MATERIALS AND METHODS

Technical Note DOER-C8

July 1999

commercially and was the subject for comparisons with the H4IIE cell-based assay (subsequently

referred to as EROD assay).

MATERIALS AND METHODS

NYDMMP Sediments. Nine subsamples of sediments collected for the New York Dredged

Material Management Plan (NYDMMP) (Simmers and Lee 1998) representing harbors in New

York and New Jersey and a composite of the nine samples were tested for dioxin activity using the

P450RGS and EROD assays. The sediments were given comprehensive chemical analysis sepa-

rately. The sediment subsamples were extracted using the Dionex Accelerated Solvent Extraction

(ASETM) system under high temperature and pressure following U.S. Environmental Protection

Agency (EPA) Method 3540 guidelines for soils and sediments. A portion of each extract was

cleaned on a sulfuric acid/silica gel (SA/SG) column to remove PAHs. Both crude and SA/SG-

cleaned extracts were solvent-exchanged to iso-octane before testing. Results obtained for both

assays were expressed as TCDD TEQs (pg/g) based on 2,3,7,8-TCDD standard curves. Results were

plotted against TCDD TEQs (pg/g) generated from gas chromatography/mass spectrometry

(GC/MS) chemical analysis data, which were calculated from the summation of TEQs derived from

individual congeners of PCDDs/PCDFs and PCBs and/or PAHs.

Miscellaneous Sediments. An additional set of 15 SA/SG-cleaned sediment extracts from

samples collected in various other waterways were also analyzed. The samples were extracted by

either ASETM or Soxhlet methods and cleaned on a SA/SG column. The ASETM and Soxhlet

extraction methods have been shown to provide comparable results (McFarland et al. 1998). Results

were expressed as described above.

Testing Procedures. The modified testing protocol for the EROD assay in 96-well microtiter

plate format was performed according to the protocol described in McFarland et al. (1998). Briefly,

5 L of a sample extract in iso-octane was added to each well of a 96-well plate containing 5,000

H4IIE cells. Iso-octane controls and a series of TCDD concentrations were also tested. The test

mixtures were incubated for 72 hr, after which cells were measured for protein and resorufin

simultaneously using a spectrofluorometer.

The detailed procedure for the P450RGS assay is described in American Society for Testing and

Materials (ASTM) (1997). Twenty-five thousand 101L cells per well were seeded in six-well plates

and incubated for 72 hr. Five microliters of the sample extract in iso-octane were then added to

each test well. Iso-octane blanks and TCDD standards were also tested. Following a 6-hr or 16-hr

incubation, the cells were washed, lysed, scraped, and the contents transferred into a microcentrifuge

tube. After centrifugation, the supernatant was analyzed for luminescence. Fold induction was

calculated as the number of times the light units of the test sample exceeded the light units of the

iso-octane blanks. The TCDD TEQ of each sample was derived from the TCDD standard curve

generated separately. All the results used in making comparisons with the EROD assay were

obtained from the 16-hr test. Readings were also taken at 6 hr as a qualitative check on the influence

of PAHs relative to the chlorinated compounds in the samples.