International Proceedings of Chemical, Biological and Environmental Engineering, V0l. 101 (2017) DOI: 10.7763/IPCBEE. 2017. V101. 4

Organic Fluorescent Characteristics of Five Tributaries in Basin in Dry Season

Guan-Ling Chen 1, Jing-Wen Cao 2, Chun-Yen Chiu 3, Lih-fu Chen 4, Wen-Liang Lai 5,* 1 Graduate School of Environmental Management, Master student, Tajen, Pingtung 907, , ROC (E- mail: [email protected]) 2 Graduate School of Environmental Management, Assistant, Tajen University, Pingtung 907, Taiwan, ROC (E-mail: [email protected]) 3 Graduate School of Environmental Management, Associate Professor, Tajen University, Pingtung 907, Taiwan, ROC (E-mail: [email protected]) 4 College of Liberal Education, Shu-Te University, Associate Professor, 82445, Taiwan, ROC (E- mail: [email protected]) 5 Graduate School of Environmental Management, Professor, Tajin University, Pingtung 907, Taiwan, ROC, (E-mail: [email protected]) *Corresponding author

Abstract. In this research, fluorescence excitation emission spectrometry (Excitation emission fluorescent matrix, EEFM) and total organic carbon Analyzer were main instruments applied into evaluating the property and amount of natural organic matter in dry season for Gaoping River basin located in Southern Taiwan. Dissolved non-volatile organic carbon in water (non-purgeable dissolved organic carbon, NPDOC) was measured and SUVA, (UV254/NPDOC), BIX (Biological index, BIX) were compared in whole samples belonging to five segments. Results showed that NPDOC values of whole samplings in Gaoping River basin were less than the criteria of Source Water Standard of Drinking Water except for Jiouru bridges. SUVA values and BIX for whole samplings were less than 1, indicating that the source pollution in the Gaoping River Basin was mainly attributed to the human activity and microbial organic matter produced in the River. The variance of peak fluorescence locations and intensity, and the difference of fluorescence intensity among five fluorescent organic groups revealed that the significant divergences existed in the five segments in Gaoping River Basin. Peak fluorescent intensity belonging to humic-like substance, those values in Qishan River, Laonong River and were less than 100. Peak fluorescent intensity belonging to fulvic-like substance, only Laonong River was below 100. The average fluorescence intensity in Gaoping River mainstream was the highest value followed by Mino River; however, the lowest existed in Ailiao River. Keywords: excitation emission fluorescent matrix (EEFM), non-purgeable dissolved organic carbon (NPDOC), SUVA, (UV254/NPDOC), Biological index (BIX), Fulvic-like substance, humic-like substance

1. Introduction Dissolved organic matter (DOM), a heterogeneous mixture of organic compounds in water bodies, is composed of myriads of organic compounds which play pivotal ecological and biogeochemical roles in the environment [1]. Part of DOM, named as Chromophoric DOM (CDOM) in aquatic DOM can be divided into two major classes: humic-like substances and protein-like substances [2]-[3]. Humic-like substances with complex properties consisted of aromatic and aliphatic compounds mainly derived from the decay of organic matter; however, protein-like substances are associated with high biological activities. For past researches, biochemical oxygen demand (BOD) and chemical oxygen demand (COD) were usually applied into the organic concentration in river; however, these parameters could not show the

  Corresponding author. Tel.: +886-8-7624002 ext.2607 E-mail address: [email protected] 26 variation of organic characteristics. EEFM (Excitation emission fluorescent matrix) applied on the dissolved organic matter have several advantages including keeping the integrity of water sample, a little volume analysed, less labour and less time consumed [4]. Fluorescence spectroscopy has been increasingly used for on-line monitoring of DOM [5]. In this research, fluorescence spectrometry as well as total organic carbon analyser was applied to evaluate the property and amount of natural organic matter in dry season for Gaoping River basin located in Southern Taiwan. Dissolved non-volatile organic carbon in water (non-purgeable dissolved organic carbon, NPDOC) was measured and SUVA (UV254/NPDOC), BIX (Biological index, BIX) were compared in whole samples belonging to five segments. 2. Material and Methods 2.1. Sampling Locations In this study, respective samples from Gaoping River on Dec.2016 were selected to compare to distinguish the organic property owing to dry season. The procedure and storage of water sampling were according to the regulation of Environmental Protection Administration in Taiwan. The relative locations were labelled in Fig.1. The distance of sampling location from upstream to downstream was near to 171 km.

