Impact of Ocean Acidification on carbonate production by

large benthic foraminifera Marginopora vertebralis in coastal

waters of Fiji

by

Roselyn Naidu

A thesis submitted in fulfillment of the

requirements for the degree of Master

of Science in Marine Science

Copyright ©2015 by Roselyn Naidu

School of Marine Studies

Faculty of Science, Technology and Environment

The University of the South Pacific

October, 2015

ii

DECLARATION

Statement by Author

I, Roselyn Naidu, declare that this thesis is my own work and that, to the best of my

knowledge, it contains no material previously published, or substantially overlapping

with material submitted for the award of any other degree at any institution, except

where due acknowledgement is made in the text.

Signature: Date: 10 September 2015

Name: Ms. Roselyn Naidu.

Student ID No: s11031942

Statement by Supervisor

The research in this thesis was performed under my supervision and to my knowledge is

the sole work of Ms Roselyn Naidu.

Signature: Date: 10 September 2015

Name: Dr. Matakite Maata

Designation: Senior Lecturer in Chemistry

iii

ACKNOWLEDGEMENTS

Firstly, I would like to acknowledge the constant support and encouragement of my ex-

supervisor, Dr. Susanne Pohler, School of Marine Studies, The University of the South

Pacific. My current supervisor, Dr. Matakite Maata, School of Biological and Chemical

Sciences, The University of the South Pacific, Professor Jonathan Erez, School of Earth

Sciences, The Hebrew University of Jerusalem. I also wish to acknowledge Dr. Helene

Jacot Des Combes for her support as the co-supervisor. I would like to acknowledge

PACE-SD for their financial support, without which the completion of this project would

have been difficult.

My sincere thanks to who made the work possible through use of the facilities of the

confocal microscope, electron probe and LA-ICPMS at the Hebrew University of

Jerusalem, Israel. I am grateful to Prof Jonathan Erez for the hospitality during the

duration of the attachment with Hebrew University.

My gratitude goes to Dr. William Camargo and Dr Delphine Dissard for their comments

and advice.

Particular acknowledgement is due to the staff and postgraduate students of the Marine

Studies Programme, University of the South Pacific, for their co-operation and

assistance during data collection and analysis. I would also like to acknowledge

Professor Pamela Hallock Muller from the University of South Florida and Dr. Antoine

N’Yeurt from The University of the South Pacific.

Last but not least, I would like to thank Mr Vijay Rajnesh Prasad for his support and my

parents Subarmani and Parwati for bringing me to this stage in my life.

iv

ABSTRACT

Increased CO2 emissions into the atmosphere lead to increased concentrations of

dissolved CO2 in the ocean. A chemical reaction between the dissolved CO2 and

seawater produces HCO3- , CO3

2- and H+ ions. These H+ ions increase the acidity of

seawater and decrease the pH. Increased acidity and decreased availability of CO32- ion

affect calcite and aragonite production by marine calcifiers in the ocean. Large benthic

foraminifera, such as Marginopora vertebralis Quoy and Gaimard, 1830, produce calcite

with high magnesium (Mg) content and have an important role in sand building in the

Pacific Islands. Foraminifera also have the ability to integrate trace elements and

isotopes from the seawater into their calcareous shells and serve as paleoceanographic

archives. It is thus important to better understand the biomineralization processes in

foraminifera to predict their calcification responses to ocean acidification.

To assess the response of a porcelaneous benthic foraminifer with algal endo-symbionts

to changing CO2 levels, M. vertebralis were cultured at pH 7.5, pH 7.8 and pH 8.1

(ambient seawater). The fluorescent compound Calcein (̴ 40 �M) was added to the

culture tanks to mark the calcite produced prior to the experiment. The specimens grown

in the laboratory were analysed using Laser Ablation-Inductively Coupled Plasma Mass

Spectroscopy (LA-ICPMS) and Electron Probe Micro Analyser (EPMA) to measure

elemental compositions, providing data to calculate boron and strontium isotope ratios

and Mg/Ca, Sr/Ca and S/Ca ratios. Shell growth, in terms of both radius and weight,

decreased with decreasing pH, as did the boron isotope ratios. The Mg/Ca ratios

decreased, while Sr/Ca ratios increased, with decreasing pH. The S/Ca ratio also

increased with a decreasing pH. Isotopic ratios for B (δ 10/11B) and Sr (δ 88/86 Sr) both

increased with decreasing pH. Since it is possible to calibrate the shell composition

against the controlling factors, foraminiferal trace elements and isotopic ratios can

provide researchers with vital and relevant proxies to investigate the physical, biological

and chemical changes in the ocean.

