Characterizing the interactions between sediment dissolved organic matter and zinc using multispectroscopic techniques

Journal Pre-proof Characterizing the interactions between sediment dissolved organic matter and zinc using multispectroscopic techniques

Lü Weiwei, Yao Xin, Ren Haoyu, Deng Huanguang, Yao Min, Zhang Baohua PII:

S0269-7491(19)34145-4

DOI:

https://doi.org/10.1016/j.envpol.2019.113644

Reference:

ENPO 113644

To appear in:

Environmental Pollution

Received Date:

27 July 2019

Accepted Date:

16 November 2019

Please cite this article as: Lü Weiwei, Yao Xin, Ren Haoyu, Deng Huanguang, Yao Min, Zhang Baohua, Characterizing the interactions between sediment dissolved organic matter and zinc using multispectroscopic techniques, Environmental Pollution (2019), https://doi.org/10.1016/j.envpol. 2019.113644

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Journal Pre-proof

Journal Pre-proof Characterizing the interactions between sediment dissolved organic matter and zinc using multispectroscopic techniques Lü Weiweia, Yao Xina,b*, Ren Haoyua, Deng Huanguanga, Yao Mina, Zhang Baohuaa

a

School of Environment and Planning, University of Liaocheng, Liaocheng 252000, China

b

Taihu Laboratory for Lake Ecosystem Research, State Key Laboratory of Lake Science and

Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008, China

*Corresponding author. Present address: School of Environment and Planning, University of Liaocheng, Hunan Road 1, Liaocheng 252000, China E-mail address: [email protected]

Abstract Sediment DOM was collected in November of 2018 from Lake Dongping, China. The lake was divided into the entrance of the Dawen River, the open area of the lake, the tourism district and the macrophyte-dominated region based on principal component analysis (PCA) results. Multispectroscopic tools were used to investigate the binding of zinc (Zn) with four kinds of DOM collected from the entrance of the Dawen River (EDOM), the open area of the lake (ODOM), the tourism district (TDOM) and the macrophyte-dominated region (mainly dominated by Potamogeton crispus L.) (PDOM). Three fluorescent components, the humic-like (components 1 and 3) and protein-like (component 2) components, were found by excitation-emission matrix 1

Journal Pre-proof spectra with parallel factor analysis. The EDOM, ODOM and TDOM were controlled by protein-like components, and the PDOM was controlled by humic-like components. Different components respond differently to Zn addition. The binding order of the tyrosine-like fraction > the tryptophan fraction > the humic-like fraction was identified by Synchronous fluorescence (SF) spectra and two-dimensional correlation spectroscopy (2D-COS). The fluorescence intensity of the protein-like component was quenched, and the humic-like component was enhanced with the addition of Zn. The effective quenching constants (log K) of the protein-like component in PDOM were clearly higher than those in the EDOM, ODOM and TDOM, indicating higher metal binding potential in PDOM than in other kinds of DOM in Lake Dongping. The %Fmax of the humic-like components exhibited a gradual increase in all kinds of DOM with the addition of metal, suggesting that the addition of Zn increased the humification of DOM. These results highlighted the significant impact of heavy metals on DOM spectral properties and the effect of DOM on the behavior, mobility and bioavailability of heavy metals in lake sediments. Key words: dissolved organic matter (DOM); zinc; metal binding; 2D-COS; PARAFAC

1. Introduction Heavy metals play a fundamental role in many different physiological processes for living organisms, but an excess amount of heavy metals can pose potential harm (Chen et al., 2019a; Chen et al., 2019b). The mobility, toxicity, and bioavailability of heavy metals are influenced by their speciation in the aquatic environment (Yuan et al., 2015). Previous studies have reported that dissolved organic matter (DOM) can form DOM-metal complexes with heavy metals and strongly affect heavy metal speciation in diverse aquatic environments (Yuan et al., 2015). Therefore, 2

Journal Pre-proof insight into DOM-metal binding is essential for understanding the biogeochemistry of heavy metals. Numerous studies have investigated DOM-metal binding in eutrophic freshwater lakes (Liu et al., 2015; Xu et al., 2019). In contrast, information on DOM-metal binding in macrophyte-dominated lakes, mainly distributed in northern China, remains lacking. With the high development of economy, the northern macrophyte-dominated lakes in China are receiving increasingly more agricultural runoff and industrial discharges, which causes high concentrations of total nitrogen and phosphorus and increases the amount of chemical oxygen demand, DOM and heavy metals (Wang et al., 2015). Lake Dongping, located in Shandong Province, northern China, is a typical macrophyte-dominated lake. It is a key drinking water supply, joining in both the east route of the South-to-North Water Diversion East Route Project of China and the water transmission from West to East of Shandong Province. Generally, the biomass of submerged plants reaches a peak in May, is largely dominated by Potamogeton crispus L. (P. crispus) and covers more than 80% of the lake (Deng et al., 2016). Numerous studies have reported that submerged plants can take up and absorb Cr, Zn, Pb, Mn, Hg, Ni and Cu in their growing periods (Sivaci et al., 2008; Xu et al., 2010). However, submerged plants begin to die and decompose in May or July, and discharge heavy metals back into the water (Deng et al., 2016). Thus, additional studies on DOM-metal binding in macrophyte-dominated lakes are required. In addition, Lake Dongping has several distinct regions with different contents, sources, and molecular weights of DOM. Aquatic DOMs with different molecular weight distributions might display different binding behaviors to heavy metals (Xu et al., 2019). Comparison of the different DOM-metal binding behaviors in different lake regions is necessary for acquainting their 3