Fig. 1: Locations for whole samples relative to Gaoping River

2.2. Analytical Parameters 2.2.1. EEFM (Excitation Emission Fluorescent Matrix). Regarding to the organic characteristic of water samples, fluorescent spectrometry (F-4500, Hitachi, Japan) was applied in this research. Whole samples must be filtered by 0.45 µm membrane filter (Mixed cellulose ester, Advantec MFS Inc., USA) before fluorescent measurement. EEFMs of whole samples were obtained by deducting from the background signal of pure water. 2.2.2. UV-VIS.

27 Ultra-violet and visible measurements were carried out with 1 cm quartz UV-visible cells at room temperature (22~24℃)by UV-visible double beam spectrophotometer (U-2900, Hitachi, Japan). UV-vis absorption spectrum of water samples was obtained at wavelength 200 to 600 nm. Before analyzed, all samples were filtered by 0.45 µm membrane filters and filtrate were put into one cm path of the cell to measure. 2.2.3. NPDOC (Non-purgeable dissolved organic matter). All water samples for DOC measurement were prefiltered through a 0.45 µm membrane sand filter to decrease the interference from microorganisms. The filtered water sample was acidified to pH of 2 by adding an appropriate volume of 85 % phosphoric acid. Inorganic carbon was then degassed with highly pure nitrogen. The DOC of water sample was finally quantified by a total organic carbon analyser (Lotix, TELEDYNE TEKMAR, USA.) equipped with high-temperature (680 ℃) combustion and a non-dispersive infrared absorption detector. The value of DOC measured was called NPDOC. Additionally, calibration curves were prepared by using a series of standard solutions consisting of anhydrous potassium biphthalate

(C8H5KO4). 3. Results and Discussion 3.1. NPDOC and SUVA Figure 2 shows the variations of NPDOC and SUVA of whole samplings in Gaoping River basin. Observed from this figure, maximal NPDOC values of 5.27 mg-C/L occurred in Jiouru bridges located in main Gaoping River (Figure 2 A). High organic content exceeding the standard of source water as drinking water, may be attributed to the pollutant drainage of livestock industry. NPDOC values of samplings respectively belonging to five side-rivers basin looked so different, revealing that the difference pollutants existing in point or non-point source steadily flew into rivers in dry season.

Fig. 2: (A) NPDOC (B) SUVA values of whole sampling locations of five tributaries located in Gaoping River Basin

-1 The ratios of UV 254 (cm ) to NPDOC (mg-C/L), named as SUVA, were plotted in Figure 3 B. Usually, SUVA larger than 4 represents the organic substance with hydrophobic property like humic fraction. Contrarily, SUVA less than 2 reveals organic substance with hydrophilic property like non-humic fraction [6]. Whole SUVA values in Gaoping River basin were less than 1, as shown in Fig.2 B, proving that organic matter with hydrophilic property was attributed to artificial activity, especially on the sampling locations belonging to Laonong River and Ailiao River where most of their values were declined to the 0.4. 28 Apparently, this area development affects the water quality, and even changes its status of utilization by water source planning.

