Figure 3. Adding reagents to a 96-well plate using a multichannel pipet

ERDC TN-DOER-C10

March 2000

25 percent and the expendable supplies cost by

about 65 percent compared with the 6-well plate

method, and produce a slight increase in sensitivity

(Table 1).

Significant additional savings in time and cost were

gained by modifications to the extraction method.

Combining the SA/SG cleanup step with extraction

reduced sample preparation time from 17 hr/sample

to 2.5 hr/sample with no loss in extraction effi-

ciency. The extraction efficiencies were deter-

mined for both ASE and ASE one-step methods

using 3H-TCDD and were found to be similar:

87.2 1.8 percent recovery for the standard ASE,

and 80 6.2 percent for the ASE one-step method.

An additional advantage of the ASE one-step

Figure 3. Adding reagents to a 96-well plate

method is that the amount of waste solvent requiring

using a multichannel pipet

disposal is reduced from 270 mL/sample for ASE

with separate cleanup to 130 mL/sample for ASE one-step. The efficacy of SA/SG removal of PAHs

for the two methods was tested by spiking a clean sediment with a PAH standard prior to analysis

with the H4IIE cell line. No response due to PAH addition was detected after cleanup with either

method (Table 2).

To test whether samples containing high levels of

Table 2

PAHs could overload the SA/SG column and allow

H4IIE Maximal Responses of

PAH breakthrough, a harbor sediment highly con-

PAH-Spiked Sediments Extracted

by ASETM

taminated with PAH was extracted with the ASE

standard method, resulting in an extremely high

Response, ng

TEQ (15,125 2,850 pg TCDD TEQ/g dry sedi-

resorufin/g protein

Method

ment, n = 5), which dropped to below detection

ASE, no cleanup

540

limits following SA/SG column cleanup. Spiking

ASE, with SA/SG

Below LOD

the extract with PAHs prior to column cleanup did

cleanup

not affect the results, indicating that the SA/SG

ASE one-step

Below LOD

column was not overloaded. Extracts of the same

sample prepared by the ASE one-step method did

not produce any detectable TCDD TEQ as

measured by the H4IIE cell-based assay, indicating

that this method is as effective in removing

PAHs as the more laborious column cleanup.

CONCLUSIONS: The ASE one-step extraction/cleanup procedure in combination with the

96-well plate adaptation of the P450RGS assay provides a very cost-effective and rapid alternative

to conventional analytical chemistry for screening sediments for dioxin and dioxinlike activity. By

scheduling the separate operations properly, it is possible for one person to extract and test three

replicates of 24 sediment samples in two workweeks. PAHs in sediments may also be assayed using

these methods. These methods are currently being applied in a field demonstration in which a

Federal channel with a historic point source of creosote contamination is being dredged, and samples