evolution of cell density and toxin production of a harmful algal bloom species in marine microcosms

55 Pages·2011·1.31 MB·English

FACULTY OF BIOSCIENCE ENGINEERING
CENTER FOR ENVIRONMENTAL SANITATION
Academic year: 2010 -2011

EVOLUTION OF CELL DENSITY AND TOXIN PRODUCTION OF A
HARMFUL ALGAL BLOOM SPECIES IN MARINE MICROCOSMS

Amanual Hadera Tesfay

Promoters:
Prof.dr.C. Janssen
Dr. Michiel Vandegehuchte
Tutor:
Dr.Frederik De Laender

Master’s dissertation submitted in partial fulfillment of the requirements for

the Master of Environmental Sanitation.
Copy right
For personal or any purpose use of this thesis requires the authorization of the author and
promoters. Reproduction of any part of this thesis without the permission of the author and
promoters is subject to copyright protection.

The Promoters: The author:
Name Signature Name Signiture
Prof. Dr. C.Janssen..…………….………. Amanual Hadera …………..…….
Dr. Michiel Vandegehuchte………………
The Tutor:
Dr.Frederik De Laender……………………
Abstract
Harmful Algal blooms are algal blooms that can cause environmental problems by producing
toxins or depleting oxygen from the aquatic ecosystem (Boesch et al., 1996). HABs comprise
of different species, including dinoflagellates, diatoms and raphidopytes. This study dealt with
the dinoflagellate species Prorocentrum lima, which produces a toxin called okadaic acid
(OA) which is responsible for the cause of diarrhetic shellfish poisoning (DSP).
There are various factors that favor the development of HABs such as nutrients, temperature,
light and salinity. This study intended to investigate the effect of nutrients on harmful algal
bloom (Prorocentrum lima) on cell density and toxin (OA) production in marine microcosms.
Marine water from the North Sea, near the Belgian coast, was treated with six different
treatments to assess the effect of nutrients and initial cell density of P. lima on toxin
production and cell growth. Two nutrient concentrations (enriched and background nutrient
conditions, particularly nitrate and phosphate level: 0.05 mg PO -P/l and 0.58 mg NO -N/l in
4 3
enriched nutrient conditions and no addition in background nutrient conditions) and three
HAB concentrations (high, low and no HAB cells concentration: 1500, 150 and 0 cells/ml,
respectively) were used.
Both the high and background nutrient conditions showed a growth of P.lima cells at the 6th
day for each HAB treatment (low and high HABs). The growth was higher in the enriched
nutrient conditions than background nutrient conditions .For example, at day 6 in the low
HAB treatments, 1851 and 1349 cells/cm² were observed in the high and background nutrient
conditions, respectively. In general, higher toxin concentrations were observed in the high
HAB treatments than in the corresponding low HAB treatments and enriched nutrient
conditions also showed higher toxin concentrations than background nutrient conditions. The
statistical analysis explained that nutrient (particularly total nitrogen nutrients) did not have
effect on toxin production (P-value = 0.3806) but cell density had an effect on the toxin
production (P-value < 0.0001).
Therefore; cell growth or cell density evolutions depends on the initial cell density and
nutrient condition where as toxin production is depends on the cell density.
:
Key words Harmful Algal Blooms, Prorocentrum lima, toxins (okadaic acid), nutrients,

North Sea
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Acknowledgements
First of all, I would like to express my deepest gratitude to my promoter Prof.dr.Colin Janssen
for his permission for letting me to work in his laboratory and fully support and
encouragement to finish my work.
Next I would like to extend my heartfelt gratitude to my promoter Dr. Michiel Vandegehuchte
and tutor Dr.Frederik De Laender for their essential support, sharing experiences, providing
constructive suggestion, comments, invaluable time and practical involvement from the
beginning of the experiment till the completion of this work.
My thanks again goes to all staff of at the Laboratory of Environmental Toxicology and
Aquatic Ecology, Department of Applied ecology and environmental biology, especially for
Nancy De Saeyer for her indispensible technical supports and valuable advises on handling
nutrient measurements and results. I would also to acknowledge Dr. Lynn Vanhaecke for her
support in measuring toxins using liquid chromatography coupled with mass spectrometry
(LC-MS/MS) at the laboratory of Chemical Analysis, Department of Veterinary Public Health
and Food Safety, UGent.
My heartfelt thanks goes to Dr.Karen Klein, a team of General Practitioners, skeleton staff in
student clinic at Ghent University, for her motherly treatment and indispensible consultations
during my health problem and vital encouragement to push up throughout my studying
including this work. I would like also to thank to my friend Bruh Asmelash for his
constructive advices and encouragement during my health problems.
I would like to express my heartfelt gratitude to my family who stood beside me throughout
my course studies and encourage me all the time to be successful in my career. I would like
also to thank my friend Tewodros Tilahun for his sharing experience and helping me reading
and editing of this work.
Last but not least I would to thank VLIR-OUS for the financial support without that which is
impossible to complete my study and for all staff of Center Environmental Sanitation for their
genuine and kind cooperation and support to have a conducive environment throughout my
course studies and other administrative issues.