3.2. EEFM and Characteristic Peaks 400 1 6 11 350

300

IV V IV V IV V 250

I II III I II III I II III 200 2 7 12 350

300 7200 IV V IV V IV V

) 250 6600 I II III

m I II III I II III

200 6000 n

( 5400 8 13 n 3 4800

o 350 i

t 4200

a 300

t 3600 i IV V IV V IV V

c 250 3000 x I II III I II III I II III 2400 E 200 1800 4 9 14 1200 350 600 300 0

IV V IV V IV V 250

I II III I II III I II III 200 5 10 15 350

300

IV V IV V IV V 250

I II III I II III I II III 200 300 350 400 450 500 300 350 400 450 500 300 350 400 450 500 Emission(nm) Fig. 3: EEFM for Gaoping River at different sampling locations

Table 1 Peak locations and fluorescent intensities at different sampling locations of five tributaries located in Gaoping River Basin Excitation nm/Emission(Fluorescence intensity) River I II III IV V 224-230/ 226-230/ 244/ 280/ 252/ Qishan 295-316 337-346 417-436 337-340 442 (160-190) (183-193) (124-217) (95-166) (88) 226/ 228-230/ 236-238/ 278-280/ 320-328/ Mino 292-295 340-346 418-424 340 412-418 (245-265) (294-309) (279-296) (212-241) (197-199) 224-226/ 226-228/ 234-236/ 278/ Laonong 289-292 328-343 409-421 331-346 No data (126-211) (82-112) (69-80) (43-52) 226/ 242-246/ 278-284/ 306-328/ Ailiao 295-328 No data 436-442 334-337 409-424 (159-164) (48-132) (46-103) (28-86) 228-230/ 228-232/ 232-246/ 274-280/ 254-328/ Gaoping 295-313 331-343 385-433 331-343 409-436 (122-464) (175-8116) (97-612) (101-1843) (55-454)

I:200-250/ 280-330 nm, Aromatic protein (Tyrosine) ；II:200-250/330-380 nm, Aromatic protein (BOD5)；III:200- 250/380-540 nm, Fulvic acid；IV:250-340/280-380, Soluble microbial by-product-like；V: 250-400/380-540 nm, Humic acid-like.

EEFMs of whole samplings were shown in Figure 3. Based on the classification of related research, dominant components could be divided into five groups, group I of aromatic protein (Tyrosine)：200-250/

280-330 nm, Group II of aromatic protein (BOD5)：200-250/330-380 nm, Group III of Fulvic acid：200- 250/380-540 nm, Group IV of soluble microbial by-product-like：250-340/280-380 and group V of humic acid-like：250-400/380-540 nm [7]. Related peak locations and intensity were listed in Table 1. Table 1 29 shows minor divergences for peak locations among Qishan River, Mino River and Laonong River, Ailiao River and Main Gaoping River. Although significant fluctuation on fluorescent intensity was observed, those values in Qishan River, Laonong River and Ailiao River were lower than those in other streams. Peak fluorescent intensity belonging to humic-like substance, those values in Qishan River, Laonong River and Ailiao River were less than 100. Peak fluorescent intensity belonging to fulvic-like substance, only Laonong River was below 100. Regarding to peak fluorescent intensity of soluble microbial by-product (SMP), Laonong River and Ailiao River were close to 100. For Main Gaoping River, the variance of peak fluorescent intensity in whole organic classification became larger than other streams in this basin, especially for Group II (Aromatic protein (BOD5) and SMP. 3.3. Fluorescent Organic Classification and Their Distribution Figure 4 reveals total fluorescence intensity and fluorescence intensity of each kind of organic fluorescent matter in whole sampling points in the Gaoping River basin. For total fluorescence intensity, the biggest difference between sampling points in each branch is the Gaoping River mainstream, and the lowest happened in Mino River. The average fluorescence intensity in Gaoping River mainstream was the highest value followed by Mino River; however, the lowest existed in Ailiao River. Actually, the variance of fluorescent intensity and maximal fluorescent intensity for five groups of fluorescent organic matter were compared in each tributary; maximal values were found in Gaoping River mainstream, and the minimal values was appeared in Laonong River. In other words, organic pollution in Laonong River was lower than that in Gaoping River mainstream. Apparently, less development in upstream than downstream was related with current results.

