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Hylebos Waterway Fish I njury Studies

Individual Data and Quality Assurance Results

CASE NARRATIVE

Reproductive Toxicology in Flatfish

Reproductive Steroid RlAs-Plasma 17-b Estradiol Analyses

Added explanation for changes in standard curve range for all test dates to the case narrative.

Added explanations for the following test dates to the case narrative:

Date: 6/27/95; Analyzed by: SS;

Date: 7/6/95; Analyzed by: LC;

Date: 7/18/95; Analyzed by: SS.

Tables:

Cleaned up tables, comments and significant digits are now more consistent.

Changes made to Table 3:

specimen# 3406: Non-target species, removed from the table;

specimen# 3528: Non-target species, removed from the table;

specimen# 3638: Non-target species, removed from the table;

specimen# 3721: Non-target species, removed from the table;

specimen# 3594A: Non-target species, removed from the table;

specimen#: 3271, test date 6/22/95: Changed E2 final from 858.03 to875.73(pg/ml);

specimen#: 3532, test date 6/22/95: Changed E2 final from 131.04 to146.75(pg/ml);

specimen# 3540, test date 7/18/95: Changed E2 final from 3520.18 to3726.95(pg/ml);

specimen# 3568, test date 7/10/95: 13% avg 48.33 changed to 31.86;

specimen# 3501, test date 7/14/95: This specimen# tested on 6/22/95 did

not meet the QA criteria. It was rerun on 7/14/95, however, not enough plasma

was available for triplicate test. The value previously reported, 393.55pg/ml,

was deleted because it was calculated based on a single analysis.
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Hylebos Waterway Fish Injury Studies

Individual Data and Quality Assurance Results

CASE NARRATIVE

Reproductive Toxicology in Flatfish

ReRroductive Steroid RIAs-Plasma--17-β Estradiol Analyses

Calibrations

Standard curve: As stated in the SAP, standards for this assay typically range from 0 - 2000 pg

estradiol 17-b/100μl; to reflect the typical range of the sample concentrations, the standard curve

was modified to range from 0 to 500pg/estradiol 17-b/100μl. Therefore, all final estradiol

concentrations are slightly modified from those originally submitted to DARC on 5/30/1996.

The data used to quantitate plasma 17-b estradiol (Table 1) met the calibration criteria outlined in

the Hylebos SAP, Table 4, except as following:

Date: 6/22/95

Analyzed by:S.S.

Two of the triplicate NSB samples analyzed were outside the range typically observed, and deleted.

RSD for 250pg/100ml standard (20.62%) exceeded maximum RSD of 200% outlined in the SAP.

Date: 6127/95

Analyzed by:S.S.

Radioactive Liquid scintillation counter (LSC) broke down in the middle of the count. Remaining

samples including standards were recounted on another LSC, and plasma estradiol concentrations of

the samples were calculated on the new standard curve generated.

Date: 7/6/95

Analyzed by: LC

RSD for 250pg/100ml standard (22.95%) exceeded maximum RSD Of

20% outlined in the SAP.

Date: 7/10/95

Analyzed by: LC

Duplicate rather than triplicate samples were used to calculate NSB for the standard curve.

Date 7/18195

Analyzed by SS

3.9pg/100ml standards were not used in the calculation of standard curve because the values

obtained were outside the range typically observed.
451

Date: 3/13/96

Analyzed by: B.B.

The standard curve regression coefficient, 0.94, was lower than the acceptable limit, 0.95, outlined

in the SAP. Nevertheless, other criterias outlined in the SAP were satisfied.

An assay performed on 6/22/95, and two assays performed on 7/10/95 had lower Bo than the

acceptable range outlined on Hylebos SAP, Table 4. However, these values were accepted because

the binding percentage of the standard curve and samples’ (B%) are calculated based on the Bo. The

change in Bo shifts the effective range of the assay, not the plasma estradiol concentrations. The

change in Bo produced only minor changes in the effective range of the assay, therefore, the assays

were not rerun.

Quality Control Sample

All quality, control samples tested (Table 2) met the criteria outlined in the Hylebos SAP, Table 4.

