Manual for Phytoplankton Sampling and Analysis in the Black Sea Dr. Snejana Moncheva Dr. Bill Parr Institute of Oceanology, Bulgarian UNDP-GEF Black Sea Ecosystem Academy of Sciences, Recovery Project Varna, 9000, Dolmabahce Sarayi, II. Hareket P.O.Box 152 Kosku 80680 Besiktas, Bulgaria Istanbul - TURKEY Updated June 2010 2 Table of Contents 1. INTRODUCTION........................................................................................................ 5 1.1 Basic documents used............................................................................................... 1.2 Phytoplankton – definition and rationale .............................................................. 1.3 The main objectives of phytoplankton community analysis ........................... 1.4 Phytoplankton communities in the Black Sea ..................................................... 2. SAMPLING ................................................................................................................. 9 2.1 Site selection................................................................................................................. 2.2 Depth ............................................................................................................................... 2.3 Frequency and seasonality ....................................................................................... 2.4 Algal Blooms................................................................................................................. 2.4.1 Phytoplankton bloom detection in the field.................................................. 2.4.2 Phytoplankton bloom measures...................................................................... 2.4.3 Evaluation forms .................................................................................................. 2.5 Equipment.................................................................................................................. 11 2.6 Methodology.................................................................................................................. 2.6.1 Qualitative sampling ........................................................................................... 2.6.2 Quantitative sampling......................................................................................... 3. SAMPLE PRESERVATION AND STORAGE..................................................... 14 3.1 Fixatives/preservatives .............................................................................................. 3.2 Advantages and disadvantages of formaldehyde............................................... 3.2.1 Advantages............................................................................................................ 3.2.2 Disadvantages ...................................................................................................... 3.3 Advantages and disadvantages of Lugol’s........................................................... 3.3.1 Advantages............................................................................................................ 3.3.2 Disadvantages ...................................................................................................... 3.4 Sample Storage ............................................................................................................ 3.4.1 Living samples..................................................................................................... 3.4.2 Preserved samples .............................................................................................. 4. PHYTOPLANKTON ENUMERATION AND DENSITIES.................................. 18 4.1 Equipment...................................................................................................................... 4.2 Sub-sample preparation............................................................................................. 4.2.1 Sample homogenisation .................................................................................... 4.3 Cell counts using an inverted microscope ........................................................... 4.3.1 Surface water removal........................................................................................ 4.3.2 Filtration ................................................................................................................. 4.3.3 Settling chambers................................................................................................ 4.2.3 Counting procedure ............................................................................................ 4.4 Cell counts using a conventional light compound microscope ..................... 4.4.1 Surface water removal........................................................................................ 4.4.2 Filter-transfer-freeze methodology ................................................................. 4.4.3 Centrifugation ....................................................................................................... 4.5 Recording the number of algal cells....................................................................... 4.6 Density calculations.................................................................................................... 5. SEMI-QUANTITATIVE ANALYSIS OF PHYTOPIANKTON SAMPLES........ 32 5.1 Inverted micoscope..................................................................................................... 5.2 Conventional light compound microscope........................................................... 6. TAXONOMIC IDENTIFICATION........................................................................... 33 6.1 Introduction ................................................................................................................... 6.2 Identification books in use within Black sea countries..................................... 6.3 Additional references of recent taxonomic developments .............................. 7. BIOMASS DETERMINATION ............................................................................... 39 7.1 Diatoms........................................................................................................................... 7.2 Calculation of a cell’s reference biovolume ......................................................... 8. RECORDING DATA............................................................................................ 4141 8.1 Bench Sheets ................................................................................................................ 9. PHYTOPLANKTON-RELATED ENVIRONMENTAL STATUS INDICATORS............................................................................................................ 43 9.1 Introduction ................................................................................................................... 9.2 Black Sea-specific phytoplankton-related environmental status indicators ........................................................................................................................... 10. QUALITY CONTROL AND QUALITY ASSURANCE (QC/QA) ...................... 45 10.1 Introduction ................................................................................................................... 10.2 Equipment...................................................................................................................... 10.3 Sampling protocol ....................................................................................................... 10.4 Sample preservation ................................................................................................... 10.5 Sub-sampling – validation of homogenization .................................................... 10.6 Sample analysis ........................................................................................................... 10.6.1 Biovolume/biomass estimation........................................................................ 10.6.2 Phytoplankton enumeration.............................................................................. 10.6.3 Taxonomy............................................................................................................... 10.6.4 Re-analysis ............................................................................................................ 10.6.5 Training and inter-laboratory comparability testing .................................. 10.6.6 Repeatability and reproducibility..................................................................... 10.6.7 Uncertainty............................................................................................................. 10.7 Reporting/data storage........................................................................................... Annex A. STANDARDISED LIST OF BLACK SEA PHYTOPLANKTON TAXA AND REFERENCE BIOVOLUMES ...................................................................... 49 Annex B. CONTROL CHARTS FOR BIOLOGICAL MEASUREMENTS........................ 56 Annex C. MEASUREMENT UNCERTAINTY....................................................................... 58 Annex D. LIST OFPARTICIPANTS…………………………………………………………62 11. REFERENCES CITED IN THE MANUAL ....................................................................... 64 4 This document is intended for use as a guide to harmonize the procedures among the different labs in the Black Sea countries for phytoplankton monitoring. The methods for phytoplankton microscope