology proposed in a previous study (Kothawala et al., 2013). Briefly, LOR was calculated for

130

each pair of excitation-emission wavelengths from the average value (Fblank(λex,λem)) and the

131

standard deviation (SD) of 10 individual blank EEMs, according to Eq. 1.

132


 , =  (, ) + 10 ∙ ( (, ) )

133

EEM of the calculated LOR values is shown in the Data Article (Sgroi et al., n.d.). Analysis of

134

water quality parameters reported in Table 1 were accomplished according to standard methods.

135

Total chlorine was measured using Hach DPD kit.

136

2.3. Filter material tests

137

To assess interferences in fluorescence measurements related to sample filtration at 0.7/0.45 µm,

138

blank samples were generated filtering Milli-Q water by different filter materials. Particularly,

139

100 mL of Milli-Q water were filtered by 0.7 µm glass microfiber filters (diameter 47 mm)

140

(Whatman, Clifton - NJ), other 100 mL of Milli-Q water were filtered by 0.45 µm polyvinyli-

141

dene fluoride (PVDF) membrane (diameter 47 mm) (EMD Millipore), and further 100 mL of

142

Milli-Q water were filtered by 0.45 µm cellulose nitrate membrane (diameter 47 mm) (Whatman,

143

Clifton - NJ). Filtration was accomplished by a laboratory filtration apparatus. In this case, 100

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mL represents a typical volume of sample filtered for laboratory analysis. In addition, 10 mL of

145

Milli-Q water were filtered by 0.45 µm polyethersulfone (PES) syringe filter (diameter 25 mm)

146

(Whatman, Clifton - NJ). A smaller volume of water was used to test filtration by PES mem-

147

brane because syringe filters are generally used to filter small amount of water. All described

148

tests were performed in triplicate using three different filter units for each tested material. UV

149

absorbance and fluorescence spectra were acquired for all the produced blank samples.

150

Finally, the described tests were repeated following the same procedure, but using pre-washed

151

filters. Particularly, glass microfiber filters, PVDF membrane filters and cellulose nitrate mem-

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brane filters were pre-washed filtering 100 ml of Milli-Q water, whereas PES syringe filters were

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pre-washed by 20 ml of Milli-Q water.

(1)

154

2.4. Sample storage tests

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Five fluorescence peaks were selected across an EEM as representative indices of different DOM

156

components by peak-picking method (Coble, 1996; Sgroi et al., 2017a). The excitation/emission

157

wavelength positions (λex/λem nm) of the selected fluorescence peaks were I1 = 225/290 nm; I2 =

158

230/355 nm; I3 = 245/440 nm; I4 = 275/345 nm; I5 = 345/440 nm. Description of the selected

159

fluorescing components is reported in the Data Article (Sgroi et al., n.d.). Effect of time and

160

temperature storage in fluorescence measurements was evaluated for these selected five fluores-

161

cence peaks as well as for total fluorescence (ΦT). ΦT was calculated integrating the volume un-

162

der the whole EEM surface according to published literature (Chen et al., 2003).

163

After each sampling event, the collected water was immediately brought to the laboratory, and

164

split in four amber glass bottles of 1L volume. Water stored in two of these amber glass bottles

165

was previously filtered at 0.7 µm by glass microfiber filters. UV absorbance and fluorescence

166

spectra were acquired the same day of the collection for filtered and unfiltered samples. Then,

167

one bottle of filtered water and one bottle of unfiltered water were stored at 4°C in the dark. The

168

remaining two bottles were stored at room temperature (~ 25 °C) in the dark. Further 20 mL ali-

169

quots of filtered and unfiltered water were stored in freezer at -20 °C. The obtained filtered and

170

unfiltered samples, which were stored at different temperatures, were denoted as “Unfiltered

171

Room”, “Unfiltered 4 °C”, “Filtered Room”, “Filtered 4°C”, “Unfiltered -20 °C”, “Filtered -20

172

°C”. The described procedure was accomplished for each of the investigated water qualities.

