Aisha S. Chiandet*, R. Keith Sherman*,. James A. Rusakâ and E. Todd Howellâ . * Severn Sound Environmental Association. â Ontario Ministry of Environment ...
Comparing fluorometric measurements of phytoplankton with conventional methods
Aisha S. Chiandet*, R. Keith Sherman*, James A. Rusak† and E. Todd Howell† * Severn Sound Environmental Association † Ontario Ministry of Environment and Climate Change
Why Measure Chlorophyll a?
Chloroplast
Green algae cell (Chlamydomonas)
Chlorophyll a molecule
• All algae contains chl a • Provides proxy for algal abundance without time consuming counts • Can act as a surrogate for measures of ecosystem function (production) • Widely used, easy to measure, useful across large gradients • Long history of measurement; value in long term datasets #2 of 19
Introduction • Need for technology that can rapidly and accurately measure algal bloom composition and distribution • The Fluoroprobe (FLPB, bbe Moldaenke) is increasingly being used to determine the composition and spatiotemporal extent of algal communities in situ • The FLPB provides rapid results with high vertical spatial resolution, accuracy is still in question (e.g. Kring et al. 2014, Catherine et al. 2012) • Evaluations of the instrument have been mixed; need for more comparisons between in situ and in vivo Fluoroprobe-estimated chl a (FLPB), spectrophotometrically determined chl a (SPEC), and biovolume obtained by microscopy #3 of 19
DETAIL
Method
Basis for Analysis
Matrix
Information Obtained
Known Issues
Solvent Extracted Chlorophyll
Spectrophotometry, Fluorometry
Sample or in situ profile
Proxy for abundance, no taxonomic info
Interference from other pigments (Reimann 1978), issues with low pH samples
High Performance Liquid Chromotography (HPLC)
Chromotography (Mass Spec method being developed that will also separate pigments)
Sample
[Pigment], proxy for abundance of high level taxonomy (division)
Difficulty separating pigment groups in complex communities due to interference
Accessory Pigment Fluorometry E.g. Fluoroprobe
Fluorometry
Sample or in situ profile
[Pigment], proxy for abundance of high level taxonomy (division)
Doesn’t handle colonies/filaments well Difficulty separating pigment groups in complex communities
Cell Counters E.g. FlowCAM
Flow Cytometry, imaging
Sample
Concentration; taxa ID (level depends on calibration)
Non-target particles may get counted or target particles may get missed without careful calibration
Microscopy
Count
Sample
Abundance; species ID
Not all species preserve well, bias inherent to taxonomist
DNA Barcoding
Genetic sequencing
Sample
Species ID; no abundance
Hybridization limits use of standard set of genes for algae
#4 of 19
Objectives • Determine relationship between FLPB and SPEC chl a using data from Severn Sound and inland lakes over varying ranges of productivity • Examine influence of sample complexity (discrete depth vs. integrated depth samples) • Explore factors influencing FLPB accuracy (as measured by slope and intercept of FLPB vs. SPEC chl regression) – Spatial variability/Influence of DOC
• Assess FLPB’s ability to distinguish phytoplankton groups by comparisons to microscope counts #5 of 19
Study Area • Severn Sound, Georgian Bay (SS data-3 sites, max SPEC chla=26 g/L) • Lake Simcoe and lakes in Dorset area of South-Central Ontario (MOE data-24 sites , max SPEC chla=45 g/L) #6 of 19
Chlorophyll a by Spectrophotometry (SPEC) (SS and MOE samples) • Integrated depth (2x Secchi depth) and discrete depth samples
Algal Biovolume by Utermöhl Microscopy (SS samples) Jason Oyadomari
Chrysosphaerella brevispina
#7 of 19
• Integrated: all taxa ID’d & counted • Discrete: all taxa ID’d, dominant taxa counted
Fluorometry Measurements Fluoroprobe
Algal Lab Analyzer #8 of 19
SS Dataset: • In situ FLPB profiles taken biweekly through ice free season, 2011-2014 • For each date and location, data were corrected for background fluorescence; spectral group fingerprints left at factory settings MOE Dataset: • The Algal Lab Analyzer was used to measure [chl a] in vivo • All MOE data acquired using factory settings, not corrected for background fluorescence
How does the Fluoroprobe work? 370
450
525
570 590 610
Spectral Group
Greens
Bluegreens
Pigment Group
Chl a/b, Xanthophyll
Divisions included
Figures adapted from bbe Moldaenke
Chlorophytes, Euglenophytes
Diatoms
Cryptophytes
Yellow Substances
Total Chlorophyll
Phycobilisomes Chl a/c, (Phycocyanin) Xanthophyll
Chl a/c Phycobilisomes (Phycoerythrin)
Chromophoric DOM
Total Chl a
PC-rich Cyanophytes
Cryptophytes, PE-rich Cyanophytes
Diatoms, Chrysophytes, Dinophytes
All groups
FLPB vs. SPEC Chlorophyll a – SS Data
1:1 line
Fitted regression
•
In situ FLPB chl was correlated with SPEC chl (r2=0.58) across full range of chl values (integrated and discrete depth samples pooled)
•
The FLPB overestimated chl compared to SPEC method (avg % error = 81%)
•
With outlier points removed (n=5): variance explained was slightly greater (r2=0.64), slope was closer to 1, intercept was further from 0
#10 of 19
FLPB vs. SPEC Chlorophyll a – MOE Data 10
60
FLPB Chl a (g/L)
50
TotalChl_IVF_ug/L
TotalChl_IVF_ug/L
FLPB Chl a (g/L)
70
40
30
20
FLPB = 1.27SPEC + 0.04 r2 = 0.82, df = 266
