Preliminary Toxicological Analysis of the Effect of Coal 

Slurry Impoundment Water on Human Liver Cells 

By Joseph E. Bunnell 

Open-File Report 2008–1143 

U.S. Department of the Interior 

U.S. Geological Survey 
U.S. Department of the Interior 

DIRK KEMPTHORNE, Secretary 

U.S. Geological Survey 

Mark D. Myers, Director 

U.S. Geological Survey, Reston, Virginia 20192 

 2008 

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Bunnell, J.E., 2008, Preliminary toxicological analysis of coal slurry impoundment Water on human liver
 

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ii 
Contents 

Background and experiment ................................................................................................................................1
 

References Cited...................................................................................................................................................2
 

Tables 

1. HepG2 cell viability assay 4-13-07, optical density measurements @ 570 nm. ...............................................2
 

2. HepG2 cell viability assay 4-13-07, optical density measurements @ 630 nm. ...............................................4
 

iii 
Background and experiment 

Coal is usually “washed” with water and a variety of chemicals to reduce its content of

sulfur and mineral matter. The “washings” or “coal slurry” derived from this process is a viscous

black liquid containing fine particles of coal, mineral matter, and other dissolved and particulate

substances. Coal slurry may be stored in impoundments or in abandoned underground mines.

Human health and environmental effects potentially resulting from leakage of chemical

substances from coal slurry into drinking water supplies or aquatic ecosystems have not been

systematically examined. Impoundments are semipermeable, presenting the possibility that

inorganic and organic substances, some of which may be toxic, may contaminate ground or surface

water. The Agency for Toxic Substances and Disease Registry, part of the Centers for Disease 

Control and Prevention, has concluded that well water in Mingo County, West Virginia, constitutes

a public health hazard (ATSDR, 2005). Residents of the Williamson area (Mingo County),

especially children, have been subjected to chronic exposure to certain inorganic compounds from

their private wells (ATSDR, 2005). These elements may have been mobilized to their well water

from nearby coal extraction activities. The presence of and exposure to organic compounds, some

of which are known genotoxins, carcinogens, and teratogens, that may also be mobilized from

mining sites to ground water, were not, however, covered under the scope of the ATSDR report.

As a first step in examining the potential toxicity of coal slurry impoundment water to

human liver tissue, a preliminary experiment using slurry impoundment water collected in 2006

from Mingo County, West Virginia, was conducted to determine its effect on HepG2 cells (ATCC,

Manassas, VA). Low passage number cells were maintained and grown at 37° C and 5% CO

experiments were conducted in 96-well plates. Twelve replicates of each slurry concentration (1%,

2, and

5%, and 10%) were included in the experiment, and 24 untreated controls were included in the

analysis. The outer perimeter of wells was not treated or included in the analysis to minimize

variability and provide thermal insulation for the assayed wells. Optical density measurements

from the study for all treatments and controls are reported in Tables 1 and 2, for measurement at

570 nm and 630 nm, respectively. Concentration-dependent effects were observed, with 1%, 5%,

and 10% exposures resulting in 13.5%, 14.0%, and 15.4% inhibition of cell viability, respectively,

as compared to untreated controls using the AlamarBlue (Biosource International, Carlsbad, CA)

oxidation-reduction colorimetric indicator. This cytotoxicity assay was selected because it has

been demonstrated to have relatively low sensitivity with HepG2 cells, generating relatively

conservative results (e.g., Jondeau and others, 2006). 

1

 USGS Eastern Energy Resources Team, Reston, VA. 

Preliminary Toxicological Analysis of the Effect of 

Coal Slurry Impoundment Water on Human Liver Cells 

By Joseph E. Bunnell1 

1 
References Cited 

Adeline Jondeau, Laurence Dahbi, Marie-Hélène Bani-Estivals and Marie-Christine Chagnon, 

2006, Evaluation of the sensitivity of three sublethal cytotoxicity assays in human HepG2 

cell line using water contaminants: Toxicology, v. 226, p. 218-228. 

ATSDR, 2005, Health consultation: Private well water quality, Williamson WV sites (aka 

Williamson Area): Atlanta, Ga., Agency for Toxic Substances and Disease Registry, Public 

Health Service, U.S. Department of Health and Human Services, 31 p. 