v

TABLE OF CONTENTS

DECLARATION .............................................................................................................. ii

ACKNOWLEDGEMENTS ...........................................................................................iii

ABSTRACT ....................................................................................................................iii

FIGURE CAPTIONS ....................................................................................................viii

TABLE CAPTIONS ......................................................................................................... x

Chapter 1 INTRODUCTION .......................................................................................... 1

1.1 AIM ..................................................................................................................... 1

OBJECTIVES .................................................................................................................. 1

HYPOTHESIS .................................................................................................................. 1

1.2 INTRODUCTION ............................................................................................... 2

1.3 BASICS OF OCEAN CHEMISTRY .................................................................. 3

1.3.1 Seawater carbonate chemistry ...................................................................... 4

1.3.2 Importance of carbon sinks in changing climate .......................................... 6

1.3.3 Climate change and its impacts .................................................................... 6

1.3.3.1 Contribution of atmospheric carbon dioxide to ocean acidification......... 8

1.3.3.2 Implications for ocean acidification and carbon sequestration ................ 9

1.4 FORAMINIFERA ............................................................................................... 9

1.4.1 Classification of foraminifera....................................................................... 9

1.4.2 Life cycles of foraminifera ......................................................................... 14

1.4.3 Distribution and ecology of foraminifera ................................................... 16

1.4.4 Carbonate production by benthic foraminifera .......................................... 17

1.4.5 Previous studies of Ocean Acidification .................................................... 18

1.4.5.1 Previous studies of responses of foraminifera ........................................ 18

1.4.5.2 Culture setups for ocean acidification studies ........................................ 19

1.4.5.3 Measuring growth (Weighing method and Calcein marking) ................ 21

1.4.5.4 Elemental concentrations & ratios .......................................................... 21

1.5 STUDY GOALS ............................................................................................... 22

Chapter 2 GROWTH OF Marginopora vertebralis IN DIFFERENT pH TREATMENTS .............................................................................................................. 23

vi

2.1 INTRODUCTION ............................................................................................. 23

2.2 METHODOLOGY ............................................................................................ 23

2.2.1 Sample collection ....................................................................................... 23

2.2.2 Experimental set-up.................................................................................... 25

2.2.3 Measuring growth ...................................................................................... 28

2.2.3.1 Fluorescent Marker ................................................................................. 28

2.2.3.2 Weighing Method ................................................................................... 30

2.3 RESULTS .......................................................................................................... 31

2.3.1 Other observations...................................................................................... 38

2.4 DISCUSSION .................................................................................................... 40

Chapter 3 CARBONATE CHEMISTRY OF CULTURE MEDIUM ....................... 42

3.1 INTRODUCTION ............................................................................................. 42

3.1.1 Purpose ....................................................................................................... 42

3.1.2 Inorganic Carbon in Seawater .................................................................... 42

3.1.3 Benthic foraminifera under ocean acidification ......................................... 46

3.2 METHODOLOGY ............................................................................................ 47

3.2.1 Determination of total alkalinity in seawater ............................................. 47

3.2.2 Analysis using CO2SYS ............................................................................ 48

3.3 RESULTS .......................................................................................................... 49

3.4 DISCUSSION .................................................................................................... 52

Chapter 4 ELEMENTAL RATIOS USING EPMA (ELECTRON PROBE MICRO-ANALYSER)................................................................................................................... 54

4.1 PURPOSE/INTRODUCTION .......................................................................... 54

4.2 METHODOLOGY ............................................................................................ 55

4.2.1 Embedding of specimens ........................................................................... 55

4.3 RESULTS .......................................................................................................... 60

4.3.1 Images from EPMA (Electron Probe Micro Analyzer) ............................. 60

4.3.2 EPMA Data Analysis ................................................................................. 62

4.3.3 Variations in elemental ratios in-relation to [CO32-] .................................. 67

4.4 DISCUSSION .................................................................................................... 69

Chapter 5 ELEMENTAL CONCENTRATIONS USING LA-ICPMS .................... 71

vii

5.1 INTRODUCTION ............................................................................................. 71

5.1.1 Purpose ....................................................................................................... 71

5.1.2 Background ................................................................................................ 71

5.2 METHODOLOGY ............................................................................................ 72

5.2.1 LA ICP MS analysis with Sills .................................................................. 76

5.3 RESULTS .......................................................................................................... 79