Journal Pre-proof environmental fates in Lake Dongping. The interactions between heavy metals and DOM have been carried out using kinds of techniques, e.g., solid-phase microextraction, synchronous fluorescence quenching (SF), passive dosing, and dialysis equilibrium (Xu et al., 2013; Bai et al., 2017; Xu et al., 2019). Thereinto, SF has been widely used due to its effective, sensitive nature and rapid (Liu et al., 2015). However, the one-dimensional spectra usually contains some overlapping peaks, restricting the application of finding details (Liu et al., 2015). Two-dimensional correlation spectroscopy (2D-COS) can solve the problem of peak overlap and identify the sequence of metal-DOM spectrum changes (Liu et al., 2015). Additionally, excitation-emission matrix and parallel factor analysis (EEM-PARAFAC) could investigate the response of DOM to the addition of metal (Bai et al., 2017). In the present work, the metal Zn, which is common in freshwater lakes, is used as the representative metal. By using fluorescence titration, EEM-PARAFAC, UV-Vis spectra, and 2D-COS, we aim to provide an in-depth understanding of the heterogeneous binding characteristics of Zn onto DOM at the molecular level. The main purposes of this study are as follows: (1) to compare the different Zn-DOM binding affinities in different lake regions and (2) to further explore the binding sequence of Zn with different DOM components.

2. Materials and methods 2.1. Sample collection The sediment samples were collected in November of 2018 from Lake Dongping (35°30′– 36°20′N, 116°00′–116°30′E), the second largest freshwater lake in Shandong Province, China. 4

Journal Pre-proof Twelve sites, with 3 sites at the entrance of the Dawen River, open area of Lake Dongping, tourism district and the Macrophyte-dominated region, were evenly distributed in the lake (Fig. 1). Surface sediments (0-5 cm) samples from Lake Dongping were collected by grab sampler. The collected samples were sifted through a 2 mm screen (Xu et al., 2019). The sifted samples were admixed with deionized water (solid-liquid ratio 1:15) and placed on a shaking bed for 24 h to ensure complexation equilibrium (Xu et al., 2019). After 4000 × g centrifugation for 25 min, the mixture was filtered with 0.45 µm cellulose acetate membrane to gain the DOM (Xu et al., 2019). 2.2. Fluorescence quenching titration Fluorescence titration was carried out with reference to Yamashita and Jaffe (2008) and Ohno et al. (2008). A certain amount of Zn2+ solution was added to a brown sealed bottle containing 0.45m filtrate 50 mL. The pH values of the solutions were maintained at 6.0 to avoid precipitation (Liu et al., 2015). After the addition of Zn2+, all samples were shaken for 24 h. Ultimately, EEM, UV-Vis absorption and synchronous fluorescence (SF) spectra of the solutions were acquired. The UV-Vis absorption spectra of 200 and 800 nm were measured by UV-Vis spectrophotometer, matching 5 cm quartz cells at 1 nm intervals. EEMs and SF spectra were acquired using fluorescence spectrometer (Bai et al., 2017). The scanning emission (Em) and excitation (Ex) wavelengths of EEMs are from 250 nm to 550 nm with 5 nm intervals. In the range of Ex wavelengths of 200-450 nm, SF spectra were obtained with constant offset (60 nm). 2.3. Data analysis 2.3.1. 2D-COS and PARAFAC analysis 5

Journal Pre-proof To study Zn-DOM binding behavior, 2D-COS was carried out by SF spectroscopy with the addition of Zn. The details of 2D-COS and PARAFAC have been described (Noda and Ozaki, 2005; Lü et al., 2019). 2.3.2 Fluorescence index Three fluorescence indices, FI (fluorescence index), BIX (biological index) and HIX (humification index), were used in the present study and the details of the three fluorescence indices have been described elsewhere (Lü et al., 2019). 2.3.3. Complexation modeling To estimate the conditional stability constants between metals and PARAFAC components, the modified Stern-Volmer equation (Hays et al., 2004) was used: 𝐹0 𝐹0 ― 𝐹

=

1 𝑓𝐾𝑀𝐶𝑀

+

1 𝑓

(1)