2 
Table 1. HepG2 cell viability assay 4-13-07, optical density measurements @ 570 nm 

[Rows B – G, Columns 4 and 5 were treated with 1.0 μL impoundment water; Rows B – G, Columns 6 and 7 were treated with 5.0 μL impoundment water; and

Rows B – G, Columns 8 and 9 were treated with 10.0 μL impoundment water; all other wells were untreated] 

Column Column Column Column Column Column Column Column Column Column Column Column 

Row A 1 2 3 4 5 6 7 8 9 10 11 12 

Sample 

ID T1 T9 T17 T25 T33 T41 T49 T57 T65 T73 T81 T89 

Position A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 

Well 

Label T1 T9 T17 T25 T33 T41 T49 T57 T65 T73 T81 T89 

OD 

Results 0.653 0.609 0.593 0.585 0.583 0.582 0.575 0.56 0.569 0.563 0.56 0.59 

Column Column Column Column Column Column Column Column Column Column Column Column 

Row B 1 2 3 4 5 6 7 8 9 10 11 12 

Sample 

ID T2 T10 T18 T26 T34 T42 T50 T58 T66 T74 T82 T90 

Position B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 

Well 

Label T2 T10 T18 T26 T34 T42 T50 T58 T66 T74 T82 T90 

OD 

Results 0.665 0.581 0.546 0.531 0.529 0.537 0.512 0.481 0.463 0.491 0.515 0.553 

Column Column Column Column Column Column Column Column Column Column Column Column

Row C 1 2 3 4 5 6 7 8 9 10 11 12

Sample

ID T3 T11 T19 T27 T35 T43 T51 T59 T67 T75 T83 T91

Position C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 

Well 

Label T3 T11 T19 T27 T35 T43 T51 T59 T67 T75 T83 T91 

OD 

Results 0.658 0.542 0.54 0.523 0.528 0.51 0.523 0.522 0.517 0.522 0.533 0.601 

Column Column Column Column Column Column Column Column Column Column Column Column

Row D 1 2 3 4 5 6 7 8 9 10 11 12

Sample

ID T4 T12 T20 T28 T36 T44 T52 T60 T68 T76 T84 T92

1 
Position D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 

Well 

Label T4 T12 T20 T28 T36 T44 T52 T60 T68 T76 T84 T92 

OD 

Results 0.603 0.533 0.53 0.517 0.522 0.528 0.516 0.515 0.521 0.524 0.528 0.587 

Column Column Column Column Column Column Column Column Column Column Column Column 

Row E 1 2 3 4 5 6 7 8 9 10 11 12 

Sample 

ID T5 T13 T21 T29 T37 T45 T53 T61 T69 T77 T85 T93 

Position E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 

Well 

Label T5 T13 T21 T29 T37 T45 T53 T61 T69 T77 T85 T93 

OD 

Results 0.637 0.535 0.53 0.534 0.517 0.526 0.528 0.535 0.523 0.528 0.546 0.605 

Column Column Column Column Column Column Column Column Column Column Column Column 

Row F 1 2 3 4 5 6 7 8 9 10 11 12 

Sample 

ID T6 T14 T22 T30 T38 T46 T54 T62 T70 T78 T86 T94 

Position F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 

Well 

Label T6 T14 T22 T30 T38 T46 T54 T62 T70 T78 T86 T94 

OD 

Results 0.672 0.563 0.531 0.527 0.539 0.519 0.516 0.534 0.523 0.521 0.526 0.587 

Column Column Column Column Column Column Column Column Column Column Column Column 

Row G 1 2 3 4 5 6 7 8 9 10 11 12 

Sample 

ID T7 T15 T23 T31 T39 T47 T55 T63 T71 T79 T87 T95 

Position G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 

Well 

Label T7 T15 T23 T31 T39 T47 T55 T63 T71 T79 T87 T95 

OD 

Results 0.593 0.53 0.54 0.528 0.526 0.538 0.542 0.605 0.525 0.533 0.544 0.556 

Column Column Column Column Column Column Column Column Column Column Column Column 

Row H 1 2 3 4 5 6 7 8 9 10 11 12 

Sample 

ID T8 T16 T24 T32 T40 T48 T56 T64 T72 T80 T88 T96 

2 
3

Position H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12

Well

Label T8 T16 T24 T32 T40 T48 T56 T64 T72 T80 T88 T96

OD

Results 0.581 0.599 0.598 0.605 0.595 0.582 0.599 0.593 0.578 0.589 0.599 0.599

Table 2. HepG2 cell viability assay 4-13-07, optical density measurements @ 630 nm

