1
Supplementary Material
Comparative assessment of the adverse outcome of wastewater effluents by integrating oxidative
stress and histopathological alterations in endemic fish
Palas Samantaa, Hyungjoon Ima, Jisu Yooa, Hwanggoo Leeb, Nan-Young Kimc,
Wonky Kimd, Soon-Jin Hwangc, Woo-Keun Kime, Jinho Junga
a Division of Environmental Science & Ecological Engineering, Korea University,
Seoul 02841, Republic of Korea
b Department of Biological Science, Sangji University, Wonju 26339, Republic of Korea
c Department of Environmental Health Science, Konkuk University,
Seoul 05029, Republic of Korea
d Ensol Partners Co., Ltd., Kunpo 15853, Republic of Korea
e System Toxicology Research Center, Korea Institute of Toxicology,
Daejeon 34114, Republic of Korea
*Corresponding author: Jinho Jung
Telephone: + 82-2-3290-3066
Fax: + 82-2-3290-3509
E-mail: [email protected]
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Section 1. Histopathological alterations in fish
Hypertrophy of the gill epithelium, fusion, and curling in C. auratus were a more common
feature of all the upstream, MZ, and downstream sites of the Eungcheon (Fig. S4). Additionally,
hyperplasia, dilation of the marginal channel, rupture of chloride cells and lamellar epithelium,
epithelial lifting and lesions, were prominent pathological characteristics in gills collected from the
MZ and downstream site on the Eungcheon. For Z. platypus, upstream sites in all streams yielded an
almost natural gill lamellae appearance in all three months, although gill lamellae fusion and gill
epithelium hypertrophy were observed (Fig. S5). In the MZ, the most notable changes were gill
epithelium hypertrophy, epithelial lifting, curling, chloride cell damage, rupture in secondary gill
lamellae, edema, and telangiectasia in secondary gill lamellae. Fish gills from the Mihocheon
downstream site showed comparatively lower pathological alterations, although the symptoms were
relatively similar to MZ lesions, including hypertrophy, curling, fusion, and chloride cell damage.
Zacco koreanus secondary gill lamellae collected from the Busocheon upstream site showed fusion,
hypertrophy, and telangiectasia (Fig. S6). In the MZ, fusion, epithelial lifting, curling, necrosis, and
telangiectasia in secondary gill lamellae represented the prominent changes. Fish from the
downstream site exhibited hypertrophy, gill lamellae fusion, chloride cell damage, and epithelial
lifting in some places.
Degenerated hepatocytes, sinusoids, acentric and hypertrophied nuclei, irregular-shaped cells,
irregular-shaped nuclei, nuclear hypertrophy, and cytoplasm vacuolation were profound changes
observed in livers of C. auratus collected from the Eungcheon MZ (Fig. S7). Degenerated hepatocytes,
acentric and hypertrophied nuclei, irregular-shaped cells, nuclear hypertrophy, and cytoplasm
vacuolation were also common in the downstream site, but the extent of alterations was comparatively
less. Cytoplasmic vacuolation and cellular hypertrophy were also common features in fish from the
upstream site. In Z. platypus, livers from the upstream site exhibited very few morphological changes,
including damage to hepatocytes, and cytoplasmic vacuolation (Fig. S8). Fish collected from the MZ
and downstream site of the Mihocheon showed higher prevalence of pathological alterations, such as
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degenerating hepatic cells, sinusoidal appearances, hypertrophied nuclei, vacuolation, irregular-shaped
cells, nuclear hypertrophy, cytoplasmic degeneration, and pyknotic nuclei. Hepatic cells with a
prominent nucleus around the central vein had an almost normal appearance in Z. koreanus from the
Busocheon upstream site (Fig. S9). Fish collected from the MZ showed severe pathological alterations,
including degenerated hepatocytes, sinusoids, cytoplasmic damage in the hepatopancreas, detachment
of the hepatopancreas from hepatocytes, and cytoplasmic vacuolation, whereas vacuolations and
irregular-shaped cells were more prominent in downstream samples.