173

Sub-sampling from the bottles was performed after established times of storage. Particularly,

174

sub-sampling from bottles of the Lentini primary effluent was performed after 1, 5, 8, 12, 16, 21

175

days of storage. Sub-sampling for Bronte tertiary wastewater effluent was performed after 1, 5,

176

8, 14, 19 days of storage. Sub-sampling for Paternò secondary wastewater effluent was per-

177

formed after 2, 4, 9, 14, 21 days of storage. Sub-sampling for Pozzillo surface water was per-

178

formed after 1, 4, 8, 11, 15, 21 days of storage. After sub-sampling, the water was allowed to

179

warm up to room temperature prior to perform spectroscopic measurements. Samples stored at -

180

20°C were analyzed for fluorescence and UV absorbance measurements after 21 days of storage

181

for all the waters (19 days for Bronte wastewater). Even in this case, the water was allowed to

182

warm up to room temperature prior to performing spectroscopic measurements.

183

2.5. Chlorination tests

184

Stock solution of sodium hypochlorite (NaClO) was prepared in the laboratory at concentration

185

of 1000 mg/L diluting in Milli-Q water commercial NaClO solution (10-14% weight percent-

186

age). Then, small volumes of prepared NaClO solution, ranging from 50 µL to 800 µL, were

187

spiked in 100 mL of wastewater samples to achieve chlorine concentration in water of 0.5, 1, 2,

188

5, 8 mg/L. Addition of such small volumes of NaClO solution in 100 mL of wastewater samples

189

had negligible effects on DOM concentration. After 5 h reaction time, when all the added chlo-

190

rine was depleted, wastewater samples were filtered at 0.7 µm by glass microfiber filters. Thus,

191

spectroscopic measurements were accomplished for all the produced samples. Spectroscopic

192

measurements were also accomplished using unchlorinated filtered samples for comparison.

193

For the described chlorination tests, wastewater samples were collected from the unchlorinated

194

secondary effluents at Paternò WWTP and Lentini WWTP. At Lentini WWTP, a further sample

195

was collected from the chlorinated final effluent to compare chlorination performed in the labor-

196

atory to chlorine disinfection accomplished at full scale.

197

3. Results and discussion

198

3.1. Filter material interference in fluorescence measurements

199

As previously described, to assess interferences in fluorescence measurements related to sample

200

filtration at 0.7/0.45 µm, defined volumes of Milli-Q water were filtered by different filter mate-

201

rials. Three different filter units were tested for each of the selected filter materials. In the Data

202

Article (Sgroi et al., n.d.) are reported fluorescence spectra for all the replicate tests, whereas UV

203

absorbance spectra are shown in Figure 1. Leaching of fluorescing substances was evident for

204

each tested material, and the fluorescing intensities of filter leachate was different even between

205

filters of the same material. It was probably related to presence of different amounts of manufac-

206

ture impurities in different filters. In Figure 2 are shown the differences between EEM of filtered

207

Mill-Q water and calculated LOR. To calculate these differences, the EEM that showed the high-

208

est fluorescence intensities between triplicate measurements for each investigated material was

209

used. These calculated differences can give important indication about the extent of interference

210

related to sample filtration in fluorescence measurements.

211

The highest intensity for fluorescing filter leachate was measured after filtration of Milli-Q water

212

by PES syringe filter, where fluorescence intensities greater than 1.5 RU were measured (Sgroi

213

et al., n.d.). Measured fluorescence intensities were very high in the leachate of these filters and

214

comparable with fluorescence values measured in natural waters (Sgroi et al., 2017b). Reason of

215

the high measured fluorescence intensity may be also related to the use of a smaller volume of

216

water for filtration (10 mL) compared to other filters tested in this study, and, thus, to a low dilu-

217

tion in Mill-Q water of leaching substances. However, syringe filters are designed to filter small

218

sample volumes, and, thus, the use of this filter can cause severe distortion of fluorescence

219

measurement of aquatic DOM. Samples of Mill-Q water filtered by PES membrane showed a

220

significant absorption band in the range of wavelengths 200 - 250 nm as well (Figure 1).