Sample Triplicate and Performance Evaluation

All samples were analyzed in triplicate. Samples in which the estradiol concentration was below

effective concentration of the assay (B% > 80%) were retested at a higher concentration (i.e.

maximum plasma volume of 100μI of plasma/tube), or given a value equal to half of lower detection

limit. Samples in which the estradiol concentrations was above effective concentration of the assay

(B% < 20%) were retested at a lower concentration (i.e. minimum volume of 5μI of plasma/tube). A

few samples were deleted because reliable measurement could not be obtained with amount of

samples available. Samples that did not meet the criteria outlined in the Hylebos SAP, Table 4, i.e.

RSD greater than 20%, were reanalyzed, or the outlier of the triplicate was removed. However, some

samples in which B% was slightly higher than 80% (i.e. 80.41), and or, RSD slightly higher than

20% (i.e. 21.40) were accepted.

Reanalysis

Samples that were reanalyzed are noted in Table 3.
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Reproductive Toxicology in Flatfish

Reproductive Steroid RlAs-Plasma 17:β Estradiol Analyses

Table 1 Notes

Total: counts per minute (cpm) of tritiated 17-β estradiol (label) added to each tubes.

NSB (non specific binding): binding to label in the absence of antigen and steroid.

0 standard: binding to label and antigen, no steroid present.

Bo: binding efficiency of the antibody to labeled antigen.

((0 standard (cpm) - NSB(cpm))/(Total(cpm) - NSB(cpm)))xl00.

B%: binding percentage of sample and standards (3.9 - 500pg/ml 17-β estradiol) relative to Bo.

((0 tan ( ) ( ))/( ( ) ( )

(( ( ) ( ))/ ( ) ( )))

s dard cpm NSB cpm Total cpm NSB cpm

sample cpm NSB cpm Total cpm NSB cpm 







− −

− −

EC80: Lower limit of the assay, where B% of 80 occurs.

EC20: Upper limit of the assay, where B% of 20 occurs.

RSD (relative standard deviation): standard deviation (SD) of each triplicate

analyses divided by mean of each triplicate multiplied by 100.
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Reproductive Toxicology in Flatfish

Reproductive Steroid RIAs-Plasma 17-β Estradiol Analyses

Table 2- Notes

B%: binding percentage of sample relative to total, NSB and 0 standard.

Total: counts per minute (cpm) of tritiated 17-β estradiol (label) added to each tubes.

NSB (nonspecific binding): binding to label in the absence of antigen and steroid.

0 standard: binding to label and antigen, no steroid present.

B% is calcuated as following:

((0 tan ( ) ( ))/( ( ) ( )

(( ( ) ( ))/ ( ) ( )))

s dard cpm NSB cpm Total cpm NSB cpm

sample cpm NSB cpm Total cpm NSB cpm 







− −

− −

Plasma 17-β estradiol concentrations (pg/ml plasma) are generated by automatic interpolation

from the standard curve fit of the mean B% of protein standards (logistic regression).

RSD (relative standard deviation), unless otherwise noted, is standard deviation (SD) of each

triplicate analyses divided by mean of each triplicate multiplied by 100.
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Reproductive’ Toxicology in Flatfish

Rel2rodugtive Steroid RlAs-Plasma 17-β Estradiol Analyses

TabIe 3 Notes

B%: binding percentage of sample relative to total, NSB and 0 standard.

Total: counts per minute (cpm) of tritiated 17-β estradiol (label) added to each tubes.

NSB (non specific binding): binding to label in the absence of antigen and steroid.

0 standard: binding to label and antigen, no steroid present.

13% is calculated as following:

((0 tan ( ) ( ))/( ( ) ( )

(( ( ) ( ))/ ( ) ( )))

s dard cpm NSB cpm Total cpm NSB cpm

sample cpm NSB cpm Total cpm NSB cpm 







− −

− −

Plasma 17-β estradiol concentrations (pg/ml plasma) are generated by automatic interpolation from the standard

curve fit of the mean B% of protein standards (logistic regression).

RSD (relative standard deviation), unless otherwise noted, is standard deviation (SD) of each triplicate analyses

divided by mean of each triplicate multiplied by 100.

Intra assay RSD is RSD within a run.

Inter assay RSD is RSD between runs.
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