Kidneys of C. auratus from the Eungcheon MZ and downstream sites showed varied
pathological alterations associated with kidney tubules and hematopoeitic tissues (Fig. S10). However,
upstream samples of kidney tubules (proximal and distal convoluted tubule), Bowman’s capsule, and
glomerulus had an almost normal appearance. Common alterations observed in fish kidneys collected
from the MZ and downstream site include degeneration in the glomerulus and proximal convoluted
tubule (PCT) and distal convoluted tubule (DCT) damage, cellular hypertrophy, dilation of
glomerulus capillaries, glomerulus enlargement, reduction of Bowman’s space, and cytoplasmic
vacuolation. However, the degree of change was more severe in the MZ. Zacco platypus kidneys from
the Mihocheon exhibited various pathological lesions at all three sites (Fig. S11). Far fewer
pathological alterations, such as PCT, DCT, and glomerulus damage were noted at the upstream site.
There was higher prevalence of PCT, DCT, and glomerulus damage, glomerulus enlargement and
cytoplasmic vacuolation recorded from the MZ sampling. Similar symptoms were also observed for
downstream sampling, but less than for the MZ sampling. Zacco koreanus kidneys collected from the
Busocheon upstream site showed damage in the proximal and distal convoluted tubules and
glomerulus, as well as cytoplasmic vacuolations (Fig. S12). In the MZ, prominent changes observed
were fragmented, dilated, or enlarged glomerulus, degenerative changes in the PCT and DCT,
cytoplasmic vacuolations, and cellular hypertrophy. Fish from the downstream site also exhibited PCT,
DCT, glomerulus damage and enlargement, and cytoplasmic vacuolations, but the damage severity
was lower than that to MZ.
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Table S1. Physicochemical properties of water samples collected at upstream (US), mixing zone (MZ), and downstream (DS) sites of the Eungcheon
stream and effluent (EF) samples.
Parameters December January February DL*
US MZ DS EF US MZ DS EF US MZ DS EF Temperature (℃) 8.6 10.7 11.3 16.5 5.8 7.9 7.5 15.0 5.0 6.5 6.9 12.5 NA** pH 8.0 7.6 7.8 7.2 8.0 7.6 7.6 7.6 7.2 6.9 6.7 7.5 NA DO (mg/L) 10.34 9.10 9.08 7.14 10.35 9.03 7.31 7.74 10.65 6.42 8.33 7.46 NA EC (μS/cm) 438 451 433 545 449 496 450 560 380 465 473 623 NA SS (mg/L) 25.2 5.8 4.7 NA 20.6 5.2 7.6 4.8 80.8 20.8 17.0 8.8 NA BOD (mg/L) 2.1 3.8 2.5 NA 2.8 2.1 1.7 3.1 1.6 1.8 1.5 2.6 NA COD (mg/L) 3.1 5.9 3.5 NA 4.9 4.7 4.0 7.5 4.6 5.1 4.6 7.8 NA TN (mg/L) 3.67 8.02 5.62 NA 3.83 5.32 5.72 9.17 3.09 8.14 6.89 8.56 NA TP (mg/L) 0.11 0.09 0.04 NA 0.11 0.05 0.07 0.05 0.21 0.07 0.17 0.17 NA DEHP (mg/L) 0.0006 0.0014 0.0091 0.0014 0.0008 0.0007 0.0023 0.0007 0.0069 0.0039 ND*** ND 0.0006 Chloroform (mg/L) ND ND ND ND ND ND ND ND 0.0066 ND 0.0020 0.0020 0.0018
Cu, Pb, As, Hg, CN, Cr(VI), Cd, Ni, Zn, Se, dichloromethane, trichloroethylene, perchloroethylene, phenols, benzene, 1,2-dichloroethane,
organophosphorus compound, carbon tetrachloride, 1,1-dichloroethylene, 1,4-dioxane, vinyl chloride, acrylonitrile, and bromoform were not detected. * Detection limit; ** Not available; *** Not detected
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Table S2. Physicochemical properties of water samples collected at upstream (US), mixing zone (MZ), and downstream (DS) sites of the Mihocheon
stream and effluent (EF) samples.