5.4 DISCUSSION .................................................................................................... 84

CONCLUSIONS ............................................................................................................ 88

REFERENCES ............................................................................................................... 90

viii

FIGURE CAPTIONS

Figure.1.1: Carbon cycle with different reservoirs ............................................................ 3

Figure 1.2: The pH Scale.................................................................................................... 4

Figure 1.3 The absorption of CO2 and the chemical reactions........................................... 5

Figure 1.4 CO2 and temperature over the last 420,000 years ............................................. 7

Figure 1.5 Principal types of chamber arrangement ........................................................ 10

Figure 1.6 Principal types of aperture .............................................................................. 11

Figure 1.7 Taxonomy of Marginopora vertebralis .......................................................... 13

Figure 1.8: Growth stages of Marginopora vertebralis ................................................... 14

Figure 1.9 Life cycles of Foraminifera............................................................................. 15

Figure 2.1: (A) Map of Laucala Bay ................................................................................ 24

Figure 2.2 Experimental set-up model ............................................................................. 27

Figure 2.3 M.vertebralis treated in 40 µmol/L of Calcein ............................................... 28

Figure 2.4: Basic setup of a confocal microscope ............................................................ 29

Figure 2.5. Confocal microscopy image of a specimen from pH treatment 7.5. ............. 32

Figure 2.6. Confocal microscopy image of a specimen from pH treatment 7.8. ............. 32

Figure 2.7 Confocal microscopy images of specimens from two replicate tanks of pH treatment 8.1. .................................................................................................................... 33

Figure 2. 8 Weight increase of 25 specimens of M. vertebralis in each replicate for each pH treatment. .................................................................................................................... 34

Figure 2. 9 Relative weight gain by 25 individuals of M. vertebralis at each pH treatment level .................................................................................................................................. 35

Figure 2.10 Relationship between increase in shell radius and in average shell weight of Marginopora vertebralis .................................................................................................. 36

Figure 2.11 Net like structure of pseudopodia. ................................................................ 38

Figure 2.12 Reproduction of Marginopora vertebralis at pH treatment 8.1 .................... 39

Figure 3.1: pCO2 in seawater of two pH treatments (7.5 and 7.8) and control (8.1). ...... 50

Figure 3.2: Mean increase in shell weight of 25 M. vertebralis, grouped by pH level at three pCO2 levels. ............................................................................................................. 51

Figure 3.3: Mean shell weight (mg) of 25 M. vertebralis, grouped by treatment against CO3

2- (mmol kg-1 SW)...................................................................................................... 51

ix

Figure 4.1 Growth was measured in pH 7.5; pH 7.8; pH 8.1 using a confocal microscope .......................................................................................................................................... 56

Figure 4.2 How the specimens were cut for embedding using epoxy mixture and then for viewing under EMPA ....................................................................................................... 57

Figure 4.3: Preparation of sample for EPMA: ................................................................. 58

Figure 4.4: A typical arrangement of a probe lab ............................................................ 59

Figure 4.5: Growth or dissolution was determined by observing the number of chambers formed or dissolved .......................................................................................................... 61

Figure 4.6: Outline of areas of each specimen cultured at pH 7.5 ................................... 63

Figure 4.7: Outline of areas of each specimen cultured at pH 7.8 ................................... 64

Figure 4.8: Outline of areas of each specimen cultured at pH 8.1 ................................... 65

Figure 4.9: Peaks generated by EPMA while analyzing M. vertebralis cultured at pH 7.5; pH 7.8; pH 8.1. ................................................................................................................. 66

Figure 4.10 Mean Mg/Ca (mmol/mol) against CO32- (a) and mean S/Ca (mmol/mol)

against CO32- (b). .............................................................................................................. 68

Figure 5.1 Example of M. vertebralis specimen (a) before (b) after analysis with LA-ICPMS. ............................................................................................................................. 73

Figure 5.2 Shows a laser – ablation inductively – coupled – plasma mass – spectrometry .......................................................................................................................................... 74

Figure 5.3 Typical transient LA–ICP–MS signal from different elements while analyzing for elemental concentration in Marginopora vertebralis ................................................. 75

