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Hylebos Waterway Fish Injury Studies

Individual Data and Quality Assurance Results

CASE NARRATIVE

Juvenile Salmon Injury

Cytochrome P4501A - 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

Each sample set had a mean blank (calculated from 2 sets of duplicates) of 50

dpm 14C or less meeting the QAP guidelines.

Performance Evaluation

As stated in the QAP in order to meet an RSD of less than 30% for triplicate

analyses of each sample, any outlying replicate was omitted from calculations

of final AHH activity. In addition all values were greater than 50 pmol/mg

microsomal protein/minute.
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Juvenile Salmon Injury

Analyses of 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 Final Liver Comp# is 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 CID values of protein standards.
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Juvenile Salmon Injury

Analyses of Cytochrome P4501A [measured as aryl hydrocarbon

hydroxylase(AHH) Activity]

Table 2 Notes

Method Blank - AHH Assay

The numbers under columns Microsome # (column abbr. μsome #), AHH # and Sample # are

internal lab use numbers and for identification only. Column Final Liver Comp# is 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 (μome

#) 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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Juvenile Salmon Injury

Analyses of Cytochrome P4501A [measured as aryl hydrocarbon

hydroxylase(AHH) Activity]

Table 3 Notes

Performance Evaluation Final - AHH Activity

Microsome # (column abbr. μsome #), AHH # and Sample # are internal lab use numbers and

for identification only. Column Final Liver Comp# is 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 PM2, 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 prote in 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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