Parameters December January February DL*
US MZ DS EF US MZ DS EF US MZ DS EF
Temperature (℃) 8.8 12.7 11.3 19.0 6.8 12.8 8.9 17.7 7.4 11. 9 11.6 14.5 NA**
pH 7.9 8.2 8.2 8.0 8.0 8.2 6.1 8.0 6.3 7.0 7.1 6.9 NA
DO (mg/L) 13.05 11.10 10.75 8.42 16.40 12.10 11.05 8.37 13.90 9.76 11.15 8.92 NA
EC (μS/cm) 335 629 531 1115 428 886 438 1274 720 911 906 1071 NA
SS (mg/L) 0.5 8.2 2.2 NA 2.8 7.6 7.2 5.4 6.5 14.5 5.8 3.8 NA
BOD (mg/L) 2.6 4.5 3.3 NA 2.2 3.8 3.1 4.8 3.9 2.4 2.7 3.7 NA
COD (mg/L) 3.6 6.4 4.8 NA 4.6 7.2 6.4 9.0 11.4 7.3 8.2 8.2 NA
TN (mg/L) 6.99 7.54 7.20 NA 10.19 8.68 10.35 7.37 8.75 8.28 8.61 6.84 NA
TP (mg/L) 0.44 0.24 0.33 NA 0.65 0.32 0.32 0.05 0.62 0.18 0.24 0.03 NA
DEHP (mg/L) ND*** ND ND ND 0.0034 ND ND ND ND 0.0039 ND 0.0039 0.0006
Chloroform (mg/L) ND ND ND ND ND ND ND ND ND ND ND ND 0.0018
Cu, Pb, As, Hg, CN, Cr(VI), Cd, Ni, Zn, Se, dichloromethane, trichloroethylene (TCE), perchloroethylene (PCE), phenols, benzene, 1,2-dichloroethane,
organophosphorus compound, carbon tetrachloride, 1,1-dichloroethylene, 1,4-dioxane, vinyl chloride, acrylonitrile, and bromoform were not detected * Detection limit; ** Not available; *** Not detected
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Table S3. Physicochemical properties of water samples collected at upstream (US), mixing zone (MZ), and downstream (DS) sites of the Busocheon
stream and effluent (EF) samples.
Parameters December January February DL*
US MZ DS EF US MZ DS EF US MZ DS EF
Temperature (℃) 5.0 8.1 7.6 22.9 1.7 3.0 4.1 17.5 1.7 7.8 3.3 24.0 NA**
pH 7.9 7.9 7.8 7.8 8.4 7.2 8.2 7.8 6.9 6.9 6.9 7.0 NA
DO (mg/L) 13.65 12.61 12.58 6.99 16.72 12.61 13.76 8.31 13.47 15.57 13.49 7.79 NA
EC (μS/cm) 103 120 104 338 86 114 149 273 99 201 142 314 NA
SS (mg/L) 0.3 4.5 6.8 1.0 0.3 0.7 1.3 1.2 1.7 7.8 1.0 0.5 NA
BOD (mg/L) 0.6 0.9 1.0 2.5 0.7 0.5 0.9 1.8 0.7 0.9 0.7 0.6 NA
COD (mg/L) 1.2 2.2 1.8 3.1 1.6 1.5 3.0 4.1 1.8 0.9 2.0 1.9 NA
TN (mg/L) 1.41 2.67 2.57 6.24 2.09 2.15 3.24 7.77 1.34 4.10 2.78 5.86 NA
TP (mg/L) 0.01 0.08 0.06 0.25 0.004 0.02 0.16 0.24 0.01 0.28 0.10 0.47 NA
DEHP (mg/L) ND*** 0.0016 0.0047 0.0016 ND ND 0.0008 ND ND ND ND ND 0.0006
Chloroform (mg/L) ND ND ND ND ND ND ND ND ND ND ND ND 0.0018
Cu, Pb, As, Hg, CN, Cr(VI), Cd, Ni, Zn, Se, dichloromethane, trichloroethylene (TCE), perchloroethylene (PCE), phenols, benzene, 1,2-dichloroethane,
organophosphorus compound, carbon tetrachloride, 1,1-dichloroethylene, 1,4-dioxane, vinyl chloride, acrylonitrile, and bromoform were not detected * Detection limit; ** Not available; *** Not detected
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(a)
(b)
(c)
Fig. S1. Levels of catalase (CAT) in the gills, liver, and kidneys of (a) Carassius auratus, (b) Zacco platypus, and (c) Z.
koreanus collected from the Eungcheon, Mihocheon, and Busocheon streams, respectively. Data represent mean ±
standard deviation (n = 3). Different letters above the columns indicate significant differences (p < 0.05).