Figure 5.4 Shows UP193-FX Excimer laser ablation system .......................................... 75

Figure 5.6 Shows a SAMPLE (Unknown) window when the unknowns are loaded in SILLS. ............................................................................................................................... 76

Figure 5.5 Shows a STANDARD window when the standards are loaded in SILLS. ..... 76

Figure 5.7 Shows spike elimination window ................................................................... 76

Figure 5.8 Calibration Graphs window ............................................................................ 77

Fig 5.8: Mean B/Ca plotted against pH of culture media. B/Ca data represent means of all measurement for specimens from that treatment ........................................................ 81

Fig 5.9: Mean Mg/Ca plotted against pH of culture media. Mg/Ca data represent means of all measurement for specimens from that treatment. ................................................... 81

Fig 5.10: Mean Sr/Ca plotted against pH of culture media. ............................................. 82

Fig 5.11: The δ 11B of M. vertebralis increases with increasing seawater acidity............ 82

Fig 5.12: The δ 88/86Sr of M. vertebralis increases with increasing seawater acidity ...... 83

x

TABLE CAPTIONS

Table 1.1: Ocean acidification studies and the response of lower pH on benthic foraminifera. ..................................................................................................................... 19

Table 2.1: Mean increase in radius of shells of specimens in each replicate of each treatment after 11 weeks in culture. ................................................................................. 31

Table 2.2: Linear regression parameters for the increase in wet weight (g) of each treatment and replicate ..................................................................................................... 35

Table 2.3 The mean increase in shell radius and the mean increase in shell weight ....... 36

Table 2.4 Mean growth rates for group of specimens at each pH treatment and their replicates........................................................................................................................... 37

Table 3.0 Major Seawater Constituents ........................................................................... 45

Table 3.1 Carbonate system parameters pertinent to the experiment. ............................. 49

Table 3.2 Mean pCO2 (matm) and mean [CO32-] (mmolkg-1 SW) calculated from pH and

alkalinity measurements and mean increase in weight (g) for 25 specimens. ................. 50

Table 4.1 Relative concentrations of Mg, S, Ca, P, Na and Sr extracted from each spectral analysis................................................................................................................ 67

Table 5.1: Laser Ablation ICP-MS operating parameters ................................................ 74

Table 5.2 Mean values of isotopes of selected elements in M. vertebralis in shell produced under three different pH treatments.................................................................. 79

Table 5.3. Mean values of Element/Ca ratios of M. vertebralis in shell produced under each pH treatment............................................................................................................. 80

Table 5.4 Mean values of the Sr and B isotopic compositions of M. vertebralis cultured at three different pH treatments. ....................................................................................... 82

1

CHAPTER 1 INTRODUCTION

1.1 AIM

The primary goal of this thesis was to study the impact of declining pH (acidification) of

seawater on calcification of Marginopora vertebralis Quoy and Gaimard, 1830, a reef-

dwelling species of Foraminifera.

OBJECTIVES

� To experimentally determine the response of M. vertebralis to increased ocean

acidification.

� To determine how selected elemental compositions and elemental ratios in shells

of these foraminifera change in response to increased ocean acidification.

� To better understand how ocean acidification will affect calcification and

calcification rates.

HYPOTHESIS

The hypothesis of the study is that the increased partial pressure of carbon dioxide

(pCO2) in the atmosphere leads to increased pCO2 in the ocean, which causes increased

acidification of seawater, thus decreasing the calcification rate and altering the shell

chemistry in the large benthic foraminiferal species, Marginopora vertebralis.

2

1.2 INTRODUCTION

Ocean acidification (the decrease in pH, and subsequently in the carbonate ion

concentration in seawater) in response to rising anthropogenic atmospheric pCO2 is

generally expected to reduce calcification of the main marine calcifiers, with potentially

severe implications for coral-reef ecosystems (Kleypas et al., 1999). Algal symbiont-

bearing, reef-dwelling foraminifera are one of the most important primary carbonate

producers in coral reefs (Muller 1974; Smith and Wiebe 1977; Hallock, 1981). They

mainly produce high-Mg calcite shells, whose solubility can exceed that of aragonite

produced by corals, making them among the “first responders” in coral reefs to the

decreasing carbonate (CO32-) saturation state of seawater (Fujita, 2008). As acidity and

sea temperature increase, the ocean’s actual ability to absorb atmospheric CO2 is being

reduced, thus exacerbating the rate of climate change. The rate at which the acidification

of the ocean occurs depends mainly on the chemical exchange reactions with the

atmosphere. Gaining knowledge of ocean conditions in the past enhances understanding

of the present climate and also provides insight into future climate changes (Feely et al.,

2009). “Proxies” (often assessed using foraminiferal shells) are used to develop methods

to obtain information about oceanic changes.

