**3. Results and Discussion**

### *3.1. Comparison of Water Quality and Biotic Indices*

Water quality parameters and biotic indices were compared in nine subwatersheds between May and September. In the temporal scale, the nitrogen level was slightly higher in May than in September (Figure 3). It appeared that a significant amount of nutrients entered the stream by summer rainfall (see Figures 2 and 3). We speculate that incoming nutrients are more dissolved forms of nitrogen and phosphorus than particulate, because we observed frequent increases of NH3–N, NO3–N, and PO4–P (Figure 3). The previous studies have reported that the high flux of NO3–N could be driven by specific agricultural activities, such as manure applications and conservational tillage [37,38]. Nonetheless, TN concentrations exhibited a gradual decrease from upstream to downstream (Figure 3), and specifically exhibited approximately from 2 mg L−<sup>1</sup> at sites SDU, SDL, and OS, to 1 mg L−<sup>1</sup> at sites SG and SL. A study by Ahn [39] reported that unexpectedly high nutrient concentrations in headstreams are often observed because of the relatively undeveloped wastewater treatment and septic tanks in the rural area of South Korea, thereby inducing an increase of BOD. In contrast to TN and NO3–N, NH3–N concentrations were very high at the SC site, regardless of time (Figure 3). Given the largest fraction (49%) of cropland among nine subwatersheds, the high ammonia concentrations could be associated with agricultural activities. In China, which is geographically close to Korea, it was reported that agricultural nitrogen accounted for more than 40% of the variability of TN, and subsequently it drives ammonia increases [40,41].

The phosphorus pattern was spatially similar to the nitrogen pattern in May. The level of TP concentrations gradually decreased from upstream to downstream, except at the headstream SDU site in May (Figure 3). However, it was comparatively notable that phosphorus concentrations rebounded in the lower part of the river, such as at the BS and SL sites. This longitudinal trend could be consistent with the fact that the lower part of the Seomjin River was dominated by agricultural lands (38.8% at the IS, SC, YC, BS, and SL sites) and urban areas (6.6% at the YC and BS sites) relative to the upper part (27% at the SDU, SDL, SG, and JD sites, and 3.3% at the SDU, SDL, and JD sites) (see Figure 1). In relation to agricultural managemen<sup>t</sup> practices (e.g., manure applications) and urbanization, a large amount of dissolved phosphorus could be generated [42,43].

Chl-*a* concentrations were also slightly higher at the upper part than at the lower part of the river. However, based on the current level of Chl-*a* concentrations, the Seomjin River changed from being oligotrophic to mesotrophic (Chl-*a* < 10 μg <sup>L</sup>−1). The BOD values mostly ranged from 1 to 3 mg <sup>L</sup>−1, and appear to differ from the spatial pattern of nutrient concentrations (Figure 3). Additionally, we also compared five biotic indices, and most of the values of biotic indices were not statistically significant across all the study sites (Figure 3). Thus, it was difficult to distinguish the spatial pattern of benthic communities based merely on the biotic indices.

**Figure 3.** Comparison of water quality and biological indices between May and September. Error bars indicate standard errors. Asterisks represent statistical significances at *P* < 0.05. (**a**) TN; (**b**) NH3-N; (**c**) NO3-N; (**d**) TP; (**e**) PO4-P; (**f**) Chl-*a*; (**g**) BOD; (**h**) DI; (**i**) EI; (**j**) H'; (**k**) RI; (**l**) BMI.

### *3.2. Spatial Distribution of Benthic Macroinvertebrate Communities Before and After Summer Rainfall*

The distribution of benthic macroinvertebrate communities was spatially distinct (Figure 4). At all nine of the subwatersheds, the abundance of benthic macroinvertebrates decreased after the summer rainfall in September. A decreasing level of benthic communities differed from the location of subwatersheds. It was a particularly remarkable pattern that while the abundance of *Gammarus* spp. (Amphipoda) slightly increased, the abundance of *Asellus* spp. (Isopoda) dramatically decreased after the heavy rainfall (Figure 4). Moreover, the relative abundance decreased from 41% to 3.3% (Table 1). However, in this respect, *Gammarus* spp. also dramatically increased in terms of relative abundance (26.6% in May to 69.6% in September, Table 1). This pattern was clearer at the SDU site, which was a forest-dominated area. We could not find evidence to support their inverse abundance pattern associated with rainfall, especially in a forest area. To understand their relationships and ecological

interactions, a long-term monitoring is highly required to depict the inter-annual variation in specific land-use coverage.

**Figure 4.** Average abundance of benthic macroinvertebrate communities based on (**a**) order and (**b**) subcategory (family, genus, and species) in Seomjin River.