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(a)
(b)
(c)
Fig. S2. Levels of glutathione S-transferases (GST) in the gills, liver, and kidneys of (a) Carassius auratus, (b) Zacco
platypus, and (c) Z. koreanus collected from the Eungcheon, Mihocheon and Busocheon streams, respectively. Data
represent mean ± standard deviation (n = 3). Different letters above the columns indicate significant differences (p < 0.05).
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(a)
(b)
(c)
Fig. S3. Levels of lipid peroxidation (LPO) in the gills, liver, and kidneys of (a) Carassius auratus, (b) Zacco platypus,
and (c) Z. koreanus collected from the Eungcheon, Mihocheon and Busocheon streams, respectively. Data represent mean
± standard deviation (n = 3). Different letters above the columns indicate significant differences (p < 0.05).
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Fig. S4. Histological micrographs of Carassius auratus gills collected from the Eungcheon stream at upstream (US),
mixing zone (MZ), and downstream (DS) sites in December (4.1–4.3), January (4.4–4.6), and February (4.7–4.9). (4.1)
Upstream specimen, showing normal gill structure and fusion of secondary gill lamellae (SGL) in some places (broken
arrow); (4.2) mixing zone, showing hypertrophy (broken arrow), damage in pillar cells (white arrow) and chloride cells
(red arrow), and SGL damage; (4.3) downstream, showing hypertrophy (broken arrow), and epithelial lifting (red arrow);
(4.4) upstream, showing normal gill structure, and hypertrophy (broken arrow); (4.5) mixing zone, showing hypertrophy
(broken arrow), fusion (white arrow), epithelial lifting (black arrow), and chloride cell damage (arrow); (4.6) downstream,
showing chloride cell damage (white arrow), gill epithelial rupture (oval), and epithelial lifting (arrow); (4.7) upstream,
showing normal gill lamellae and hypertrophy (broken arrow); (4.8) the mixing zone, showing hypertrophy (broken arrow),
chloride cell damage and rupture (oval), fusion (black arrow), and epithelial lifting (white arrow); and (4.9) downstream,
showing gill epithelial rupture (oval) and epithelial lifting (arrow).
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Fig. S5. Histological micrographs of Zacco platypus gills collected from the Mihocheon stream at upstream (US), mixing
zone (MZ), and downstream (DS) sites in December (5.1–5.3), January (5.4–5.6), and February (5.7–5.9). (5.1) Upstream,
specimen, showing normal gill structure with fusion of secondary gill lamellae (arrow) in some places; (5.2) mixing zone,
showing hypertrophy (broken arrow), curling (square), chloride cell damage (oval), and SGL hypertrophy (white arrow);
(5.3) downstream, showing fusion (arrow), epithelial lifting (broken arrow), and gill epithelium damage; (5.4) upstream,
showing normal gill structure, and hypertrophy (arrow); (5.5) mixing zone, showing hyperplasia, curling (square), fusion
(arrow), and chloride cell damage; (5.6) downstream, showing chloride cell damage (oval), and epithelial lifting (broken
arrow); (5.7) upstream, showing normal gill lamellae and fusion in some places (black arrow), and chloride cell damage
(white arrow); (5.8) mixing zone, showing fusion (broken arrow), and chloride cell damage and rupture (oval); and (5.9)
downstream, showing hypertrophy (arrow), and fusion (broken arrow).
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Fig. S6. Histological micrographs Zacco koreanus gills collected from the Busocheon stream at upstream (US), mixing
zone (MZ), and downstream (DS) sites in December (6.1–6.3), January (6.4–6.6), and February (6.7–6.9). (6.1) Upstream
specimen, showing fusion (black arrow), curling of SGL (square), aneurysm (red arrow) and telangiectasia in secondary
gill lamellae (broken arrow); (6.2) mixing zone, showing fusion (black arrow), epithelial lifting (white arrow), and
telangiectasia in secondary gill lamellae (broken arrow); (6.3) downstream, showing chloride cell damage and hypertrophy
(black arrow); (6.4) upstream, showing hypertrophy (arrow), and chloride cell damage (oval); (6.5) mixing zone, showing
fusion (black arrow), hypertrophy, hyperplasia (broken arrow), and aneurism in SGL (white arrow); (6.6) downstream,
showing fusion (black arrow), epithelial lifting (white arrow), and hypertrophy (broken arrow); (6.7) upstream, showing
normal gill lamellae with fusion (arrow); (6.8) mixing zone, showing hyperplasia and chloride cell damage and rupture
(oval), and rupture in gill lamellae; and (6.9) downstream, showing hypertrophy (arrow).
