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Hylebos Waterway Fish Injury Studies

Individual Data and Quality Assurance Results

CASE NARRATIVE

Toxicopathic conditions in Flatfish

Cytochrome P4501LA - measured as aryl hydrocarbon hydroxylase (AHH)

Activity (Tables 1-3)

Microsomal Protein Standard Curve Calibration

Both criteria of the QAP were met for data used in this calibration. Standard curves for each sample

set had regression coefficent (r2) of ≥0.990 and each protein standard run in triplicate had RSD <

20%.

Method Blank

Two sample sets had a mean blank (calculated from 2 sets of duplicates) of 50 dpm 14C or less. In

order to meet the QAP guidelines outlined in Table 4, one sample set with mean blank of > 50 dpm

14C, was rerun. Previously unthawed microsome samples were used in the rerun set. The resulting

mean dpm of blanks was less than 50 dpm 14C for the rerun set, thus these data were used.

Performance Evaluation

Triplicate analyses of each sample met the criteria as stated in the QAP of less than 30% RSD, for

all values > 50 pmol/mg microsomal protein/minute. Sample RS-CP-C3, which was < 50 pmol/mg

microsomal protein/minute, had a SD of < 20 pmol/mg microsomal protein/minute, as outlined in

Table 4 of the QAP.
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Toxicopathic conditions in Flatfish

Cytochrome P4501A [measured as aryl hydrocarbon hydroxylase (AHH)

Activity]

Table 1 Notes

Microsomal Protein Standard Curve Calibration

The Microsome # (column heading abbr. μsome #). Protein sample #(column heading abbr.

prot, sample# and Sample #, are internal lab use numbers and for identification only. Column

Sample # is species-internal site abbr. composite # (the official number assigned to each

composited sample).

The number under Plate # column is the number assigned to each assay plate.

The concentrations of total protein were calculated using Bovine Albumin Serum (BSA) as the

standard.

Column for Protein Standard concentrations (column abbr. Prot. Std. Conc # ) identify each

standard concentration dilution used : 2.0, 4.0, 8.0, 16.0,20.0, and 30.0 mg/ml, with all standards

loaded on plate as triplicates (e.g. 2.0-1, 2.0-2, 2.0-3 etc).

The Prot. Std OD value column is the absorbance of the sample measured as Optical Density

(OD) at a wavelength of 620nm.

Percent coefficient of variation ( column OD-%CV) is standard deviation (SD) of each triplicate

analyses (reported in Prot. Std OD value) divided by mean of each triplicate, multiplied by 100.

Correlation Coefficient (column abbr. Corr, Coeff. ) is generated from linear standard curve fit

of mean OD values of protein standards.

Sample Protein concentrations are given in column mg Prot, /ml and are generated by automated

interpolation from the standard curve fit of the mean OD values of protein standards.
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Toxicopathic conditions in Flatfish

Cytochrome P4501A [measured as aryl hydrocarbon hydroxylase (AHH)

Activity]

Table 2 Notes

Method Blank - AHH Assay

The Microsome # (column heading abbr. μsome #). Protein sample #(column heading abbr.

prot. sample#) and Sample #, are internal lab use numbers and for identification only. Column

Sample # is species-internal site abbr.-composite # (the official number assigned to each

composited sample).

The AHH Assay Date column is the day sample set was run. The column AHH Set Blk (μsome

#) is the sample run as the blank for each set.

Substrate blank is reaction mixture without substrate ( 14C-Benzo[a]Pyrene added. Boiled blank

is reaction mixture with an aliquot of boiled microsome sample. Both blank sets run as duplicates

(i.e. DPM1 and DPM2). All 4 blanks are then averaged to give AHH Assay Set Blk Avg DPM.
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Toxicopathic conditions in Flatfish

Cytochrome P4501A [measured as aryl hydrocarbon hydroxylase (AHH)

Activity]

Table 3 Notes

Performance Evaluation Final AHH ActivityThe Microsome # (column heading abbr. μsome #). Protein sample #(column heading abbr.

prot. sample#) and Sample #, are internal lab use numbers and for identification only. Column

Sample # is species-internal site abbr.-composite (the official number assigned to each composited

sample).

The AHH Assay Date column is the day sample set was run. The AHH Set Blk (μsome #) is the

sample run as the blank for each set.

Samples are run in triplicate. The percent coefficient of variation (%CV of DPM column) is the

standard deviation (value reported in column DPM SD) of each triplicate (reported in columns

:AHH DPM1, AHH DPM2, AHH DPM3) divided by mean of each triplicate (column AHH

DPM Avg) multiplied by 100.

Triplicate sample outlier indicated by •, was not used in final calculation to meet criteria of

%CV < 30%.

The column Avg AHH minus set Blk, is AHH DPM Avg, minus the value calculated in Table

2 (AHH set Blk) for each set.

Final AHH Activity is normalized for total microsomal protein i.e. “Avg AHH minus set Blk”

divided by “mg prot/ ml” (from Table 1).

Final units for AHH activity are picomoles BaP metabolized per minute per mg microsomal

protein.
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