In relation to unfavorable benthic macroinvertebrates in polluted environments, it was found that Diptera (Chironomidae and *Simulium* spp.) prevailed across the Seomjin River watershed (Figure 3). However, it was also observed that their abundance was significantly low in September after summer rainfall. Chironomidae abundance consistently decreased across all of the study sites except for SDL. A slight increase (21.9% to 24.1%) of the abundance might be longitudinal flush effects, since the SDL site was located downstream of the Seomjin Dam. *Simulium* spp. also decreased after summer rainfall, but their low abundance appeals to further surveys over a long term.

On the other hand, the key benthic macroinvertebrates such as Coleoptera and Ephemeroptera were scrutinized. Interestingly, the abundance patterns of *Elmidae* spp. and *Eubrianax* spp. were opposite between May and September. The former was commonly higher in May, while the latter was higher in September (Table 1). Ephemeroptera were slightly more abundant in September. Particularly, *Ecdyonurus* spp. abundance was distinctly high in September relative to the other Ephemeroptera (Table 1). The most dominant Ephemeroptera, *Baetis* spp., showed irregular spatial pattern in their abundance. Interestingly, the longitudinal pattern of abundance looked like a zigzag, which implies that *Baetis* spp. (e.g., larva) could be influenced serially from upstream to downstream by hydrological factors. Another key group Trichoptera showed higher abundance, particularly at the SDL and OS sites (Table 1). *Hydropsyche* spp. appeared to be sensitive to summer rainfall, while *Cheumatopsyche* spp. seem to be more tolerant.





### *3.3. Association among Benthic Macroinvertebrates, Land-Use Coverage, and Ambient Water Quality*

The CCA simplified the relationships among the variables of our interest (Figure 5). The CCA results explicitly accounted for 61% of the relational variability with two primary ordination axes; the first axis (45.5%) was related to land-use factors, and the second axis (15.5%) appeared to be related to water quality. The first ordination axis characterized the gradient of land-use coverage (Figure 5). The land-cover gradient was mainly separated by forest and agricultural/cropland areas. Urban land is topographically placed in the middle between forest and cropland. Wetland is close to cropland, which is reasonable because extensive agricultural areas have been converted from riverine wetlands by constructing levees in South Korea [44,45]. The second ordination axis depicted the gradient of water quality parameters. On the whole, the upper part of plot is closely associated with the parameters related to water quality deterioration, such as the higher concentration level of BOD, Chl-*<sup>a</sup>*, TP, and TN (Figure 5). It was notable that the ammonia (NH3–N) concentration showed an inverse pattern against the other water quality parameters. This pattern appears to be associated with agriculture and pasture. In contrast, the NO3–N concentration showed a weak relationship with agricultural activities including grassland/pasture, while the PO4–P concentration had close association (Figures 5 and 6a). As previous mentioned, higher PO4–P concentrations were apparently observed in the lower part of the river (Figure 3). Thus, the CCA ordination depicted the disparate responses of nitrogen and phosphorus dynamics in the lower part of the Seomjin River.

**Figure 5.** Results of data ordination based on canonical correspondence analysis (CCA). Comparison of land use and water quality with benthic macroinvertebrate communities; (**a**) 14 groups, and (**b**) eight Ephemeroptera groups.

**Figure 6.** (**a**) CCA data ordination associated with data samples and ambient environmental factors. (**b**) Dissimilarity of data attributes between May and September.

In the ordination pattern of key benthic macroinvertebrates (e.g., EPTC taxa: E = Ephemeroptera, P = Plecoptera, T = Tricoptera, and C = Coleoptera), the major Coleoptera groups, *Elmidae* spp.

and *Eubrianax* spp., were distinctly di fferentiated according to water quality. These two genera, especially the ri ffle beetles *Elmidae*, are typically known as a large group of aquatic Coleoptera that are generally indicators of good water quality, because of their sensitivity to changes in the surrounding environmental conditions [46,47]. Nonetheless, the association of *Elmidae* with several water quality signals to eutrophic conditions (PO4-P, Chl-*<sup>a</sup>*, and BOD) was unexpected (Figure 5a). This counterintuitive pattern may be a confounding e ffect stemming from the spatial migration of larvae. However, there have been some evidence that *Elmidae* can be distributed in a wide range of nutrient conditions [48]. We speculate that *Elmidae* can prevail to some extent, since the Seomjin River watershed is mainly dominated by forest (48% on average, see Figure 1). The Tripcoptera groups, *Hydropsyche* spp. and *Cheumatopsyche* spp., were separately characterized. It appears that *Hydropsyche* is more related to Chl-*a* than *Cheumatopsyche*. There have been several literatures on food preferences and niche partitioning among these species [49,50]. We understand the separate pattern of these species, in the sense that Hydropsyche larvae prefer higher-velocity microhabitats and mainly digest detritus and benthic diatoms [51]. Another key benthic macroinvertebrate Ephemeroptera generally tended to inhabit good water quality conditions (Figure 5b). However, they seemed to be apart from mountainous areas (i.e., forest areas). Within the same family Baetidae, *Baetiella* spp. was more associated with wetland habitat, while *Baetis* spp. was more associated with ammonia concentration. Particularly, it was found that *Baetis* spp. was less sensitive to ammonia toxicity than other mayflies [52]. We infer that the linkage between *Baetis* spp. and ammonia concentration is highly associated with their tolerance. In this context, *Ephemera* spp. and *Caenis* spp. seem to inhabit in a similar water quality condition (Figure 5b). The spatial ordination of *Labiobaetis* spp. was clearly distinct, which appears to be related to good water quality. The other Ephemeroptera, such as *Choropterpes* spp., *Epeorus* spp., and *Ecdyonurus* spp., were placed on a mixture of wetland, cropland, and urban areas. However, there was little evidence to advocate their association with surrounding environmental conditions.