Here F0 and F are the fluorescence intensities of PARAFAC components in the absence and presence of Zn, respectively. KM and f represent the effective quenching constant and the initial spectral intensities, respectively. 2.3.4. Absorption coefficient Absorption coefficients were applied (Zhou et al., 2015): [𝑎(𝜆)] = 2.303 × [𝐷(𝜆)]/𝑟

(2)

Here D(λ) and r are the corrected absorbance of wavelength λ and the path length, respectively. The absorption coefficient of 350 nm (a(350)) is chosen to indicate the abundance of DOM in this study. The molecular weight parameter M is inversely proportional to the molecular weight. M is the ratio of the absorption coefficients at 250 to 365 nm (Guo et al., 2017): 6

Journal Pre-proof

𝑀=

𝑎(250) 𝑎(365)

(3)

2.3.5. Principal component analysis Principal component analysis (PCA) in this study was conducted to divide the lake into the entrance of the Dawen River, open area of the lake, tourism district and macrophyte-dominated regions based on 9 DOM-related parameters, including a(350); M value; total fluorescence intensity of DOM; relative abundance of components 1, 2, and 3; FI; BIX; and HIX. PCA in the present study was performed using MATLAB 2017b software. 2.4 Statistical analyses Statistical analyses, including the one-way analysis of variance (ANOVA), SNK-q test, mean value and standard deviation were performed using SPSS 21.0 software. Correlation plots were carried out using MATLAB 2017b software. Spatial distributions of sampling sites and different DOM-related parameters were made by ArcGIS 10.2 software. The results of p<0.05 in the present study indicate significant during linear regression and SNK-q test analyses.

3. Results 3.1. PCA results and characteristics of DOM in surface sediments of Lake Dongping The two components (PC1 and PC2) obtained by PCA explain 47.56% and 34.45% of the total variance, respectively (Fig. 2). PC1 and PC2 could be explained as DOM composition and concentration, respectively (Zhou et al., 2015). The colors of the dots in Fig. 2a suggest that PC1 is likely superimposed on different DOM compositional variables. A significant correlation was found between the %Fmax of humic-like material and PC1 (r2=0.982, p<0.001). A significant correlation was found between PC2 and a(350) (r2=0.889, p<0.001), indicating that PC2 might act 7

Journal Pre-proof as DOM abundance. Based on these two components, four lake regions, including the entrance of the Dawen River, the open area of the lake, the tourism district and the macrophyte-dominated region, were determined (Figs. 1 and 2). Obvious differences in EEM spectral were recorded among samples collected from the four distinct lake regions (Fig. 3). DOM derived from the entrance of the Dawen River (EDOM), the open area of the lake (ODOM), and the macrophyte-dominated region (PDOM) had one more fluorescent peak compared to that from the tourism district (TDOM). PDOM was dominated by humic-like fluorophores, and EDOM, ODOM and TDOM were dominated by protein-like fluorophores. Values of a(350) of all samples ranged from 0.37 to 1.25 m-1 and had a mean (mean ± standard deviation) value of 0.72 ± 0.35 m-1. The mean EDOM, ODOM, PDOM and TDOM a(350) values were 0.96±0.45, 0.40±0.10, 0.96±0.25, and 0.58±0.20 m-1, respectively, indicating that the DOM concentrations in the entrance of Dawen River and macrophyte-dominated region were higher than those in the open area of Lake Dongping and the tourism district (Fig. 4). M values ranged from 4.06 to 7.09 with a mean of 5.47±1.19. The mean M values of EDOM, ODOM, PDOM and TDOM were 5.24±1.24, 7.09±0.10, 4.91±0.67, and 4.65±0.58, respectively. A significantly higher value of M was found in the open area of the lake than in the other regions of Lake Dongping (p<0.05), implying smaller molecules in ODOM (Fig. 4). Three DOM components were identified in PARAFAC model (Fig. 5). Components in C1 (Ex/Em = 260(300)/413 nm) were ascribed to humic-like components and might be influenced by microbial degradation products of plant residues (Yamashita et al., 2008; Williams et al., 2010). Components in C2 (Ex/Em = 240(275)/340 nm) can be classified as protein-like components and 8