221

Glass microfiber filters leached substances with fluorescing intensities little higher than LOR in

222

the tryptophan-like region of EEM measured at excitation wavelength lower than 250 nm,

223

whereas PVDF membrane produced a fluorescence signal slightly higher than LOR in the tyro-

224

sine-like and in the humic-like region of EEM. On the contrary, filtration by cellulose nitrate

225

membrane produced fluorescence intensities that affected in a significant extent tyrosine-like,

226

tryptophan-like and humic-like regions of EEM (Figure 2). When comparing UV absorbance

227

spectra, the lowest values were measured for glass microfiber filters (Figure 1).

228

However, pre-washing with Mill-Q water was able to reduce leaching of UV light absorbing

229

substances and fluorescing substances from all tested filter materials. Particularly, EEMs of Mil-

230

li-Q water filtered after pre-washing have always showed fluorescence signals significantly re-

231

duced and with intensities lower than calculated LOR values. As an example, in the Data Article

232

(Sgroi et al., n.d.) are reported fluorescence and UV absorbance spectra of Mill-Q water filtered

233

by a pre-washed PES syringe filter. To sum up, filter pre-washing by Mill-Q water is mandatory

234

prior to performing spectroscopic measurements of DOM containing water/wastewater samples.

235

In rare case, when pre-washing is not possible, it is advisable to filter samples by glass fiber fil-

236

ters, which produced the lowest interferences in fluorescence and UV absorbance measurements

237

among the tested filters.

238

3.2. Effect of storage condition on fluorescence EEM

239

Aim of this study was to investigate sample preservation in different storage environments and

240

conditions to assure accurate fluorescence and UV absorbance measurements. Particularly, ef-

241

fects of 0.7 µm filtration and storage temperature were tested to define the best practices for

242

sample preservation. Investigations were performed on very different wastewater qualities, in-

243

cluding primary, secondary and tertiary wastewater effluents, and on a wastewater impacted sur-

244

face water. In the Data Article (Sgroi et al., n.d.) are reported several figures that depict for all

245

the investigated water qualities the comparison of value changes of fluorescence peaks I1, I2, I3,

246

I4, I5 (i.e., fluorescence indexes indicative of different DOM components) and ΦT with respect to

247

time in samples stored filtered or unfiltered at different temperatures (i.e., room temperature,

248

4°C, -20°C). In the Data Article (Sgroi et al., n.d.) are shown changes observed in the UV ab-

249

sorbance spectra for the same waters and storage conditions as well. In Table 2 are reported the

250

coefficient of variation (%) for the abovementioned fluorescence indexes and UV absorbance at

251

254 nm (UV254) calculated for the measurements accomplished during the testing period and for

252

each storage condition.

253

For all tested waters, smaller changes of UV absorbance and fluorescence intensities were ob-

254

served in filtered samples compared to unfiltered samples (Table 2). Indeed, filtration is known

255

to be able to remove bacteria from water (Koivunen et al., 2003) and, thus, to reduce degradation

256

processes of organic matter. Particularly, filtration was the preeminent factor to reduce changes

257

in spectroscopic measurements due to storage as can be observed in Table 2. A temperature stor-

258

age of 4 °C improved samples preservation compared to storage at room temperature. These ob-

259

servations were validated for all the investigated fluorescence peaks, ΦT and UV absorbance

260

measurements (Table 2). Bronte wastewater was the water that showed the smallest changes of

261

fluorescence indexes and UV absorbance under different storage conditions and over the entire

262

testing period. Bronte WWTP is able to perform high oxidation rate during biological treatment,

263

and produces an effluent of good quality, which is further treated by sand filtration (Table 1).

264

The DOM in this wastewater is mainly derived from microbial soluble product, which is charac-

265

terized by low biodegradability (Tran et al., 2015). On the contrary, high variations of spectro-

266

scopic measurements were observed in unfiltered samples of the other investigated water quali-

267

ties, even after one day of storage. These latter water qualities have a greater fraction of rapidly

268

biodegradable organic matter and higher bacteria concentration, which were not removed by fil-

269

tration. Thus, it is very important to filter samples for these latter waters immediately after col-

270

lection to assure effective preservation. If a research laboratory is interested in performing spec-

271

troscopic measurements of particulate organic matter in water (Lee et al., 2019), these measure-

272

ments should be accomplished the same day of collection. Spectroscopic measurements accom-

273

plished at different time of storage showed both increasing and decreasing changes (Sgroi et al.,

274

n.d.).

