Preliminary Toxicological Analysis of the Effect 

of Coal Slurry Impoundment Water on Human 

Liver Cells 

By Joseph E. Bunnell 

Report Series 2008–1143, version 1.1

U.S. Department of the Interior 

U.S. Geological Survey 
 ii

U.S. Department of the Interior 

DIRK KEMPTHORNE, Secretary 

U.S. Geological Survey 

Mark D. Myers, Director 

U.S. Geological Survey, Reston, Virginia: 2008 

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Suggested citation: 

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

cells: U.S. Geological Survey Open-File Report 2008–1143, 7 p., 2 tables, available online at 

http://pubs.usgs.gov/of/2008/1143/ver. 1.1 

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copyright owners to reproduce any copyrighted material contained within this report. 
Contents 

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

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

Tables 

1. HepG2 cell viability assay 4-13-07, optical density measurements @ 570 nm. ........................3 

2. HepG2 cell viability assay 4-13-07, optical density measurements @ 600 nm. ........................5 

Footnote to Open-File Report 2008-1143, Ver. 1.1 

This document supersedes the previous version of Open-File Report 2008-1143 because the following 

corrections have been made: 

• throughout the text and in the Table 2 caption, a typographical error of “630” has been replaced with the 

correct wavelength used in the analysis of 600 nm; 600 nm was used in all calculations, so the table of 

values for the 600 nm run (Table 2) presented in the initial report is correct. 

• location of sample collection has been correctly identified as Raleigh County, rather than Mingo County, 

WV 

• “for purposes of analytical methods development” has been added to the first sentence of paragraph 3 to 

further emphasize the rationale for conducting the preliminary experiment 

• plural form has been corrected to singular in second sentence of paragraph 3 

• inhibition of cell viability results has been updated. Previous values used 36 untreated control wells; 

updated values reflect stated exclusion of outer perimeter wells, giving 24 untreated control wells. 

Relative differences and trends are unchanged from previous version. 

 iii
Preliminary Toxicological Analysis of the 

Effect of Coal Slurry Impoundment Water on 

Human Liver Cells 

By Joseph E. Bunnell1

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 Raleigh County, West Virginia, was conducted for purposes of analytical methods 

development to determine its effect on HepG2 cells (ATCC, Manassas, VA). Low passage number 

cells were maintained and grown at 37° C and 5% CO2, and the experiment was conducted in a 96-

well plate. Twelve replicates of each slurry concentration (1%, 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 600 nm, respectively. 

Concentration-dependent effects were observed, with 1%, 5%, and 10% exposures resulting in 

0.827%, 1.37%, and 2.96% inhibition of cell viability, respectively, as compared to untreated 

controls using the AlamarBlue (Biosource International, Carlsbad, CA) oxidation-reduction 

 

1

 USGS Eastern Energy Resources Team, Reston, VA 

 1
 2

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). 

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 226: 218-228. 

ATSDR, 2005. Health Consultation: Private Well Water Quality, Williamson WV Sites (aka 

Williamson Area), Agency for Toxic Substances and Disease Registry, Public Health 

Service, U.S. Department of Health and Human Services, Atlanta, GA, 31 pp. 
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 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 

Sample ID T1 T9 T17 T25 T33 T41 T49 T57 T65 T73 

Position A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 Well Label T1 T9 T17 T25 T33 T41 T49 T57 T65 T73 OD Results 0.653 0.609 0.593 0.585 0.583 0.582 0.575 0.56 0.569 0.563 

Row B 

Column 1 

Column 2 

Column 3 

Column 4 

Column 5 

Column 6 

Column 7 

Column 8 

Column 9 

Column 10 

Sample ID T2 T10 T18 T26 T34 T42 T50 T58 T66 T74 

Position B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 Well Label T2 T10 T18 T26 T34 T42 T50 T58 T66 T74 OD Results 0.665 0.581 0.546 0.531 0.529 0.537 0.512 0.481 0.463 0.491 

Row C 

Column 1 

Column 2 

Column 3 

Column 4 

Column 5 

Column 6 

Column 7 

Column 8 

Column 9 

Column 10 

Sample 

ID T3 T11 T19 T27 T35 T43 T51 T59 T67 T75 

Position C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 

Well Label T3 T11 T19 T27 T35 T43 T51 T59 T67 T75 

OD Results 0.658 0.542 0.54 0.523 0.528 0.51 0.523 0.522 0.517 0.522 

Row D 

Column 1 

Column 2 

Column 3 

Column 4 

Column 5 

Column 6 

Column 7 

Column 8 

Column 9 

Column 10 

Sample ID T4 T12 T20 T28 T36 T44 T52 T60 T68 T76 

 3
Position D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 

Well Label T4 T12 T20 T28 T36 T44 T52 T60 T68 T76 

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

Row E 

Column 1 

Column 2 

Column 3 

Column 4 

Column 5 

Column 6 

Column 7 

Column 8 

Column 9 

Column 10 

Sample ID T5 T13 T21 T29 T37 T45 T53 T61 T69 T77 

Position E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 Well Label T5 T13 T21 T29 T37 T45 T53 T61 T69 T77 OD Results 0.637 0.535 0.53 0.534 0.517 0.526 0.528 0.535 0.523 0.528 