Journal Pre-proof are associated with amino acid-like substances (Murphy et al., 2008; Yamashita et al., 2008). Components in C3 (Ex/Em = 270(365)/456 nm) can be defined as humic-like components that are derived primarily from organic-rich soil substances (Williams et al., 2010; Zhou et al., 2016). The mean total fluorescence intensities of EDOM, ODOM, PDOM and TDOM were 14.87±2.30, 9.15±0.58, 14.32±0.99, and 12.60±2.71 QSU, respectively, showing that the DOM concentration decreased from the entrance of the Dawen River and macrophyte-dominated region to the open area of the Lake Dongping and tourism district (Fig. 4). The total fluorescence intensities exhibited a similar pattern of variation to that seen with the absorption coefficients a(350). Significantly higher ratios of the humic-like component (C1) to the total fluorescence intensity (%Fmax) were found in ODOM and PDOM than in EDOM and TDOM (p<0.01). The %Fmax of the protein-like component C2 in EDOM, ODOM and TDOM were significantly higher than in PDOM (p<0.01). Based on the %Fmax values (Table 1), the EDOM, ODOM and TDOM were dominated by protein-like component C2, and the PDOM was dominated by the humic-like component C1, which was consistent with the original EEMs (Fig. 3). FI, BIX and HIX had ranges (mean values) of 1.69 - 2.44 (2.07 ± 0.22), 0.52 - 0.99 (0.80 ± 0.16) and 0.99 - 4.13 (2.37 ± 0.90), respectively (Fig. 4). Significantly higher FI values were found in ODOM and PDOM than in EDOM and TDOM (p<0.05). Previous research explained that DOM has endogenous characteristics with higher FI indicates (Zhou et al., 2015). Relatively high FI values of water samples taken from the open area of the lake and the macrophyte-dominated region indicate that ODOM and PDOM have strong endogenous characteristics. Previous studies (Zhou et al., 2015) suggested that a lower BIX indicates DOM with stronger exogenous characteristics. Significantly lower BIX was found in the entrance of the 9

Journal Pre-proof Dawen River than in other regions of Lake Dongping (p<0.05). This indicates that the Dawen River inputs a large amount of terrestrial DOM into Lake Dongping. Research has reported that HIX suggests the degree of DOM humification (Zhou et al., 2015). The HIX value in PDOM was significantly higher than those in EDOM, ODOM and TDOM (p<0.05). Relatively high HIX was found in the macrophyte-dominated region, indicating a higher degree of humification of PDOM. 3.2. Binding behaviors of sediment DOM with trace metals The absorbance of EDOM, ODOM, PDOM and TDOM exhibited a gradual increase with the incremental addition of the metal titrant (Fig. 6). This indicates that Zn changed the microbial environment of DOM and suggests an increase in the molecular weight of DOM with the addition of Zn. The results were consistent with previous findings that the absorbance of both macrophyteand algal-dominant DOM increased with the gradual addition of metal titrants (Xu et al., 2013). SF spectra can suggest the effect of the metal on DOM and display information about the changes in DOM components (Bai et al., 2017). Fluorescence wavelength was divided into 3 regions, including tyrosine-like (TYF, wavelengths 200-250 nm), tryptophan-like (TRF, wavelengths 250-285 nm) and humic-like (HUF, wavelengths 285-450 nm) fluorescence regions (Fig. 6). Fluorescence quenching was found in all the DOM, indicating the occurrence of electronic structural changes in the DOM with the addition of Zn (Hur and Lee, 2011). However, the behaviors of spectral changes were different for the DOM, suggesting that the different behaviors may depend on the concentration of Zn and the origin of the DOM. For the EDOM, ODOM, PDOM and TDOM, fluorescence was quenched with the increase in Zn addition in both TYF and TRF regions, while the fluorescence intensities increased after Zn addition in the HUF region. 10

Journal Pre-proof The synchronous map displayed three autopeaks at 275/275, 300/300 and 350/350 nm along the diagonal line for EDOM, ODOM and PDOM, respectively, whereas two autopeaks were found at 275/275 and 350/350 along the diagonal line for TDOM (Fig. 7). The intensity of the autopeaks decreased in the order of 275, 300, and 350 nm, suggesting that tryptophan-like substances were more accessible to metal addition than the humic-like substances. One positive crosspeak (275/250 nm) and five negative crosspeaks (230/270, 230/300, 230/350, 270/300 and 270/350 nm) were identified under the diagonal line in the asynchronous maps of EDOM, ODOM and PDOM, while five negative crosspeaks (230/270, 230/300, 230/350, 270/300 and 270/350 nm) were present in TDOM. According to Noda’s rule (Noda and Ozaki, 2005), the sequential fluorescent quenching followed the order 230 > 270 > 250 > 300 > 350 for EDOM, ODOM, TDOM and PDOM. This indicated that Zn bound to DOM fractions in the following sequence: tyrosine-like fraction > tryptophan fraction > humic-like fraction. A similar phenomenon was also observed in the DOM of Lake Taihu, a eutrophic shallow lake (Xu et al., 2019). 3.3. PARAFAC-derived components Before metal titration, C2 among the six PARAFAC components had the highest fluorescent intensity in EDOM, ODOM and TDOM, followed by C1 (Fig. 8). However, for PDOM, the intensity score of C1 was higher than that of both C2 and C3. This indicated that the protein-like components were primarily located in EDOM, ODOM and TDOM, while the humic-like components were mostly distributed in PDOM, which was consistent with the original EEMs (Fig. 3). When metal addition was applied, the fluorescence intensity of protein-like component C2 11