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Table of Contents
Abstract ....................................................................................................................................... I
Acknowledgements ................................................................................................................... II
List of table and figures ............................................................................................................. V
List of tables ........................................................................................................................................ V
List of figures ...................................................................................................................................... VI
List of Abbreviations ............................................................................................................. VIII
Chapter-One: Introduction ......................................................................................................... 1
1.1. Background of the study ......................................................................................................... 1
1.2. Statement of the problem ....................................................................................................... 1
1.3. Objectives ................................................................................................................................ 2
1.3.1. General objective ............................................................................................................ 2
1.3.2. Specific objective ............................................................................................................. 2
Chapter-Two: Literature reviews ............................................................................................... 3
2.1. What are Algal blooms? What are HABs? ............................................................................... 3
2.1.1. Algal Blooms (Definition of algal Blooms ........................................................................ 3
2.1.2. Harmful Algal Blooms (HABs) .......................................................................................... 4
2.2. Harmful Algal Blooms (HABs) and their toxins ........................................................................ 4
2.2.1. Species which may form HABs ........................................................................................ 4
2.2.2. Toxins and their classifications ........................................................................................ 5
2.3. Consequences of HABs ............................................................................................................ 7
2.4. Factors that affect HABs .......................................................................................................... 7
2.4.1. Biotic factors .................................................................................................................... 7
2.4.2. Abiotic factors.................................................................................................................. 8
2.5. Approaches for managing Harmful Algal Blooms (HABs) ...................................................... 10
Chapter –three: Methods and Materials ................................................................................... 13
3.1. Experimental Setup ............................................................................................................... 13
3.2. Sampling and storing ............................................................................................................. 15
3.2.1. Nutrients and toxins (water column) ............................................................................ 15
3.2.2. Toxins and cell counting ................................................................................................ 15
3.3. Analyzing techniques ............................................................................................................. 15
3.3.1. Sample preparation for nutrient analysis ...................................................................... 15
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3.2.2. Toxin extraction ............................................................................................................. 17
3.3.3. Measurement of nutrients and toxins .......................................................................... 18
3.4. Cell counting .......................................................................................................................... 20
3.5. Data Processing ..................................................................................................................... 21
Chapter –Four: Results and Discussions .................................................................................. 22
4.1. Results ................................................................................................................................... 22
4.1.1. Nutrient concentrations ................................................................................................ 22
4.1.2. HAB cell density ............................................................................................................. 26
4.1.3. Toxin production (intracellular and extracellular) ......................................................... 29
4.2. Discussion .............................................................................................................................. 35
4.2.1. Nutrients ........................................................................................................................ 35
4.2.2. Cell biomass (Cell density) ............................................................................................. 36
4.2.3. Toxin production (intracellular and extracellular) ......................................................... 36
Chapter –five: Conclusions and Recommendation .................................................................. 38
5.1. Conclusions ............................................................................................................................ 38
5.2. Recommendations................................................................................................................. 38
References: ............................................................................................................................... 40
Appendix .................................................................................................................................. 43

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List of table and figures
List of tables
Table 1 : HAB species and their effects (Uronen, 2007) .......................................................... 6
Table 2: Concentration of nutrients in standards and spikes and measuring parameters
(wavelength, detection limits and measuring ranges). ............................................... 19
Table 3: Sedgwick-Rafter magnification factor ....................................................................... 20
Table 4: Statistical analysis for fixed effect of nutrients and cells on toxin production.
Transfer cells (ml) means the cells concentration was explained using natural
logarithms in lme (linear mixed effect models) statistical analysis. .......................... 34