Fig. 4: EEFM for Gaoping River at different sampling locations

3.4. Humification Index ( HIX) and Biological Index ( BIX) HIX was used as the basis of organic humification. It could be calculated by total fluorescent intensity of emission wavelength of 435-480 nm divided by that of 300-345 nm at constant excitation wavelength of 254 30 nm [9]. DOM with weak humification, less than 4 of HIX, was attributed to microbial activity. DOM was mainly from terrestrial sources while HIX value ranged from 10 to 16. BIX (Biological index), calculated as fixed excitation wavelength of 310 nm, the ratio of fluorescent intensity in emission wavelength of 380 nm to that in 430 nm [9], is another judgement to explain the formation duration of organic compounds in water sample. BIX ranging from 0.6 to 0.7 represents low genetic composition (the index of Recent autochthonous contribution); 0.7 to 0.8 represents degrees in genetic composition; 0.8 to 1 represents strong native content, and BIX greater than 1 represents microbial or bacterial activity.

Fig. 5: (A) HIX (B) BIX values at different sampling locations of five tributaries located in Gaoping River Basin

Figure 5 shows the comparison between HIX and BIX in five tributaries located in Gaoping River Basin. Observed from Fig 5 A, HIX values in whole sampling points were less than 4, indicating that CDOM in Gaoping River Basin with low humification was formed from biological activities. Regarding to BIX value (Fig. 5 B), most of values ranged from 0.74 to 0.99, apparently, revealing that dissolved organic matter was freshly produced by microbial metabolism in river. 4. Summary 1. SUVA values and BIX for whole samplings were less than 1, indicating that the source pollution in the Gaoping River Basin was mainly attributed to the human activity and microbial organic matter produced in the River. 2. For peak fluorescent intensity belonging to humic-like substance, those values in Qishan River, Laonong River and Ailiao River were less than 100. Peak fluorescent intensity belonging to fulvic-like substance, only in Laonong River, was below 100. 3. The average fluorescence intensity in Gaoping River mainstream was the highest value followed by Mino River; however, the lowest existed in Ailiao River. 5. Acknowledgements The authors would like to thank Ministry of Science and Technology in Taiwan, ROC for their financial support (MOST 103-2221-E-127 -001 –MY3 and MOST 104-2221-E-127 -002 -MY2). 6. References [1] Y. Wang, D. Zhang, Z. Y. Shen, J. Chen, & Feng, C. H. Characterization and spacial distribution variability of chromophoric dissolved organic matter (CDOM) in the Yangtze Estuary.2014, Chemosphere 95, 353–362. [2] M. Sgroi, P. Roccaro, G. V. Korshin, V. Greco, S. Sciuto, T. Anumol, & F. G. Vagliasindi, Use of fluorescence

31 EEM to monitor the removal of emerging contaminants in full scale wastewater treatment plants. Journal of hazardous materials 2017, 323:367-376. [3] G. Yamin, M. Borisover, E. Cohen, & J. van Rijn, Accumulation of humic-like and proteinaceous dissolved organic matter in zero-discharge aquaculture systems as revealed by fluorescence EEM spectroscopy. Water research 2017, 108: 412-421. [4] W. T. Li, S. Y. Chen, Z. X. Xu, Y. Li, C. D. Shuang, A. M. Li, Characterization of dissolved organic matter in municipal wastewater using fluorescence PARAFAC analysis and chromatography multi-excitation/emission scan: a comparative study. Environ. Sci. Technol 2014. 48:2603-2609. [5] E. M. Carstea, J. Bridgeman, A. Baker, D. M. Reynolds, Fluorescence spectroscopy for wastewater monitoring: a review. Water Res 2016, 95:205–219. [6] J. K. Edzwald, D. Q. Bunker, J. Dahlquist, L. Gillberg, T. Hedberg, Dissolved Air Flotation: Pretreatment and Comparisons to Sedimentation. In: Klute R., Hahn H.H. (eds) Chemical Water and Wastewater Treatment III.. Springer Berlin Heidelberg 1994, pp. 3-18. [7] W. Chen, P. Westerhoff, J. A. Leenheer, & K. Booksh, Fluorescence Excitation-Emission Matrix Regional Integration to Quantify Spectra for Dissolved Organic Matter. Environmental science & technology 2003, 5701- 5710.

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