Because M. vertebralis is an important source of carbonate sediments in Laucala Bay,

Fiji, we assessed its potential responses to ocean acidification in a laboratory study of the

effects of reduced pH on shell growth and shell chemistry using stable isotope

composition and elemental ratios.

The structure of this thesis is to first present, background information on ocean

chemistry, foraminiferal shells as indicators of ocean chemistry, and experimental

methods utilized in previous studies of changes in ocean chemistry on calcareous

organisms. Chapter 2 presents the methods and results of an experimental study of the

influence of seawater pH on growth and calcification of M. vertebralis. Chapter 3

describes the carbonate chemistry of the cultured medium. Chapter 4 presents analyses

of elemental ratios using EPMA (Electron Probe Micro Analyzer). Chapter 5 presents

the results of analysis of elemental concentrations using LA-ICPMS (Laser Ablation

3

Inductively Coupled Plasma Mass Spectrometry). Chapter 6 presents a synthesis of the

results from Chapters 2–5 and discussion of their significance, in context of previous

studies and projection of future ocean acidification, concluding with suggestions for

future studies.

1.3 BASICS OF OCEAN CHEMISTRY

Carbon dioxide is essential for life on Earth, including life in the ocean. The carbon

cycle (Fig 1.1) is described as a biogeochemical cycle by which carbon is recycled and

reused throughout the land, the oceans and the atmosphere. The major reservoirs of

carbon include: the atmosphere, the terrestrial environment (including living organisms,

fresh-water ecosystems and non-living organic matter), the ocean (dissolved inorganic

carbon, marine biota, and non-living forms of organic carbon), the sediments and

sedimentary rocks (including fossil fuels) and the Earth’s interior. The marine carbon

cycle includes the production and recycling of organic material and carbonate sediments

and rocks (CaCO3).

Figure.1.1: Carbon cycle with different reservoirs where carbon is stored

(http://spark.ucar.edu/imagecontent/carbon-cycle-diagram-nasa)

4

Increasing atmospheric carbon dioxide is causing a significant decline in seawater pH as

a result of the imbalance between the rate of CO2 uptake into the ocean and the ocean’s

ability to buffer the resulting increase in hydrogen ions (Caldeira and Wickett, 2003).

Since the industrial revolution, seawater pH has declined by an average of 0.1 units

(Kleypas et al., 2006; IPCC, 2013); pH is the negative logarithm of the hydrogen ion

concentration (Fig 1.2). It has been estimated by IPCC (2013) that the pH will decrease

from a current global average of approximately 8.1 to as low as 7.7 by 2100.

Figure 1.2: The pH Scale (modified from Ophardt, 2003)

1.3.1 SEAWATER CARBONATE CHEMISTRY

The carbonate system controls the acidity of seawater and acts as a regulator for the

carbon cycle. The carbonate system of the ocean plays a key role in controlling the

partial pressure of carbon dioxide in the atmosphere, which further helps in regulating

the temperature of the globe.

Seawater pH is a key parameter that reflects the speciation of the carbonate system,

nutrients and other major and trace-element species in the oceans. It is largely unknown

if, or how, various organisms will adapt to the large-scale pH changes that are

pH of seawater

5

anticipated over the next two to three centuries. At present it is not possible to determine

how the ecosystem structure will change or how these ecosystem changes might

influence future climate-feedback mechanisms. It is therefore important to develop new

research strategies to better understand the long term vulnerabilities of sensitive marine

organisms to these changes (Kleypas et al., 2006). Seawater acidification not only

affects the ecosystem components that are CO2 sensitive, but also affects those

components that interact with them as epibionts, predators, preys and competitors

(Langer et al., 2006).

Calcium carbonate production in the ocean is mostly biogenic. Photic zones have the

highest biogenic calcification rates, while the highest dissolution rates occur in the deep

ocean (Kleypas and Landon, 2006).