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Fig. S7. Histological micrograph of Carassius auratus livers collected from the Eungcheon stream at upstream (US),
mixing zone (MZ), and downstream (DS) sites in December (7.1–7.3), January (7.4–7.6), and February (7.7–7.9). (7.1)
Upstream specimen, showing normal appearance of hepatocytes (HC), and compact arrangement around central vein
(CV); (7.2) mixing zone, showing degenerating hepatic cell (black arrow), sinusoidal appearances, acentric nuclei (arrow
head), and vacuolation (white arrow); (7.3) downstream, showing almost normal appearance of hepatocytes (HC) with
distinct nucleus (N); (7.4) upstream, showing normal appearance of hepatocytes (HC) and nucleus (N); (7.5) mixing zone,
showing degenerating hepatic cell (broken arrow), and vacuolation (black arrow); (7.6) downstream, showing degenerating
hepatic cell (broken arrow) and vacuolation (white arrow); (7.7) upstream, showing normal arrangement of hepatic cords
around central vein (black arrow) and less vacuolation (white arrow); (7.8) mixing zone, showing degenerating hepatic cell
(broken arrow) and vacuolation (white arrow); and (7.9) downstream, showing degenerating hepatic cell (broken arrow).
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Fig. S8. Histological micrographs of Zacco platypus livers collected from the Mihocheon stream at upstream (US), mixing
zone (MZ), and downstream (DS) sites in December (8.1–8.3), January (8.4–8.6), and February (8.7–8.9). (8.1) Upstream
specimen, showing normal appearance of hepatocytes (HC) and compact arrangement around central vein (CV) with
prominent nucleus (N); (8.2) mixing zone, showing degenerating hepatic cell (broken arrow), sinusoidal appearances,
pyknotic nuclei (black arrow), and vacuolation (white arrow); (8.3) downstream, showing almost normal appearance of
hepatocytes (HC), and hepatic cords (black arrow) with distinct nucleus (N); (8.4) upstream, showing sinusoidal
appearances and nuclei absence (arrow); (8.5) mixing zone, showing degenerating hepatic cell (broken arrow) and
vacuolation (black arrow); (8.6) downstream, showing degenerating hepatic cell (broken arrow), and detachment of
hepatopancreas from hepatocytes (arrow); (8.7) upstream, showing normal hepatic cords (black arrow); (8.8) mixing zone,
showing degenerating hepatic cell (broken arrow) and vacuolation (black arrow); and (8.9) downstream, showing
degenerating hepatic cell (broken arrow) and vacuolation (black arrow).
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Fig. S9. Histological micrographs of Zacco koreanus livers collected from the Busocheon stream at upstream (US), mixing
zone (MZ), and downstream (DS) sites in December (9.1–9.3), January (9.4–6.6), and February (9.7–9.9). (9.1) Upstream
specimen, showing normal hepatocytes with distinct nucleus (N); (9.2) mixing zone, showing hepatic cell damage (black
arrow) and cytoplasmic vacuolation (brown arrow); (9.3) downstream, showing less vacuolation in hepatocytes (white
arrow) and pyknotic nuclei (black arrow); (9.4) upstream, showing normal hepatocytes (HC) with prominent nucleus; (9.5)
mixing zone, showing degeneration in hepatocytes (broken arrow), sinusoidal appearance, and cytoplasmic damage in
hepatopancreas (black arrow), hepatocyte damage (white arrow), and detachment of hepatopancreas from hepatocytes
(arrow head); (9.6) downstream, showing normal hepatocytes and less vacuolation; (9.7) upstream, showing normal
hepatic cells with prominent nucleus and central vein; (9.8) mixing zone, showing degenerating hepatic cell (broken
arrow), pyknotic nuclei (white arrow) and vacuolation (black arrow); and (9.9) downstream, showing degenerating hepatic
cell (broken arrow) and vacuolation (black arrow).