[Rows B – G, Columns 4 and 5 were treated with 1.0 μL impoundment water; Rows B – G, Columns 6 and 7 were treated with 5.0 μL impoundment water; and

Rows B – G, Columns 8 and 9 were treated with 10.0 μL impoundment water; all other wells were untreated]

Row A

Column

1

Column

2

Column

3

Column

4

Column

5

Column

6

Column

7

Column

8

Column

9

Column

10

Column

11

Column

12

Sample

ID T1 T9 T17 T25 T33 T41 T49 T57 T65 T73 T81 T89

Position A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12

Well

Label T1 T9 T17 T25 T33 T41 T49 T57 T65 T73 T81 T89

OD

Results 0.118 0.129 0.12 0.135 0.12 0.124 0.125 0.123 0.126 0.125 0.126 0.125

Row B

Column

1

Column

2

Column

3

Column

4

Column

5

Column

6

Column

7

Column

8

Column

9

Column

10

Column

11

Column

12

Sample

ID T2 T10 T18 T26 T34 T42 T50 T58 T66 T74 T82 T90

Position B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12

Well

Label T2 T10 T18 T26 T34 T42 T50 T58 T66 T74 T82 T90

OD

Results 0.118 0.131 0.126 0.133 0.125 0.145 0.125 0.122 0.12 0.133 0.137 0.129

Row C

Column

1

Column

2

Column

3

Column

4

Column

5

Column

6

Column

7

Column

8

Column

9

Column

10

Column

11

Column

12

Sample

ID T3 T11 T19 T27 T35 T43 T51 T59 T67 T75 T83 T91
Position C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 

Well 

Label T3 T11 T19 T27 T35 T43 T51 T59 T67 T75 T83 T91 

OD 

Results 0.128 0.127 0.127 0.133 0.128 0.126 0.127 0.136 0.135 0.133 0.138 0.135 

Column Column Column Column Column Column Column Column Column Column Column Column 

Row D 1 2 3 4 5 6 7 8 9 10 11 12 

Sample 

ID T4 T12 T20 T28 T36 T44 T52 T60 T68 T76 T84 T92 

Position D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 

Well 

Label T4 T12 T20 T28 T36 T44 T52 T60 T68 T76 T84 T92 

OD 

Results 0.124 0.131 0.131 0.128 0.127 0.133 0.124 0.135 0.132 0.129 0.138 0.133 

Column Column Column Column Column Column Column Column Column Column Column Column 

Row E 1 2 3 4 5 6 7 8 9 10 11 12 

Sample 

ID T5 T13 T21 T29 T37 T45 T53 T61 T69 T77 T85 T93 

Position E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 

Well 

Label T5 T13 T21 T29 T37 T45 T53 T61 T69 T77 T85 T93 

OD 

Results 0.13 0.129 0.128 0.139 0.128 0.139 0.128 0.138 0.143 0.147 0.145 0.138 

Column Column Column Column Column Column Column Column Column Column Column Column 

Row F 1 2 3 4 5 6 7 8 9 10 11 12 

Sample 

ID T6 T14 T22 T30 T38 T46 T54 T62 T70 T78 T86 T94 

Position F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 

Well 

Label T6 T14 T22 T30 T38 T46 T54 T62 T70 T78 T86 T94 

OD 

Results 0.126 0.138 0.129 0.132 0.132 0.131 0.125 0.136 0.141 0.14 0.15 0.134 

Column Column Column Column Column Column Column Column Column Column Column Column 

Row G 1 2 3 4 5 6 7 8 9 10 11 12 

Sample 

ID T7 T15 T23 T31 T39 T47 T55 T63 T71 T79 T87 T95 

4 
Position G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 

Well 

Label T7 T15 T23 T31 T39 T47 T55 T63 T71 T79 T87 T95 

OD 

Results 0.126 0.129 0.126 0.132 0.122 0.131 0.129 0.159 0.143 0.136 0.16 0.136 

Column Column Column Column Column Column Column Column Column Column Column Column 

Row H 1 2 3 4 5 6 7 8 9 10 11 12 

Sample 

ID T8 T16 T24 T32 T40 T48 T56 T64 T72 T80 T88 T96 

Position H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 

Well 

Label T8 T16 T24 T32 T40 T48 T56 T64 T72 T80 T88 T96 

OD 

Results 0.134 0.141 0.137 0.143 0.132 0.134 0.129 0.136 0.141 0.137 0.153 0.143 

5