275

Figure 3 depicts the comparison of changes of fluorescence intensity of peaks I1, I2, I3, I4, I5 and

276

total fluorescence (ΦT) respect to time between sample storage at 4°C and room temperature for

277

0.7 µm filtered samples of Lentini primary wastewater effluent. In this graph, changes in fluores-

278

cence intensities are compared to values corresponding to ±5% and ±10% of the fluorescence

279

values measured on the day of collection. Similar graphs for the other investigated water quali-

280

ties are reported in the Data Article (Sgroi et al., n.d.), whereas the same comparison for UV254 is

281

depicted in Figure 4. When considering fluorescence indexes for filtered samples stored at 4 °C,

282

observed changes have always been within the 10% of the original values over the entire testing

283

period of 21 days for Bronte and Pozzillo waters (exception were indexes I1 and I2 in Pozzillo

284

surface water). For Lentini wastewater, examined fluorescence indexes changed less than 10%

285

over the entire testing period if excluding measurements accomplished after 20 days of storage,

286

and peak I4. This latter index is indicative of tryptophan-like fluorescence substances, which are

287

characterized by high biodegradability (Carstea et al., 2016; Sgroi et al., 2017a), and it exceeded

288

the 10% of the original value after only 8 days of storage. In Paternò secondary wastewater ef-

289

fluent, all fluorescence indexes exceeded the 10% of the original values after 4 days of storage,

290

excluding ΦT that showed higher values after 2 days of storage, and peak I5, which, on the con-

291

trary, has never exceeded the 10% of the value measured on the day of collection. When consid-

292

ering UV absorbance measurements accomplished for filtered samples stored at 4 °C, UV254

293

changes exceeded 10 % of the original value after 5 days in Lentini and Paternò wastewater, and

294

after 11 days in Pozzillo surface water. UV254 values have always remained within the 10% of

295

the original value for filtered samples of Bronte wastewater.

296

In this study, changes of fluorescence intensities and UV absorbance after 21 days of storage at

297

-20°C (frozen samples) were also evaluated for both filtered and unfiltered samples. However,

298

obtained results showed important variation from the original values for all the investigated spec-

299

troscopic indexes, and these storage conditions were not ameliorative of storage at 4 °C (Sgroi et

300

al., n.d.). These obtained results were in agreement with studies that investigated freeze/thaw ef-

301

fects in fluorescence measurements of freshwater and seawater samples (Spencer et al., 2007;

302

Wang et al., 2015).

303

To sum up, the best practice of samples preservation for fluorescence and UV absorbance meas-

304

urements of wastewater organic matter is to filter samples at 0.7/0.45 µm immediately after col-

305

lection, and then store samples at 4 °C. However, the time of storage that assures changes of the-

306

se spectroscopic measurements that do not exceed the 10% of the original value is dependent on

307

water quality and selected wavelengths. As a general rule, we advise to perform spectroscopic

308

measurements as soon as possible after collection, and preferably to store samples no longer than

309

2 days.

310

3.3. Effect of chlorine disinfection on fluorescence EEM of wastewater organic

311

matter

312

Previous studies have evaluated the effect of chlorine addition in wastewater showing reduction

313

of fluorescence intensities after disinfection (Hambly et al., 2010; Murphy et al., 2011). Howev-

314

er, in the cited studies high doses of chlorine (i.e., super-chlorination conditions) were used for

315

final disinfection, and often wastewaters were treated by advanced treatment before the final

316

chlorination (Hambly et al., 2010; Murphy et al., 2011). In those studies reduction even higher

317

than 50% were observed for all the fluorescence components detected in the EEM spectra

318

(Hambly et al., 2010; Murphy et al., 2011). In the present study, we evaluated the effect of chlo-

319

rine disinfection in secondary municipal wastewater effluents (i.e., wastewater treated exclusive-

320

ly by conventional biological treatments with activated sludge unit) adding typical chlorine dos-

321

es, which are generally used before wastewater discharge in surface water (range 0.5 – 8 mg/L).

