2.1 UV-Visible Absorption Spectral Characteristics of DOM
The parameter SUVA254 is positively correlated with the humification level of DOM in the river water (Weishaar et al., 2003). Figure 2(a) shows that the SUVA254 of water is significantly higher during the wet season (7.20±1.22) L·(mg·m) -1 compared to the dry season's (4.29±1.34) L·(mg·m) -1 (P<0.01). During the wet season, terrestrial DOM enters the water with the rainfall, leading to an increase in the relative molecular weight, aromaticity, and humification level of DOM in the water.
The E2/E3 ratio represents the absorbance ratio at 254 nm and 365 nm. When E2/E3 < 3.5, it indicates that DOM is mainly composed of organic matter with larger molecular weights. When E2/E3 > 3.5, it indicates that DOM is mainly composed of fulvic acid with relatively smaller molecular weights(Ma and Li, 2020). Figure 2(b) shows that the E2/E3 ratio is significantly lower during the wet season (4.27±0.69) compared to the dry season (6.54±1.18) (P<0.01). Therefore, in Huai River surface water, DOM is mainly composed of fulvic acid with smaller molecular weights. Given the reduced level of biological activity in the water, there a stable presence of small molecular organic compounds, such as fulvic acid.
The parameter SR also helps indicate the source of DOM. When SR > 1, it indicates that biological sources most dominant. When SR <1, it indicates that DOM mainly originates from terrestrial animal and plant residues, and organic matter generated by human activities and other exogenous inputs (Kenneth, 2009). Figure 2(c) shows that the SR values of the Huai River are all less than 1; the SR is significantly higher during the wet season (0.97±0.18) compared to the dry season (0.86±0.074) (P<0.01). During the wet season, the increase in rainfall and runoff leads to a higher exogenous input of DOM into the Huai River surface water.
The differences in the UV-Visible absorption spectral characteristics of DOM between the dry and wet seasons are mainly attributed to seasonal hydrological dynamics and changes in exogenous inputs. The increased rainfall during the wet season leads to added organic matter with a higher degree of humification. This significantly affects the humification level and molecular weight characteristics of DOM in the water.
Characteristics of DOM Three-Dimensional Fluorescence Spectra
2.2.1 Parallel Factor Analysis (PARAFAC)
Using EEM-PARAFAC, three fluorescence components were identified in both wet and dry season water DOM. The model's reliability was confirmed through half-split validation (TTC>0.99) and residual analysis (Figure 3). For the wet season samples, Component 1 (C1) shows a single excitation and emission peak at Em/Ex = 400/240 nm; Component 2 (C2) shows an excitation and emission main peaks at Em/Ex = 450/360 nm and a secondary peak at Em/Ex = 450/260 nm; and Component 3 (C3) shows a single excitation and emission peak at Em/Ex = 290/210 nm. In contrast, for the dry season samples, C1 shows an excitation and emission main peaks at Em/Ex = 410/240 nm and a secondary peak at Em/Ex = 410/290 nm; C2 shows an excitation and emission main peaks at Em/Ex = 450/360 nm and a secondary peak at Em/Ex = 450/270 nm; and C3 shows an excitation and emission main peaks at Em/Ex = 335/280 nm and a secondary peak at Em/Ex = 335/230 nm. In the wet season samples, the C1 peak at Ex=240 nm, located in the short-wave ultraviolet region, indicates the material is an UVC-like fulvic acid, with fluorescence at the traditional "A" peak(Phoebe et al., 2019) . The C2 main peak at Ex=360 nm indicates the material is a terrestrial humic acid component; it is a visible light-range humic substance, located at the traditional "C" peak. The C2 secondary peak Ex=260 nm is similar to C1, and indicates the material is an UVC-like fulvic acid(Phoebe et al., 2019, Kim et al., 2020) . The C3 peak indicates the material is the aromatic protein tyrosine, sensitive to photochemical reactions and microbial degradation. This serves as an important indicator of human activities (Jeonghyun et al., 2021, Lee, 2020) .
