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Survey of Estrogenic Activity in the Danish Aquatic Environment

Appendix Report to "Survey of Estrogenic Activity in the Danish Aquatic Environment"

1 Pre-survey testing of procedures and equipment

• 1.1 Assessment of loss of conjugates during analysis
• 1.2 Stability of mono-conjugated steroid estrogens
• 1.3 Adsorption of conjugated estrogens to glass equipment
• 1.4 Solid Phase extraction (SPE) testing
• 1.5 Deconjugation
• 1.6 Silica gel clean-up

2 Chemical analysis

• 2.1 Overview of analytical concept
• 2.2 Sample preparation
  • 2.2.1 Particle filtering and solid phase extraction
  • 2.2.2 Deconjugation
  • 2.2.3 Silica gel clean up
  • 2.2.4 Derivatisation
  • 2.2.5 GC-MS/MS
  • 2.2.6 Identification of analytes and specification of MS-conditions
  • 2.2.7 Quantification

3 In vitro test - YES assay standard curves

• 3.1 Calculation of E2 equivalents in the YES-assay
• 3.2 How E2 equivalents were calculated
• 3.3 Explanation of data treatment exemplified in a spreadsheet
• 3.4 Six real examples
• 3.5 References

4 Quality assurance data for YES assay and chemical analysis

• 4.1 Quality assurance measures
  • 4.1.1 Internal quality control
  • 4.1.2 Precision and Limit of Detection
  • 4.1.3 Interlaboratory comparison - chemical analyses
  • 4.1.4 Interlaboratory comparison - YES assay
  • 4.1.5 Comparison between control sample results from biological and chemical measurements

5 Steroid estrogens versus estrogenic activity

• 5.1 Free versus total estrogens
• 5.2 Biological assay versus chemical analysis
  • 5.2.1 Samples with non-steroid estrogenic activity

6 Alternative presentation of study results

7 Locations and sampling scheme

8 Survey data

• 8.1 Explanations and abbreviations

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Version 1.0 Januar 2005, © Danish Environmental Protection Agency