Journal Pre-proof was quenched in all kinds of DOM. The decline in fluorescent intensities indicated an effective binding of protein-like component C2 with Zn. Further analysis displayed that the binding behaviors of protein-like component C2 in EDOM, ODOM, PDOM and TDOM exhibited obvious differences. The mean log K value of C2 in PDOM (log K=4.32±0.35) was clearly higher than that in EDOM (log K=3.95±0.42), ODOM (log K=3.59±0.09) and TDOM (log K=3.68±0.50) (Table 1). Additionally, the log K values found in the present study are comparable with those found in eutrophic shallow lakes, such as Lake Taihu. The log K values of DOM in macrophyte-dominated lakes were lower than those in algae-dominated lakes, such as Lake Taihu, China. Both Xu et al. (2014) and Liu et al. (2015) reported that the log K values of DOM derived from Lake Taihu were >4.50. This suggests that the interactions between protein-like component C2 and Zn were stronger in PDOM than in EDOM, ODOM and TDOM, and the algae-dominated DOM displayed a more effective binding to metals than macrophyte-dominated DOM. These results indicate that algae-dominated lakes would play a significant role in the bioavailability, toxicity and mobility of heavy metals. Compared with fluorescent quenching of protein-like component C2, the intensities of both humic-like components C1 and C3 exhibited an increase in EDOM, ODOM and PDOM with the addition of Zn. No obvious change was observed in the intensities of both humic-like components C1 and C3 in TDOM with the addition of Zn. These results indicated different binding mechanisms between protein- and humic-like components (Xu et al., 2019). The increase in fluorescence intensities might be related to changes in the molecular environments of humic molecules (Yamashita and Jaffe, 2008). Further analysis showed that the relative abundance of the fluorescence intensity of the 12

Journal Pre-proof humic-like components (C1+C3) compared to the total fluorescence exhibited a gradual increase in all DOM types after metal addition (Fig. 8e). The intensities of humic-like components (C1+C3) increased by 5%-14%. This result suggests that the addition of Zn increased the humification of all DOM types. This result is consistent with the UV-vis absorption spectra.

4. Discussion Our results suggest that the protein-like components of sediment DOM from macrophyte-dominated Lake Dongping were quenched and that the humic-like components were released through the interaction with Zn, which was especially pronounced in PDOM. The addition of Zn induced a decrease in fluorescence intensity in both tyrosine-like fluorescence (200-250 nm) and tryptophan-like fluorescence (285-450 nm) regions in SF spectra for all kinds of DOM (Fig. 6). The 2D-COS of the SF spectra showed that the sequential orders of fluorescent quenching for all kinds of DOM followed the order 230 > 270 > 250 > 300 > 350 (tyrosine-like fraction > tryptophan fraction > humic-like fraction), providing further evidence (Fig. 7). This suggests that protein-like substances were more accessible to Zn addition than the humic-like substances. Moreover, the decline in the fluorescent intensities of protein-like component C2 in all kinds of DOM (Fig. 8) supported Zn-induced fluorescent quenching for protein-like material. A large fraction of humic-like material accumulated in the fluorescence quenching titrations with Zn addition, and the addition of Zn increased the humification of all kinds of DOM. This is substantiated by the UV-vis absorption results, where gradual increasing absorbance in all kinds of DOM indicated the increase in the humification of DOM (Fig. 6). In the SF spectra, the increasing 13

Journal Pre-proof fluorescence intensities after Zn addition in the humic-like fluorescence region (285-450 nm) further supported the idea that humic-like material was released through the interaction with Zn (Fig. 6). The PARAFAC results provide evidence that the intensities of both humic-like components C1 and C3 exhibited increases in EDOM, ODOM and PDOM with the addition of Zn, and no obvious change was observed in the intensities of both humic-like components C1 and C3 in TDOM with the addition of Zn (Fig. 8). These results indicate the accumulation of humic-like components in EDOM, ODOM and PDOM after Zn addition and that there are different binding mechanisms in different kinds of DOM. The intensities of the humic-like components (C1+C3) increasing by 5%-14% provided further evidence of the humification of all kinds of DOM (Fig. 8e). The enhancement in the fluorescence intensities of the humic-like components might be related to changes in the molecular environments of humic molecules (Yamashita and Jaffe, 2008). Before the addition of Zn, the fluorescence of the protein-like component might have been quenched by other quenchers (excluding Zn). After the addition of Zn, the increase of fluorescence intensity in humic-like components may be due to the replacement of the original DOM-metal complexes with the newly formed and more stable DOM-Zn complexes, whereby the interactions between DOM and Zn resulted in the release of humic-like components from the DOM matrix (Yamashita and Jaffe, 2008). Thus, the intensities of humic-like components were enhanced through the interaction with Zn. The DOM derived from the macrophyte-dominated region had a higher binding ability than that derived from the entrance of the Dawen River, the open area of Lake Dongping, and the tourism district. This is substantiated by the PDOM having a higher log K value than EDOM, ODOM and TDOM (Table 1). The higher binding ability in PDOM than in EDOM, ODOM and 14