The dominant Diptera groups were Chironomidae and *Simulium* spp. in the Seomjin River basin. These two groups were clearly separated in the data ordination induced by land-use coverage rather than water quality (Figure 5a). Chironomidae were closely associated with wetland and cropland. Plenty of literature papers have reported that Chironomidae are the most abundant insects in wetlands and play an important role in wetland food webs [53,54]. In addition, it is clear that agricultural land use has influenced water quality, as evidenced by high nutrient concentrations [55]. Thus, the accumulation of organic matters subsequently fosters the colonization of Chironomidae groups, particularly in lowland agricultural areas [55,56]. In contrast, the same group of Diptera, *Simulium* spp., was correlated with nitrogen rather than phosphorus in forest areas. The aforementioned *Hydropsyche* spp. is known to prefer boulder microhabitats, which are commonly found in upstream areas (i.e., forest area in Seomjin River). It was remarkable to show a weak relationship between *Hydropsyche* spp. and forest land cover. Given the interspecific competition between *Hydropsyche* spp. and *Simulium* spp., our result was plausible to make *Hydropsyche* spp. separate in order to avoid excessive predation and competition [57].

Of the remaining benthic macroinvertebrates, *Asellus* spp. and *Gammarus* spp. were strongly identified in forest areas (Figure 5a). These two genera were clearly separated by components related to water quality, which we will discuss in relation to temporal water quality changes in the following sections. The Gastropoda groups *Radix* spp. and *Semisulcopsira* spp. were similarly ordinated in the plot (Figure 5a). Although the former was more related to grassland (i.e., pasture) and the latter was more related to wetlands, it was di fficult to characterize their habitat preferences. In this regard, further investigations are required. The Haplotaxida *Limnodrilus* spp. were closely associated with ammonia concentration in agricultural areas, and are known to be tolerant of unfavorable condition such as hypoxic and eutrophic states [58]. Thus, we suppose that *Limnodrilus* spp. is less sensitive to ammonia (NH3–N) toxicity, similar to the aforementioned *Baetis* spp. and *Ephemera* spp.

### *3.4. Identification of Spatiotemporal Characteristics in the Data from Seomjin River*

We portrayed the data ordination according to time and space (Figure 6a). It was remarkable to primarily characterize the data characteristics between May and September. Two data points were associated with forest areas. In comparison with Figure 5a, it was certain that these data points were correlated with *Asellus* spp. (Isopoda) and *Gammarus* spp. (Amphipoda), respectively. However, most data points characterized their clear separation across the vertical axis, which implies that benthic macroinvertebrate communities were a ffected by the temporal change of water quality. Through this pattern, the key macroinvertebrates Coleoptera *Elmidae* spp. and Tricoptera *Hydropsyche* spp. were relatively abundant in May, while Coleoptera *Eubrianax* spp. were relatively abundant in September (Figures 5a and 6a). Interestingly, the Ephemeroptera groups were closely associated with September compared to other key macroinvertebrates (Figures 5b and 6a).

We also question what major factors could drive the water quality changes between May and September. Given the unique climatological features of East Asia, such as summer monsoons and typhoons [18,19], we conjecture that a significant factor of water quality change would be the precipitation between two time periods (Figure 2). Since our CCA did not accommodate the precipitation data, we put more emphasis on the intensity of rainfall as a key factor for benthic macroinvertebrate communities, especially in East Asian countries. There have also been plenty of literatures related to the e ffects of flooding on benthic macroinvertebrates on a global scale [59,60].

Keeping this clear temporal pattern of data ordination in mind, we estimated the dissimilarity of data samples between May and September. Since the amount of rainfall was spatially distinct and the corresponding land-use coverage di ffers, there was a conspicuous deviation of data ordination between May and September (Figure 6b). The OS and SDU sites showed a larger disparity of data compared to the YC and SL sites. While the SDU site is in a headstream area, the SL site is near the river mouth (Figure 1). In this respect, their sensitivity to rainfall could be distinct. This pattern was clearer at the SDU site, which is a forest-dominated area and was consistent with the data ordination from CCA (Figure 5).
