New Supplementary Material · 2018. 12. 5. · tubule (PCT) and distal convoluted tubule (DCT) damage, cellular hypertrophy, dilation of glomerulus capillaries, glomerulus enlargement,

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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



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



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


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


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),


(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),


(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)


(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)


(b)


(c)


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.

Documents

New Supplementary Material · 2018. 12. 5. · tubule (PCT) and distal convoluted tubule (DCT) damage, cellular hypertrophy, dilation of glomerulus capillaries, glomerulus enlargement,