In the dry season samples, C1's main peak at Ex=410/240 nm indicates a traditional "A" peak(Phoebe et al., 2019);the secondary peak at Ex=290 nm is located at the traditional "M" peak (Dalmagro et al., 2019).Together, these results indicate the material is UV-like fulvic acid. C3's main peak at Ex=280 nm and the secondary peak at Ex=230 nm are located at the traditional "T" peak, and indicates the material is a protein-like substances, tyrosine (Jeonghyun et al., 2020).The C2 peak is similar to the wet season's C2 peak, both representing the presence of terrestrial humic acid material (Table 1).
Overall, C1 and C2 peaks indicate the DOM samples contain humic substances, primarily influenced by terrestrial inputs, aquatic biological activities, and photochemical reactions. Protein-like components, including, tyrosine in the wet season and tryptophan in the dry season (indicated by C3 peak), are mainly produced by aquatic microbial and flora activities. These are generally linked to endogenous processes in water bodies, and associated with effluents discharged from human activities.
Table1. Characterization of the fluorescence component of DOM in water samples from wet and dry seasons
Components
|
Ex/Em
|
Component type
|
Reference
|
Wet season
|
C1
|
240/400
|
UVC fulvic-like
|
(Phoebe et al., 2019)
|
C2
|
360(260)/450
|
humic-like
|
(Phoebe et al., 2019, Kim et al., 2020)
|
C3
|
210/290
|
tyrosine
|
(Jeonghyun et al., 2021, Lee, 2020)
|
Dry season
|
C1
|
240(290)410
|
UV fulvic-like
|
(Phoebe et al., 2019, Dalmagro et al., 2019)
|
C2
|
360(270)/450
|
humic-like
|
(Kim et al., 2020, Phoebe et al., 2019)
|
C3
|
280(280)/335
|
tryptophan-like
|
(Jeonghyun et al., 2020)
|
Figure 4 shows that for the wet season samples, the mean fluorescence intensities of C1, C2, and C3 were (0.33±0.08), (0.23±0.04), and (0.24±0.07) R.U., respectively. For the dry season samples, the mean fluorescence intensities were (0.42±0.05), (0.13±0.03), and (0.32±0.03) R.U., respectively. This indicates that humic substances (C1+C2) are the main contributing components of river DOM. During the wet season, increased water activity due to rainfall promotes hydrodynamic conditions in the river, leading to the migration and mixing of organic matter in the river channel. This results in significantly lower levels of components C1 and C3 during the wet season compared to the dry season (P<0.01). Component C2shows significantly higher levels during the wet season than the dry season (P<0.01), due to DOM from surrounding soils entering the water body with surface runoff. Figure 5 indicates that the Huai River DOM is primarily composed of humic substances: humic substances (C1+C2) account for 70% of the total fluorescence intensity in the wet season samples and 63% in the dry season samples. Specifically, in the wet season samples, of the total fluorescence intensity, UV-like fulvic acid (C1) accounts for 42%, terrestrial and UV-like fulvic acid (C2) accounts for 28%, and tyrosine-like protein (C3) accounts for 30%. In the dry season samples, of the total fluorescence intensity, UV-like fulvic acid and marine-derived fulvic acid (C1) account for 48%, terrestrial and UV-like fulvic acid (C2) account for 15%, and tryptophan-like protein (C3) accounts for 37%.
