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2006 Chesapeake Bay Monitoring Program Split Sample Data Analysis Phytoplankton

2006 Chesapeake Bay Monitoring Program Split Sample Data Analysis Phytoplankton Morgan State University Estuarine Research Center Ann Marie Hartsig Richard Lacouture Stella Sellner. Introduction

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2006 Chesapeake Bay Monitoring Program Split Sample Data Analysis Phytoplankton

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  1. 2006 Chesapeake Bay Monitoring Program Split Sample Data Analysis Phytoplankton Morgan State University Estuarine Research Center Ann Marie Hartsig Richard Lacouture Stella Sellner

  2. Introduction • In 2005, a standard method for phytoplankton identification and enumeration was adopted, yet, analysis of 2006 phytoplankton split-samples show major discrepancies. • Differences are observed for major groups at the high magnification counts as well as for a variety of groups at the primary (mid) magnification. • Commonly identified species such as Cylindrotheca closterium, Skeletonema costatum, and Heterocapsa rotundatum show significant differences in densities. •For 2006, MSU and ODU are examining exactly the same subvolume or % of total sample. ODU determined that 1/64 of their concentrated sample equals 7.81mls of sample settled for MSU. • Possible reasons for this discrepancy might include calculations, sample preparation techniques and resulting cellular optical properties.

  3. The Canary in the Coal Mine Cylindrotheca closterium

  4. Calculation of Normalized Densities MSU Calculation: Number of individuals counted Constant 1000 = Number of Number of Fields Counted Number of mls settled Cells/Liter X X Ex: 120cells * 1214.9 (312.5mag) X 1000= 1866683.7cells/liter 10 7.81ml ODU Calculation: Number of individuals counted Constant 1 1 = Number of Number of Fields Counted 1 concentrate volume Cells/Liter X X X Constant= Number of Fields in Chamber at magnification used (Note: Percent of sample analyzed equal between MSU and ODU for 2006)

  5. Sample Preparation Methods Pipette 1-10mls settle 2-24h MSU Fill up to 4/4 mark w/formalin water Now 1/16 dilution 48h siphon until ~40ml 72h siphon ~250ml ~40mls in vial settle 16-24h Pour ¼ of orig. into next vial Pour ¼ into chamber Now 1/64 dilution Settle in chamber 16-24h ODU

  6. Potential Errors Related to Sample Preparation Methods MSU: 1) Calibration of pipettes 2) Non-settling cells ODU: 1) Loss of cells during siphoning phases 2) Error associated with transfer of subsample during dilution process 3) Effects of dilution process on optical properties of cells (bleaching) 4) Non-settling cells

  7. Photo of Cells Found in 50mls of Supernatant Skeletonema Cylindrotheca 300X Cryptomonas sp. (Sample settled >72 hours before siphoning)

  8. Loss of Cells during Siphoning Procedure

  9. Alteration of Cellular Appearance by ‘Bleaching’ Effect of Dilution Water at 300X ODU prep 300X MSU prep 300X Heterocapsa rotundatum ODU prep 300X

  10. Alteration of Cellular Appearance by ‘Bleaching’ Effect of Dilution Water at 500X MSU prep – 500X Loss of stain Hard to see pigment Cryptomonas spp. Pyramimonas sp. and Heterocapsa rotundatum ODU prep – 500X

  11. Alteration of Cellular Appearance by ‘Bleaching’ Effect of Dilution Water 500X

  12. Canary in the Coal Mine Differences

  13. Results Summary • There are several sources of error related to the preparation of samples for each laboratory. • The pipettes used by MSU to draw off the subsample for taxonomic enumeration are macropipettes (1-5mls or 5-10mls) and could introduce an error but not likely a bias. • The siphoning of the sample during the concentration phase of ODU sample preparation may be a source of cell loss and therefore a negative bias (loss was ~ 10% for total cell densities and 3-18% for five of the dominant taxa). • The transfer of subsamples during the dilution phase of the ODU sample preparation may be a source of error but not likely a bias. • The ‘bleaching’ effect induced by the transfer of subsamples during the ODU sample preparation may be a source of cell loss and therefore a negative bias. • There may be cells that do not settle out of the subsample completely and may be a source of cell loss and therefore a negative bias.

  14. CONCLUSIONS • Several potential sources of error related to sample preparation have been identified for each laboratory. • Cell loss caused by the siphoning phase of the ODU sample preparation only accounts for a small portion of the discrepancy between the 2 labs’ total cell densities but showed higher proportional losses for several dominant taxa. • The transfer of subsample during the dilution phase of the ODU sample preparation technique may lead to a certain amount of error but this was not quantifiable in the current split sample analysis. • The source of intra-laboratory variability remains a mystery. • Several recommendations are proposed: 1) Identify other potential sources of error and a means of quantifying the potential error associated with these issues. 2) Adopt a common sample preparation technique so that the entire protocol for enumerating phytoplankton is ‘identical’ between the two laboratories. 3) Bring in outside experts in the field to assist with this standardization of methodologies.

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