Row F 

Column 1 

Column 2 

Column 3 

Column 4 

Column 5 

Column 6 

Column 7 

Column 8 

Column 9 

Column 10 

Sample ID T6 T14 T22 T30 T38 T46 T54 T62 T70 T78 

Position F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 

Well Label T6 T14 T22 T30 T38 T46 T54 T62 T70 T78 OD Results 0.672 0.563 0.531 0.527 0.539 0.519 0.516 0.534 0.523 0.521 

Row G 

Column 1 

Column 2 

Column 3 

Column 4 

Column 5 

Column 6 

Column 7 

Column 8 

Column 9 

Column 10 

Sample 

ID T7 T15 T23 T31 T39 T47 T55 T63 T71 T79 

Position G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 Well Label T7 T15 T23 T31 T39 T47 T55 T63 T71 T79 OD Results 0.593 0.53 0.54 0.528 0.526 0.538 0.542 0.605 0.525 0.533 

Row H 

Column 1 

Column 2 

Column 3 

Column 4 

Column 5 

Column 6 

Column 7 

Column 8 

Column 9 

Column 10 

Sample ID T8 T16 T24 T32 T40 T48 T56 T64 T72 T80 

 4
Position H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 

Well Label T8 T16 T24 T32 T40 T48 T56 T64 T72 T80 

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

Table 2. HepG2 cell viability assay 4-13-07, optical density measurements @ 600 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 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 

Sample ID T1 T9 T17 T25 T33 T41 T49 T57 T65 T73 

Position A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 Well Label T1 T9 T17 T25 T33 T41 T49 T57 T65 T73 OD Results 0.118 0.129 0.12 0.135 0.12 0.124 0.125 0.123 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 

Sample ID T2 T10 T18 T26 T34 T42 T50 T58 T66 T74 

Position B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 Well Label T2 T10 T18 T26 T34 T42 T50 T58 T66 T74 OD Results 0.118 0.131 0.126 0.133 0.125 0.145 0.125 0.122 0.12 0.133 

Row C 

Column 1 

Column 2 

Column 3 

Column 4 

Column 5 

Column 6 

Column 7 

Column 8 

Column 9 

Column 10 

Sample 

ID T3 T11 T19 T27 T35 T43 T51 T59 T67 T75 

 5
Position C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 

Well Label T3 T11 T19 T27 T35 T43 T51 T59 T67 T75 

OD Results 0.128 0.127 0.127 0.133 0.128 0.126 0.127 0.136 0.135 0.133 

Row D 

Column 1 

Column 2 

Column 3 

Column 4 

Column 5 

Column 6 

Column 7 

Column 8 

Column 9 

Column 10 

Sample ID T4 T12 T20 T28 T36 T44 T52 T60 T68 T76 

Position D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 

Well Label T4 T12 T20 T28 T36 T44 T52 T60 T68 T76 

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

Row E 

Column 1 

Column 2 

Column 3 

Column 4 

Column 5 

Column 6 

Column 7 

Column 8 

Column 9 

Column 10 

Sample ID T5 T13 T21 T29 T37 T45 T53 T61 T69 T77 

Position E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 Well Label T5 T13 T21 T29 T37 T45 T53 T61 T69 T77 OD Results 0.13 0.129 0.128 0.139 0.128 0.139 0.128 0.138 0.143 0.147 

Row F 

Column 1 

Column 2 

Column 3 

Column 4 

Column 5 

Column 6 

Column 7 

Column 8 

Column 9 

Column 10 

Sample 

ID T6 T14 T22 T30 T38 T46 T54 T62 T70 T78 

Position F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 

Well Label T6 T14 T22 T30 T38 T46 T54 T62 T70 T78 OD Results 0.126 0.138 0.129 0.132 0.132 0.131 0.125 0.136 0.141 0.14 

Row G 

Column 1 

Column 2 

Column 3 

Column 4 

Column 5 

Column 6 

Column 7 

Column 8 

Column 9 

Column 10 

Sample ID T7 T15 T23 T31 T39 T47 T55 T63 T71 T79 

 6
Position G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 

Well Label T7 T15 T23 T31 T39 T47 T55 T63 T71 T79 OD Results 0.126 0.129 0.126 0.132 0.122 0.131 0.129 0.159 0.143 0.136 

Row H 

Column 1 

Column 2 

Column 3 

Column 4 

Column 5 

Column 6 

Column 7 

Column 8 

Column 9 

Column 10 

Sample ID T8 T16 T24 T32 T40 T48 T56 T64 T72 T80 

Position H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 

Well Label T8 T16 T24 T32 T40 T48 T56 T64 T72 T80 

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

 7