Journal Pre-proof TDOM might be related to the death and decomposition of P. crispus. Lake Dongping has kinds of vegetation types, such as Cyperus, Trapa, Typha, Nymphea, P. crispus, Lemna, etc. (Deng et al., 2016). Lake Dongping is dominated by P. crispus and P. crispus is mainly distributed in the macrophyte-dominated region. P. crispus grows in winter and spring (December-May) and dies in autumn (September-November) (Deng et al., 2016). Over 80% of the lake area is controlled by P. crispus in May (Zhang et al., 2009). In late May and early July (early summer), P. crispus begins to die and decompose, which causes the deterioration of the aquatic ecological environment and the massive death of fishes (Deng et al., 2016). Previous studies have reported that macrophytes can generate a large amount of decomposed organic matter, especially in winter, releasing DOM into the aquatic environment and increasing water turbidity (Yuan et al., 2015; Zhang et al., 2018). In summary, our results collectively suggest that the decrease in the protein-like components in DOM with the addition of Zn is induced by the interaction between the protein-like component and Zn and the formation of DOM-metal complexes. The increase in humic-like components in DOM with the addition of Zn might be related to the replacement of the original DOM-quencher complexes by the formation of more stable DOM-Zn complexes. The macrophyte-dominated region would play a significant role in affecting the bioavailability, toxicity and mobility of heavy metals compared to the other regions in Lake Dongping..

5. Conclusions A total of three fluorescent components, humic-like (component 1 and 3) and protein-like (component 2) components, were identified by EEM-PARAFAC. The binding order of DOM-Zn is the tyrosine-like fraction > the tryptophan fraction > the humic-like fraction. The fluorescence 15

Journal Pre-proof intensity of the protein-like component was quenched, and the humic-like component was enhanced with the addition of Zn. DOM collected from the macrophyte-dominated region had a stronger quenching constant than DOM collected from other areas of Lake Dongping. Moreover, the addition of Zn increased the humification of DOM. The present study provides deep insight into the impact of heavy metals on DOM spectral properties and the effect of DOM on the behavior, mobility and bioavailability of heavy metals in lake sediments.

Acknowledgments We gratefully thank the National Natural Science Foundation of China (41301544) and Open research Foundation of the State Key Laboratory of Lake Science and Environment, Chinese Academy of Sciences (2018SKL004) for their financial support on this study.

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Journal Pre-proof of silver in Potamogeton crispus L. Journal of Hazardous Materials 173, 186–193. Yamashita, Y., Jaffe, R., 2008. Characterizing the interactions between trace metals and dissolved organic matter using excitation-emission matrix and parallel factor analysis. Environmental Science & Technology 42, 7374–7379. Yamashita, Y., Jaffe, R., Maie, N., Tanoue, E., 2008. Assessing the dynamics of dissolved organic matter in coastal environments by excitations emission matrix fluorescence and parallel factor analysis. Limnology and Oceanography 53, 1900–1908. Yuan, D.H., Guo, X.J., Wen, L., He, L.S., Wang, J.G., Li, J.Q., 2015. Detection of Copper (II) and Cadmium (II) binding to dissolved organic matter from macrophyte decomposition by fluorescence excitation-emission matrix spectra combined with parallel factor analysis. Environmental Pollution 204, 152–160. Zhang, J., Duan, D., Wang, Z., 2009. Harm and control measures for the decomposition of Potamogeton crispus in Lake Dongping. Environmental Study Monitoring 2, 31–33. Zhang, L.S., Zhang, S.H., Lü, X.Y., Zheng, Q., Zhang, Z.Q., Yan, L.Y., 2018. Dissolved organic matter release in overlying water and bacterial community shifts in biofilm during the decomposition of Myriophyllum verticillatum. Science of the total environment 633, 929–937. Zhou, Y.Q., Zhang, Y.L., Shi, K., Niu, C., Liu, X.H., Duan, H.T., 2015. Lake Taihu, a large, shallow and eutrophic aquatic ecosystem in China serves as a sink for chromophoric dissolved organic matter. Journal of Great Lakes Research 41, 597–606. Zhou, Y.Q., Zhou, J., Jeppesen, E., Zhang, Y.L., Qin, B.Q., Shi, K., Tang, X.M., Han, X.X., 2016. Will enhanced turbulence in inland waters result in elevated production of autochthonous dissolved organic matter? Science of the total environment 543, 405–415. 19