322

It was observed that changes of tyrosine-like and humic-like indexes (i.e., peaks I1, I3, I5) have

323

never exceed the 10% of the fluorescence intensity measured in the unchlorinated sample at

324

chlorine doses of 0.5 – 5 mg/L, as shown in Figure 5 and in the Data Article (Sgroi et al., n.d.).

325

On the contrary, when a chlorine dose of 8 mg/L was added in water, almost all the investigated

326

fluorescence indexes exceeded the abovementioned 10% in both the examined wastewaters. It

327

was also observed that chlorine produced the highest changes in the tryptophan-like components

328

of wastewater organic matter, as shown by the calculation of the coefficients of variation report-

329

ed in the Data Article (Sgroi et al., n.d.). Particularly, in Paternò wastewater effluent, fluores-

330

cence peaks I2 and I4 exceeded the 10% of the fluorescence intensity measured in the unchlorin-

331

ated sample at chlorine doses ≥ 1 mg/L (Figure 5). On the contrary, UV absorbance values

332

measured at 254 nm have never exceeded the 10% of the value measured in the unchlorinated

333

sample of Lentini and Paternò wastewaters (Sgroi et al., n.d.). Finally, sample of the chlorinated

334

effluent collected at Lentini WWTP showed very similar fluorescence and UV absorbance values

335

to the unchlorinated sample (Sgroi et al., n.d.). Thus, when sampling the final effluent of a

336

wastewater treatment plant, it should be considered that chlorination may have an important ef-

337

fect on the measured fluorescence spectra, particularly in the tryptophan-like region of EEM.

338

Particularly, if absorbance and fluorescence are used as surrogate to study the formation of disin-

339

fection by-products (DBPs), unchlorinated secondary effluents should be collected because very

340

small changes in organic matter cause by chlorination are associated to DBPs generation

341

(Roccaro et al., 2009; Roccaro and Vagliasindi, 2012).

342

4. Conclusions

343

In this study, several sample handling strategies were investigated in order to define useful guide-

344

lines for performing accurate fluorescence and UV absorbance measurements of wastewater organic

345

matter. Furthermore, the effect of chlorine disinfection in wastewater for these spectroscopic meas-

346

urements was evaluated. Based on the results of the study, the following conclusions can be obtained:

347

•

Leaching of fluorescing manufacture impurities in Milli-Q water was observed from 0.7

348

µm glass microfiber filter, 0.45 µm polyvinylidene fluoride (PVDF) membrane, 0.45 µm

349

cellulose nitrate membrane, and 0.45 µm polyethersulfone (PES) syringe filter. Particu-

350

larly, the highest interferences in fluorescence measurements were observed when filter-

351

ing Milli-Q water by PES syringe filter, and cellulose nitrate membrane;

352 353

•

Pre-washing of filter by Milli-Q water was able to reduce to negligible extent interferences in fluorescence measurement due to presence of filter leachate for all tested filters;

354

•

Filtration of wastewater/surface water samples at 0.7/0.45 µm immediately after collec-

355

tion, and storage at 4°C represent the best practices for sample preservation before fluo-

356

rescence and UV absorbance analyses. However, the time of storage that assures changes

357

of these spectroscopic measurements that do not exceed the 10% of the original value is

358

dependent on water quality and selected wavelengths. Generally, samples should be

359

stored no longer than 2 days;

360

•

Unfiltered samples showed very high changes of measured fluorescence intensities and

361

UV absorbance spectra even after one day from the collection, and it was independent of

362

storage temperature;

363

•

364 365

Storage of wastewater samples in freezer at -20 °C was not an ameliorative condition compared to storage at 4 °C for both filtered and unfiltered samples;

•

Chorine disinfection significantly affected tryptophan-like components of wastewater or-

366

ganic matter even at low chlorine doses (i.e., ≥ 1 mg/L). On the contrary, tyrosine-like

367

and humic-like components of EEM spectra exceed the 10% of the fluorescence intensity

368

measured in the unchlorinated sample only at chlorine doses ≥ 8 mg/L. Finally, UV ab-

369

sorbance values measured at 254 nm have never exceeded the 10% of the value measured

370

in unchlorinated samples in all the performed chlorination experiments.