During the dry season, the Huai River water is heavily affected by human activities, receiving sewage generated from surrounding industrial, agricultural, shipping, and residential sources. This results in intensified microbial activity in the water, leading to an increase in tryptophan component levels. Studies have shown that tyrosine-like components are closely related to microbial activity. There are higher microbial levels in the water during the wet season, and microorganisms in the water grow and reproduce faster due to higher temperatures(Baker, 2002). Natural water bodies contain higher levels of humic substances. During the dry season, poor water flow exacerbates the impact of human disturbances, leading to higher levels of protein-like substances in the water. During the wet season, rainfall leads to increased levels of terrestrial humic substances in the Huai River compared to the dry season; the origins include soil runoff and plant decomposition. Research by Baker et al. (2004) found that the influence of human activities causes a humic peak in river water DOM samples to gradually shift towards a protein-like peak. This indicates that human activities have a greater endogenous impact on the Huai River during the dry season (Baker and Spencer, 2004).
2.2.2 Self-Organizing Map (SOM)
Using the "somtoolbox" toolbox in Matlab 2022b, SOM models were constructed for the EEM data. A SOM was trained with 50 samples from both wet and dry seasons This resulted in a final (3×12) neuron grid based on the 5√n rule, with additional mapping units included in the U-matrix to represent different distances between neurons (Figure 7). The K-means algorithm and Davies-Bouldin index (DBI) indicated the DBI was lowest when the cluster number was set to 2. This divided the fluorescence spectra into two regions (Fig 6).
The SOM model adds labels to similar EEMs data points, and retains labels with higher hit counts. Figure 8 shows, that wet season samples are mainly distributed in the upper part of the map; dry season samples are distributed in the lower part of the map. The hit counts of neurons indicate that neurons located at the edges of the map have higher hit counts. Neuron 1, with 6 hits, represents the fluorescence characteristics of wet season water samples. Neuron 12, with 7 hits, represents the fluorescence characteristics of dry season water samples. Figure 9 shows the visual results between two neurons, and indicates similar colors in the corresponding components. This indicates a correlation between neuron 1 and neuron 12.
Neuron 1 exhibits excitation and emission peaks at Em/Ex=440/260 nm, with a secondary peak at Em/Ex=420/240 nm. Neuron 12 shows a single excitation and emission peak at Em/Ex=410/250 nm (Fig 10). The fluorescence signals of neurons 1 and 12 are located at the traditional "A" peak (Yao et al., 2011). This indicates the presence of UV-type humic substances, which are small molecular weight organic compounds with effective degradation and stable structures. These components represent humic substances of terrestrial origin, increasing in level with an increase in nutrient inputs from riverine sources.
The Huai River Basin is significantly influenced by agricultural activities. Studies have shown that applying nitrogen fertilizer in farmland can increases microbial activity in the soil enhancing the degradation capacity of DOM by releasing oxidases and accelerating the degradation of protein-like substances. This increases the level of soil humification (Waldrop, 2008). However, the Huai River basin is mainly composed of plain areas, where rainfall and agricultural runoff lead to a large amount of terrestrial DOM input into the Huai River. This leads to an increase in the fluorescence intensity of humic substances. The results of the SOM model are similar to those of the PARAFAC model, indicating that humic substances dominate the Huai River water.
2.3Fluorescence Parameter Analysis
To further analyze the spectral characteristics of DOM in the Huai River during the wet and dry seasons, this study included an analysis of fluorescence indices, including the Biological Index (BIX), Fluorescence Index (FI), and Humification Index (HIX). The results are in Table 2. The BIX (0.85±0.07) is significantly lower for wet season samples compared to dry season samples (1.02±0.05) (P<0.01). Studies have found that when the BIX value ranges from 0.6 to 0.8, external contributions to DOM are dominant. Values between 0.8 and 1.0 indicate both internal and external sources are present. When the value exceeds 1.0, internal sources are dominant(Huiying et al., 2022). In this study, the BIX values in wet season samples range between 0.8 and 1.0, indicating that the main source of DOM in the water body is from rainfall transporting humic substances from the soil into the water. This is coupled with increased microbial activity due to temperature effects, leading to increased internal sources. In contrast, the BIX values in dry season samples exceed 1.0, indicating internal sources are dominant, as the reduced flow rate during this period decreases external inputs.