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List of figures
Figure 1: Experimental set up .................................................................................................. 14
Figure 2: Counting cell (Sedgwick-Rafter) showing method of filling (Source: WHIPPLE,
1927) .......................................................................................................................... 21
Figure 3: Concentrations of NH +-N, NO --N and NO --N in background nutrient conditions
4 2 3
and no harmful algal bloom (HAB) treatments. The values that lie on the x-axis are
not zero; they are values below detection limit. Detection limit for NH +-N, NO --N
4 2
and NO --N is 0.05, 0.02 and 0.2 mg/L, respectively ................................................ 23
3
Figure 4: Concentrations of NH +-N, NO --N and NO --N in background nutrient conditions
4 2 3
and low harmful algal bloom (HAB) treatments. The values that lie on the x-axis are
not zero; they are values below detection limit. ......................................................... 23
Figure 5: Concentrations of NH +-N, NO --N and NO --N in background nutrient conditions
4 2 3
and high harmful algal bloom (HAB) treatments. The values that lie on the x-axis are
not zero; they are values below detection limit. ......................................................... 24
Figure 6: Concentrations of NH4+-N, NO --N and NO --N in enriched nutrient conditions and
2 3
no harmful algal bloom (HAB) treatments. The values that lie on the x-axis are not
zero; they are values below detection limit. Detection limit for NH +-N, NO --N and
4 2
NO --N is 0.05, 0.02 and 0.2 mg/L, respectively ....................................................... 25
3
Figure 7: Concentrations of NH4+-N, NO --N and NO --N in enriched nutrient conditions and
2 3
low harmful algal bloom (HAB) treatments. The values that lie on the x-axis are not
zero; they are values below detection limit. ............................................................... 25
Figure 8: Concentrations of NH4+-N, NO --N and NO --N in enriched nutrient conditions and
2 3
high harmful algal bloom (HAB) treatments. The values that lie on the x-axis are not
zero; they are values below detection limit. ............................................................... 26
Figure 9: The evolution of cells density in background nutrient conditions and low harmful
algal bloom (HAB) treatments. .................................................................................. 27
Figure 10: The evolution of cells density in enriched nutrient conditions and low harmful
algal bloom (HAB) treatments. .................................................................................. 27
Figure 11: The evolution of cells density in background nutrient conditions and high harmful
algal bloom (HAB) treatments. .................................................................................. 28
Figure 12: The evolution of cells density in enriched nutrient conditions and high harmful
algal bloom (HAB) treatments. .................................................................................. 28
Figure 13: Intracellular toxin concentration in background nutrient conditions and low
harmful algal bloom (HAB) treatments. The values that lie on the x-axis are not zero;
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they are values below detection limit (DL= 0.0003575 µg/ml). OA refers to okadaic
acid, which is a toxin produced by P.lima. ................................................................ 29
Figure 14: Intracellular toxin concentration in background nutrient conditions and high
harmful algal bloom (HAB) treatments. OA refers to okadaic acid, which is a toxin
produced by P.lima. ................................................................................................... 30
Figure 15: Intracellular toxin concentration in enriched nutrient conditions and low harmful
algal bloom (HAB) treatments. The values that lie on the x-axis are not zero; they are
values below detection limit (DL= 0.0003575 µg/ml). OA refers to okadaic acid,
which is a toxin produced by P.lima. ......................................................................... 30
Figure 16: Intracellular toxin concentration in enriched nutrient conditions and high harmful
algal bloom (HAB) treatments. OA refers to okadaic acid, which is a toxin produced
by P.lima. ................................................................................................................... 31
Figure 17: Toxin in water column in background nutrient conditions and low harmful algal
bloom (HAB) treatments. OA refers to okadaic acid, which is a toxin produced by
P.lima. ........................................................................................................................ 32
Figure 18: Toxin in water column in background nutrient conditions and high harmful algal
bloom (HAB) treatments. OA refers to okadaic acid, which is a toxin produced by
P.lima. ........................................................................................................................ 32
Figure 19: Toxin in water column in enriched nutrient conditions and low harmful algal
bloom (HAB) treatments. The value lies at x-axis is not zero, it is below detection
limit (0.088 µg/l). OA refers to okadaic acid, which is a toxin produced by P.lima. 33
Figure 20 : Toxin in water column in enriched nutrient conditions and high harmful algal
bloom (HAB) treatments. The value lies at x-axis is not zero. OA refers to okadaic
acid, which is a toxin produced by P.lima. ................................................................ 33

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List of Abbreviations
CRM-OA-c: Certified Reference Materials Program-Okadaic Acid-calibration
HABs: Harmful Algal Blooms
LC-MS: Liquid Chromatography –Mass Spectrometer
NRC: National Research Council Canada
OA: Okadaic acid
SPE: Solid Phase Extraction

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