371

Supplementary data

372

Supplementary data are described in the related Data Article (Sgroi et al., n.d.).

373

Acknowledgements

374

This study was partially funded by the University of Catania within the “Piano della Ricerca Di-

375

partimentale 2016-2018” of the Department of Civil Engineering and Architecture, Project “Ad-

376

vanced treatment processes for the removal of emerging contaminants from water (PACEm)”.

377

Tables

378

Table 1. Water quality parameters of tested waters. COD values for 0.7 µm filtered sam-

379

ples are shown in parentheses.

380 381

Table 2. Coefficient of variation (%) of fluorescence intensity for peaks I1, I2, I3, I4, I5, total

382

fluorescence (ΦT) and UV254 measured at different days of storage for all the investigated

383

water qualities. Calculations were accomplished using average values of replicate meas-

384

urements. Unfiltered and 0.7 µm filtered samples were stored at 4°C or at room tempera-

385

ture.

386 387 388 389 390 391 392 393 394 395

396 397 398 399 400

Figures

401

Figure 1. UV absorbance spectra of triplicate Milli-Q water after filtration with different

402

filters.

403 404

Figure 2. Difference between EEMs of filtered Mill-Q water and calculated LOR.

405 406

Figure 3. Comparison of changes of fluorescence intensity of peaks I1, I2, I3, I4, I5 and total

407

fluorescence (ΦT) respect to time between sample storage at 4°C and room temperature for

408

0.7 µm filtered samples of Lentini primary wastewater effluent. Dotted lines indicate values

409

corresponding to ± 5% from the fluorescence intensity measured the day of collection. Con-

410

tinuous lines represent values corresponding to ± 10% from the fluorescence intensity

411

measured the day of collection. Error bars indicate the minimum and maximum values

412

from duplicate measurements.

413 414

Figure 4. Comparison of changes of UV absorbance at 254 nm respect to time between

415

sample storage at 4°C and room temperature for 0.7 µm filtered samples of all the investi-

416

gated waters. Dotted lines indicate ± 5% from the fluorescence intensity measured the day

417

of collection. Continuous lines represents ± 10% from the fluorescence intensity measured

418

the day of collection. Error bars indicate the minimum and maximum values from dupli-

419

cate measurements.

420 421

Figure 5. Comparison of changes of fluorescence intensity of peaks I1, I2, I3, I4, I5 and total

422

fluorescence (ΦT) respect to addition of different doses of sodium hypochlorite (chlorine

423

concentration of 0, 0.5, 1, 2, 5, 8 mg/L) in the secondary wastewater effluent of Paternò

424

WWTP. Spectroscopic measurements were accomplished after 5 hours of reaction time.

425

Dotted lines indicate ± 5% from the fluorescence intensity measured in the unchlorinated

426

sample. Continuous lines represents ± 10% from the fluorescence intensity measured in the

427

unchlorinated

sample.

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27

Table 1. Water quality parameters of tested waters. COD values for 0.7 µm filtered samples are shown in parentheses. Water Conductvity Matrix (µS/cm) Lentini 1074 Paternò 1046 Bronte 653 Pozzillo 1135 * LOD = limit of detection

pH 7.58 7.81 7.42 8.27

Ammonia (mg/L) 31.8 < LOD* < LOD < LOD

Nitrite (mg/L) < LOD < LOD < LOD < LOD

Nitrate (mg/L) 0.3 8.8 17.3 0.4

COD (mg/L) 201 (90) 23 (15) 18 (18) 25 (12)