FI is a fluorescence index of DOM that reflects the source of humic substances(Wickland et al., 2007). When FI is less than 1.2, external humic substance sources are dominant. When FI is between 1.2 and 1.8, both internal and external sources contribute. When FI exceeds 1.8, internal sources prevail. In this study, FI values in wet and dry season samples are1.72±0.05 and 1.73±0.09, respectively. This indicates that DOM in the Huai River water is influenced by both internal and external sources. Furthermore, the HIX reflects the degree of humification of DOM(Ohno, 2002), indicating a lower humification level in the Huai River water. The HIX value (0.73±0.05) is significantly higher in wet season samples compared to dry season samples (0.70±0.03) (P<0.05). This indicates a lower humification level during the dry season compared to the wet season.
Table2. Parameters of fluorescence fractions during wet and dry seasons
|
BIX **
|
FI ns
|
HIX *
|
Wet Season
|
0.85±0.07
|
1.72±0.05
|
0.73±0.05
|
Dry Season
|
1.02±0.05
|
1.73±0.09
|
0.70±0.03
|
Note: ns indicates not relevant, * indicates P<0.05, ** indicates P<0.01
In summary, this study analyzed fluorescence indices of DOM in Huai River during wet and dry seasons, including BIX, FI, and HIX. There are significant differences seen between the two seasons. The higher BIX value in wet season samples suggests that DOM is primarily influenced by external sources, likely due to the influx of humic substances from soil caused by rainfall and increased microbial activity. In contrast, the BIX values in the dry season samples indicate a dominance of internal sources, likely influenced by reduced water flow and decreased external inputs. The FI results indicate that DOM in both the wet and dry season samples is influenced by both internal and external factors. The variation in HIX values may result from a combination of factors, such as water flow velocity, external inputs, meteorological factors, and soil humic substance inputs. This leads to a lower degree of humification during the dry season compared to the flood season.
2.4 Principal Component Analysis (PCA)
Principal Component Analysis (PCA) was performed to explore the relationship between DOM spectral parameters and physicochemical water quality indicators, using the Adonis test. A selection of parameters was analyzed. The results indicated that the Kaiser-Meyer-Olkin (KMO) measure of sampling adequacy was 0.728, and the Bartlett's test of sphericity was significant (Sig <0.001). These outcomes indicate the data were suitable for PCA. The results are shown in Figure 11. The cumulative contribution rate was 68.3%.PC1 explained 50.9% of the variance, and PC2 explained 17.4% of the variance. Figure 11 (b) shows that the Adonis multivariate analysis of variance (based on Euclidean distance) was conducted on the grouping situation with 999 permutations (P<0.001) (Iman A. Sylvain, 2019).
The figure shows that variables such as E2_E3, BIX, DO, and C1% are located in the positive loading direction of PC1 axis. During the dry season, dissolved oxygen (DO) stimulates microbial activity in the water, leading to an increase in autochthonous sources. This causes large molecular DOM to decompose into smaller molecules. During the wet season, the DO in the water decreases. Additionally, the rainfall during the wet season may dilute the organic matter and microorganisms in the water. This slows microbial activity and reducing reduces the contribution of autochthonous sources. This difference results in the distinction between the dry and wet seasons on the PC1 axis. Variables such as C2%, a254, a355, SUVA254, and pH are located in the negative loading direction of the PC1 axis.
During the wet season, rainfall washes through the soil, bringing humic substances from the surrounding soil into the water. For example, increased rainfall washes organic matter from the soil surface; with the onset of the rainy season, organic matter produced from decaying vegetation is washed into the water, increasing the level of in humic substances. The organic acids and basic functional groups in the humic substances act as buffers. When acid-base reactions occur in the water, the organic acids and basic functional groups absorb or release hydrogen ions, slowing down the pH changes. On the PC2 axis, HIX and DOC have strong positive loadings, while C3% has a strong negative loading. C3% (protein-like substances) appears to have a greater influence during the dry season.