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Fig. S10. Histological micrographs of Carassius auratus kidneys collected from the Eungcheon stream at upstream (US),
mixing zone (MZ), and downstream (DS) sites in December (10.1–10.3), January (10.4–10.6), and February (10.7–10.9).
(10.1) Upstream specimen, showing normal proximal convoluted (PCT) and distal convoluted (DCT) tubules, Bowman’s
capsule, and glomerulus; (10.2) mixing zone, showing degeneration in glomerulus (broken arrow), PCT (white arrow), and
DCT damage; (10.3) downstream, showing PCT (white arrow) and DCT (black arrow) damage; (10.4) upstream, showing
normal PCT, DCT, Bowman’s capsule and glomerulus (G); (10.5) mixing zone, showing PCT degeneration (broken arrow)
and fragmented glomerulus (black arrow); (10.6) downstream, showing PCT damage (white arrow); (10.7) upstream,
showing normal PCT and DCT, Bowman’s capsule, and glomerulus (G); (10.8) mixing zone, showing PCT degeneration
(broken arrow); and (10.9) downstream, showing PCT damage (white arrow), and reduction of Bowman’s space (black
arrow).
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Fig. S11. Histological micrographs of Zacco platypus kidneys collected from the Mihocheon stream at upstream (US),
mixing zone (MZ), and downstream (DS) sites in December (11.1–11.3), January (11.4–11.6), and February (11.7–11.9).
(11.1) Upstream specimen, showing normal proximal convoluted (PCT) and distal convoluted (DCT) tubules; (11.2)
mixing zone, showing PCT damage (arrow); (11.3) downstream, showing PCT damage (white arrow), damage in
glomerulus (black arrow) and occlusion (broken arrow); (11.4) upstream, showing normal PCT, DCT, glomerulus (G) and
damage in some places (arrow); (11.5) mixing zone, showing PCT (white arrow) and DCT (black arrow) degeneration;
(11.6) downstream, showing PCT and DCT damage (white and black arrow, respectively) (black arrow); (11.7) upstream,
showing PCT damage; (11.8) mixing zone, showing PCT and DCT damage (white arrow), and dilated glomerulus (black
arrow); and (11.9) downstream, showing PCT damage (black arrow).
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Fig. S12. Histological micrographs of Zacco koreanus kidneys collected from the Busocheon stream at upstream (US),
mixing zone (MZ), and downstream (DS) sites in December (12.1–12.3), January (12.4–12.6), and February (12.7–12.9).
(12.1) Upstream specimen, showing PCT damage (arrow); (12.2) mixing zone, showing degeneration in tubules (black
arrow), PCT damage (red arrow), and dilated glomerulus (G); (12.3) downstream, showing PCT damage (white arrow) and
degeneration in tubules (black arrow); (12.4) upstream, showing damage in the PCT (white arrow) and distal convoluted
tubule (black arrow); (12.5) mixing zone, showing PCT (white arrow) and DCT (black arrow) degeneration; (12.6)
downstream, showing DCT damage (black arrow); (12.7) upstream, showing PCT and DCT damage (black arrow); (12.8)
mixing zone, showing PCT and DCT degeneration (white arrow), and reduction of Bowman’s space (black arrow); and
(12.9) downstream, showing damage in tubules (black arrow) and dilated glomerulus (G).
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(a)
December January February
(b)
December January February
(c)
December January February Fig. S13. Star plots of the biomarker responses of oxidative stress (CAT, GST, and LPO) in (a) Carassius auratus, (b)
Zacco platypus, and (c) Z. koreanus collected from the Eungcheon, Mihocheon and Busocheon streams, respectively. The
star plots were used to compute the IBR index for each sampling site.
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(a)
December January February
(b)
December January February
(c)
December January February
Fig. S14. Star plots of the biomarker responses of histopathological alterations (DTC) in (a) Carassius auratus, (b) Zacco
platypus, and (c) Z. koreanus collected from the Eungcheon, Mihocheon and Busocheon streams, respectively. The star
plots were used to compute the IBR index for each sampling site.