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Figure captions Fig. 1. Map of Lake Dongping showing locations of sampling sites, which were divided into the entrance of Dawen River (sites 1-3), open area of the lake (sites 4-6), the macrophyte-dominated region (sites 7-9) and the tourism district (sites 10-12). Fig. 2. Four regions, the entrance of the Dawen River, the open area of the lake, the tourism district and the macrophyte-dominated region, were divided according to PC1 and PC2 scores (a). Dot color denotes the relative abundance of humic-like material (a). Correlation between relative abundance of humic-like material and PC1 (b), correlation between a(350) and PC2 (c). Dot color denotes the fluorescence intensity of DOM in both (b) and (c). Fig. 3. Examples of EEM spectra of EDOM (a), ODOM (b), PDOM (c) and TDOM (d). Fig. 4. Spatial distributions of the absorption coefficient a(350) (m-1) (a); M (arbitrary unit) (b); total fluorescence intensity (AU) (c); relative abundance of fluorescence intensities C1 (%) (d), C2 (%) (e), and C3 (%) (f); fluorescence index FI (g); BIX (h); and HIX (i) in Lake Dongping, November 2018. Fig. 5. Three fluorescent components (C1 – C3), split-half validated and calculated from the PARAFAC model. Each EEM was normalized to its maximum fluorescence prior to modeling. The spectral properties of the three components (three upper panels) and the split-half validation results (three lower panels) are given. Fig. 6. UV-vis absorption (a-d) and synchronous fluorescence spectra (e-h) of EDOM, ODOM, PDOM and TDOM with increasing concentrations of Zn. 20

Journal Pre-proof Fig. 7. Typical synchronous and asynchronous 2D fluorescence correlation maps generated from the 200 to 450 nm region of the EDOM, ODOM, PDOM and TDOM with zinc addition. (a) Synchronous map for EDOM; (b) asynchronous map for EDOM; (c) synchronous map for ODOM; (d) asynchronous map for ODOM; (e) synchronous map for PDOM; (f) asynchronous map for PDOM; (g) synchronous map for TDOM; (f) asynchronous map for TDOM. Red represents positive correlations, and blue represents negative correlations; higher color intensity indicates a stronger positive or negative correlation. Fig. 8. Variations in fluorescent intensities of the PARAFAC-derived components and in ratios of humic-like components to the total fluorescence of DOM with increasing metal addition.

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Fig. 1. Map of Lake Dongping showing locations of sampling sites, which were divided into the entrance of Dawen River (sites 1-3), open area of the lake (sites 4-6), the macrophyte-dominated region (sites 7-9) and the tourism district (sites 10-12).

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Fig. 2. Four regions, the entrance of the Dawen River, the open area of the lake, the tourism district and the macrophyte-dominated region, were divided according to PC1 and PC2 scores (a). Dot color denotes the relative abundance of humic-like material (a). Correlation between relative abundance of humic-like material and PC1 (b), correlation between a(350) and PC2 (c). Dot color denotes the fluorescence intensity of DOM in both (b) and (c).

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450 c a) EDOM

450 c b) ODOM

400

400

350

350

300 Protein-like 250 fluorophores

200

300

350

Ex. (nm)

Ex. (nm)

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Humic-like fluorophores

400 450 Em. (nm)

500

550

300

350

Humic-like fluorophores

400 450 Em. (nm)

300 350 450 400 c 350 450 300 250 200 d) TDOM

500

550

400

600

450

400

Ex. (nm)

400

Ex. (nm)

Protein-like 250 fluorophores

200

600

450 c c) PDOM

350 300 Protein-like 250 fluorophores

200

300

300

Fluorescence intensity (AU)

350

Humic-like fluorophores

350 300 250 Protein-like

Humic-like fluorophores

fluorophores

400 450 Em. (nm)

500

550

200

600

300

350

400 450 Em. (nm)

500

550

600

0 0.0630.13 0.25 0.38 0.50 0.75 1.0 1.5 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 9.5 10

Fig. 3. Examples of EEM spectra of EDOM (a), ODOM (b), PDOM (c) and TDOM (d).

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500

550

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Fig. 4. Spatial distributions of the absorption coefficient a(350) (m-1) (a); M (arbitrary unit) (b); total fluorescence intensity (AU) (c); relative abundance of fluorescence intensities C1 (%) (d), C2 (%) (e), and C3 (%) (f); fluorescence index FI (g); BIX (h); and HIX (i) in Lake Dongping, November 2018.

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Fig. 5. Three fluorescent components (C1 – C3), split-half validated and calculated from the PARAFAC model. Each EEM was normalized to its maximum fluorescence prior to modeling. The spectral properties of the three components (three upper panels) and the split-half validation results (three lower panels) are given.

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c) PDOM 2.4 2.2 2.0 1.8 1.6 1.4 Zn addition 1.2 1.0 0.8 0.6 0.4 0.2 0.0 200 250 300 350 Wavelength (nm)

Fluorescence intensity (AU)

450

400

d) TDOM 2.4 2.2 2.0 1.8 1.6 1.4 1.2 Zn addition 1.0 0.8 0.6 0.4 0.2 0.0 200 250 300 350 400 Wavelength (nm)