TSS (mg/L) 80 4 2 169

Table 2. Coefficient of variation (%) of fluorescence intensity for peaks I1, I2, I3, I4, I5, total fluorescence (ΦT) and UV254 measured at different days of storage for all the investigated water qualities. Calculations were accomplished using average values of replicate measurements. Unfiltered and 0.7 µm filtered samples were stored at 4°C or at room temperature. Unfiltered Room 4 °C Room Lentini primary wastewater effluent (n = 7) I1 80.0 63.8 111.7 I2 49.9 72.4 29.0 I3 38.2 41.0 13.5 I4 55.1 47.8 35.6 I5 23.4 31.7 9.6 ΦT 54.8 62.1 34.5 UV254 55.0 21.0 7.0 Paternò secondary wastewater effluent (n = 6) I1 16.4 14.5 7.2 I2 34.0 16.5 17.2 I3 10.0 11.1 15.6 I4 41.8 15.0 10.4 I5 6.7 7.7 8.4 ΦT 19.5 11.6 15.1 UV254 18.5 5.7 8.8 Bronte tertiary wastewater effluent (n = 6) I1 10.0 8.4 6.7 I2 11.6 7.0 9.1 I3 3.7 7.3 5.3 I4 10.3 6.4 4.3 I5 3.0 4.3 4.1 ΦT 7.4 6.8 4.5 UV254 6.8 5.2 3.7 Pozzillo surface water (n = 6) I1 55.2 20.7 29.7 I2 44.0 18.3 4.6 I3 7.0 8.7 4.2 I4 33.2 17.6 6.3 I5 6.4 8.6 6.3 ΦT 20.3 12.3 4.2 UV254 20.1 19.7 7.4 n = number of measurements utilized for the calculation of the coefficient of variation Spectroscopic index

Filtered 4 °C 8.6 9.7 6.5 14.3 6.0 6.26 7.1 8.8 9.6 9.1 8.5 6.3 12.8 8.2 4.0 4.5 3.5 4.7 1.9 2.6 3.6 18.9 7.0 3.6 4.9 4.1 3.4 8.9

Glass microfiber (0.7 µm)

PVDF (0.45 µm)

Cellulose nitrate (0.45 µm)

PES (0.45 µm)

Figure 1. UV absorbance spectra of triplicate Milli-Q water after filtration with different filters.

Glass microfiber (0.7 µm)

PVDF (0.45 µm)

Cellulose nitrate (0.45 µm)

PES (0.45 µm)

Figure 2. Difference between EEMs of filtered Mill-Q water and calculated LOR.

Figure 3. Comparison of changes of fluorescence intensity of peaks I1, I2, I3, I4, I5 and total fluorescence (ΦT) respect to time between sample storage at 4°C and room temperature for 0.7 µm filtered samples of Lentini primary wastewater effluent. Dotted lines indicate values corresponding to ± 5% from the fluorescence intensity measured the day of collection. Continuous lines represent values corresponding to ± 10% from the fluorescence intensity measured the day of collection. Error bars indicate the minimum and maximum values from duplicate measurements.

Bronte

Lentini

Paternò

Pozzillo

Figure 4. Comparison of changes of UV absorbance at 254 nm respect to time between sample storage at 4°C and room temperature for 0.7 µm filtered samples of all the investigated waters. Dotted lines indicate ± 5% from the fluorescence intensity measured the day of collection. Continuous lines represents ± 10% from the fluorescence intensity measured the day of collection. Error bars indicate the minimum and maximum values from duplicate measurements.

Figure 5. Comparison of changes of fluorescence intensity of peaks I1, I2, I3, I4, I5 and total fluorescence (ΦT) respect to addition of different doses of sodium hypochlorite (chlorine concentration of 0, 0.5, 1, 2, 5, 8 mg/L) in the secondary wastewater effluent of Paternò WWTP. Spectroscopic measurements were accomplished after 5 hours of reaction time. Dotted lines indicate ± 5% from the fluorescence intensity measured in the unchlorinated sample. Continuous lines represents ± 10% from the fluorescence intensity measured in the unchlorinated sample.

Highlights

•

Filter pre-washing eliminates interferences in fluorescence measurements

•

Wastewater filtration 0.7 µm with storage at 4°C is the best preservative condition

•

Storage effectiveness is dependent on water quality and fluorescing component

•

Wastewater should be stored no longer than 2 days

•

Chlorine disinfection (0.5–5 mg/L) mainly affected tryptophan-like components

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: