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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

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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# Contents {#contents .TOCHeading1}

Background and experiment 1

References Cited [2](#references-cited)

# Tables {#tables .TOCHeading1}

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 {#footnote-to-open-file-report-2008-1143-ver.-1.1 .TOCHeading1}

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.

Preliminary Toxicological Analysis of the Effect of Coal Slurry
Impoundment Water on Human Liver Cells

By Joseph E. Bunnell[^1]

# 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% CO~2~, 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 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

\[Rows B -- G, Columns 4 and 5 were treated with 1.0 µL impoundment
water; Rows B -- G, Columns 6 and 7 were treated with 5.0 µL impoundment
water; and Rows B -- G, Columns 8 and 9 were treated with 10.0 µL
impoundment water; all other wells were untreated\]

  ---------- -------- -------- -------- -------- -------- -------- -------- -------- -------- -------- -------- --------
  Row A      Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Column 7 Column 8 Column 9 Column   Column   Column
                                                                                              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

                                                                                                                

  Row B      Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Column 7 Column 8 Column 9 Column   Column   Column
                                                                                              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

                                                                                                                

  Row C      Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Column 7 Column 8 Column 9 Column   Column   Column
                                                                                              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

                                                                                                                

  Row D      Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Column 7 Column 8 Column 9 Column   Column   Column
                                                                                              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 0.603    0.533    0.53     0.517    0.522    0.528    0.516    0.515    0.521    0.524    0.528    0.587

                                                                                                                

  Row E      Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Column 7 Column 8 Column 9 Column   Column   Column
                                                                                              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

                                                                                                                

  Row F      Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Column 7 Column 8 Column 9 Column   Column   Column
                                                                                              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

                                                                                                                

  Row G      Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Column 7 Column 8 Column 9 Column   Column   Column
                                                                                              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

                                                                                                                

  Row H      Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Column 7 Column 8 Column 9 Column   Column   Column
                                                                                              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 0.581    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 @ 600 nm

\[Rows B -- G, Columns 4 and 5 were treated with 1.0 µL impoundment
water; Rows B -- G, Columns 6 and 7 were treated with 5.0 µL impoundment
water; and Rows B -- G, Columns 8 and 9 were treated with 10.0 µL
impoundment water; all other wells were untreated\]

  ---------- -------- -------- -------- -------- -------- -------- -------- -------- -------- -------- -------- --------
  Row A      Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Column 7 Column 8 Column 9 Column   Column   Column
                                                                                              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 0.118    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   Column   Column
                                                                                              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 0.118    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   Column   Column
                                                                                              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 0.128    0.127    0.127    0.133    0.128    0.126    0.127    0.136    0.135    0.133    0.138    0.135

                                                                                                                

  Row D      Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Column 7 Column 8 Column 9 Column   Column   Column
                                                                                              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

                                                                                                                

  Row E      Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Column 7 Column 8 Column 9 Column   Column   Column
                                                                                              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

                                                                                                                

  Row F      Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Column 7 Column 8 Column 9 Column   Column   Column
                                                                                              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

                                                                                                                

  Row G      Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Column 7 Column 8 Column 9 Column   Column   Column
                                                                                              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 0.126    0.129    0.126    0.132    0.122    0.131    0.129    0.159    0.143    0.136    0.16     0.136

                                                                                                                

  Row H      Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Column 7 Column 8 Column 9 Column   Column   Column
                                                                                              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

                                                                                                                

                                                                                                                
  ---------- -------- -------- -------- -------- -------- -------- -------- -------- -------- -------- -------- --------

[^1]: USGS Eastern Energy Resources Team, Reston, VA