450

450

e) EDOM 2200 TYF TRF HUF 2000 270 Zn 0  1800 Zn Zn 40  1600 Zn 80  addition 1400 300 Zn 120  1200 Zn 160  350 1000 Zn 200  800 Zn 240  Zn 280  600 230 400 200 200 250 300 350 400 450 Wavelength (nm)

f) ODOM TYF TRF 270 Zn 300 800 addition

1000

600 400

HUF

350

230

200 200

Fluorescence intensity (AU)

b) ODOM 2.4 2.2 2.0 1.8 1.6 Zn addition 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 200 250 300 350 400 Wavelength (nm)

Fluorescence intensity (AU)

a) EDOM 2.4 2.2 Zn 0  2.0 Zn 40  1.8 Zn 80  1.6 1.4 Zn 120  Zn addition 1.2 Zn 160  1.0 Zn 200  0.8 0.6 Zn 240  0.4 Zn 280  0.2 0.0 200 250 300 350 400 450 Wavelength (nm)

250

300 350 400 Wavelength (nm)

g) PDOM TYF TRF 270 300 1000 Zn addition 800 1200

450

HUF 350

600 400 210 200 200

Fluorescence intensity (AU)

Absorbance

Absorbance

Absorbance

Absorbance

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250

300 350 400 Wavelength (nm)

h) TDOM TYF TRF 270 1200 Zn addition 1000 1400

450

HUF

800 360

600 400 200 200

230 250

300 350 400 Wavelength (nm)

450

Fig. 6. UV-vis absorption (a-d) and synchronous fluorescence spectra (e-h) of EDOM, ODOM, PDOM and TDOM with increasing concentrations of Zn.

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Fig. 7. Typical synchronous and asynchronous 2D fluorescence correlation maps generated from 28

Journal Pre-proof the 200 to 450 nm region of the EDOM, ODOM, PDOM and TDOM with zinc addition. (a) Synchronous map for EDOM; (b) asynchronous map for EDOM; (c) synchronous map for ODOM; (d) asynchronous map for ODOM; (e) synchronous map for PDOM; (f) asynchronous map for PDOM; (g) synchronous map for TDOM; (f) asynchronous map for TDOM. Red represents positive correlations, and blue represents negative correlations; higher color intensity

0

C2

C3

50 100 150 200 250 300 Zinc concentration (M)

c) PDOM

6 5 4 3 2 50 100 150 200 250 300 Zinc concentration (M) Humic-like component / Total fluorescence

0

0.70 0.68 0.66 0.64 0.62 0.60 0.58 0.56 0.54 0.52 0.50 0.48 0.46 0.44 0.42 0.40 0.38 0.36 0.34

Fluorescent intensities score (AU)

C1

4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2

Fluorescent intensities score (AU)

a) EDOM 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0

Fluorescent intensities score (AU)

Fluorescent intensities score (AU)

indicates a stronger positive or negative correlation.

6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0

b) ODOM

0

50 100 150 200 250 300 Zinc concentration (M)

d) TDOM

0

50 100 150 200 250 300 Zinc concentration (M)

e) 1# 2# 3# 4# 5# 6# 7# 8# 9# 10# 11# 12# 0

50

100 150 200 Zinc addition (M)

250

300

Fig. 8. Variations in fluorescent intensities of the PARAFAC-derived components and in ratios of 29

Journal Pre-proof humic-like components to the total fluorescence of DOM with increasing metal addition. Table Table 1. Fluorescence intensity of three components in sediment DOM and fluorescence quenching parameters of zinc bound to C2. The quenching of C2 was fitted by a modified Stern-Volmer equation to estimate their effective quenching constants. The values of r2 showed that the model described the fluorescence quenching well.

Origin

EDOM

ODOM

PDOM

TDOM

Fluorescence

Relative

Intensity

Abundance

(AU)

(%)

C1

4.98±1.63

33±2.30

C2

7.66±1.76

52±3.43

C3

2.23±0.82

C1

Log K

r2

(Mean)

(Mean)

-

-

3.47-4.20

0.91-0.99

(3.95±0.42)

(0.96±0.04)

15±3.64

-

-

3.70±0.43

40±0.22

-

-

C2

4.08±0.29

45±1.59

3.53-3.69

0.65-0.95

(3.59±0.09)

(0.85±0.17)

C3

1.38±0.28

15±1.37

-

-

C1

6.06±0.70

42±1.79

Component

-

-

3.92-4.57

0.76-0.92

(4.32±0.35)

(0.85±0.08)

C2

5.56±0.30

39±2.74

C3

2.71±0.80

19±3.31

-

-

C1

3.34±0.71

27±3.84

-

-

C2

7.99±3.40

63±3.03

3.38-4.25

0.97-0.99

(3.68±0.50)

(0.98±0.01)

C3

1.27±0.59

10±0.95

-

-

30

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Declarations of interest: none

Journal Pre-proof 1. Different Zn-DOM binding affinities in different lake regions were investigated. 2. Lake Dongping was divided into four regions based on PCA. 3. Protein-like component was quenched and humic-like component was released with Zn addition. 4. The interaction of Zn with PDOM was stronger than with EDOM, ODOM and TDOM.