Figure 1.3 The absorption of CO2 and the chemical reactions that occur within the marine environment (Modified from Kleypas et al., 2006)

6

Four processes that are responsible for the uptake of atmospheric CO2 into the ocean are:

(i) air-sea exchange of CO2 gas and formation of carbonic acid, which later dissociates to

form bicarbonate and carbonate ions; (ii) production of CaCO3 (calcification) and its

dissolution; (iii) photosynthesis and respiration; and (iv) ocean circulation (Fig 1.3).

1.3.2 IMPORTANCE OF CARBON SINKS IN CHANGING CLIMATE

Processes that remove greenhouse gases from the atmosphere are called ‘sinks’ and

processes that put greenhouse gases in the atmosphere are called ‘sources’.

Net emissions = (emissions from sources) – (removal by sinks)

Over the two decades of the 1980s and 1990s, only about half of the CO2 released by

human activity has remained in the atmosphere, with the oceans having taken up about

30% and the terrestrial biosphere 20% (Sabine et al., 2004). Similar partitioning of

anthropogenic CO2 is expected to continue with the result that the partial pressure of

CO2 (pCO2) dissolved in the surface ocean is likely to double its pre-industrial value

within the next 50 years. Over the next millennium, the ocean will absorb about 90% of

the anthropogenic CO2 released to the atmosphere (Archer et al., 1989). Deep-ocean

injection of CO2 extracted from gases released from industrial and renewable energy

(biogas) activities could potentially mitigate global warming by removing CO2 from its

sources and pumping it in the form of gel liquid into sea-floor depressions where it could

dissolve over millennial time scales (Haugan and Drange, 1992).

1.3.3 CLIMATE CHANGE AND ITS IMPACTS

According to Nunn (2007), the people of the Pacific Island countries are likely to face

climate changes that will threaten the sustainability of their livelihoods. This will occur

through: a) increased climate variability such as amplification of the present ENSO

pattern that sees El Niño occurring every 3–5 years with the likelihood that there will be

less seasonal rain in the future; b) changes in climatic extremes, such as the recent

increased incidence of intense tropical cyclones; c) temperature rise that will impact

terrestrial ecosystem productivity and near shore ecosystems, particularly coral reefs;

and d) sea level rise that will cause coastal inundations, shoreline erosion, groundwater

7

salination in low-lying areas, and allow more large amplitude waves to cross offshore

reef barriers than at present.

The greenhouse effect causes the increase in surface temperature where the thermal

radiation from the Earth’s surface is absorbed by atmospheric greenhouse gases (GHG)

(water vapor, CO2, N2O, and CH4) and is re-radiated in all directions. Consequently,

further global warming is projected with an increase in global average temperature

between 1.4º C– 5.8º C and rise in sea-level of 20–60 cm by the end of this century, 2100

(IPCC, 2013). Over the last 420,000 years, the global atmospheric concentration of CO2

fluctuated naturally between 180 parts per million (ppm) and 280 ppm in glacial and

interglacial conditions, respectively. In the last 200 years, since the industrial revolution,

atmospheric CO2 has increased dramatically, recently reaching 400 ppm (Fig 1.4). Both

GHG emissions (CO2) and the global mean temperature are consistent with the highest

predictions rather than the middle of the range (IPCC, 2013). Thus some effects that, in

2000, were projected to occur in about 2020 are already being observed now.

Figure 1.4 CO2 and temperature over the last 420,000 years (from IPCC Fourth Assessment Report, WG4, 2007). The current global concentrations of CO2 in the atmosphere ( ̴400 parts per million) are the highest in the last 420,000 years.

8

1.3.3.1 Contribution of atmospheric carbon dioxide to ocean acidification

Carbon dioxide (CO2) is one of the most important gases in the atmosphere, affecting the

radiative heat of the Earth as well as the calcium carbonate (CaCO3) equilibrium of the

oceans. As the amount of CO2 dissolved in the ocean increases, the pH decreases, as

does the availability of carbonate (CO3-2) ions, which lowers the saturation state of the

major carbonate minerals found in shells and skeletons of marine organisms. Tripling the

pre-industrial atmospheric CO2 concentration will cause a reduction in surface ocean pH

that is almost three times greater than that experienced during transitions from glacial to

interglacial periods. This decrease in available alkalinity is often termed “ocean

acidification” because it leads to decreasing pH (Caldeira and Wickett, 2005).

Atmospheric CO2 concentrations remained between 180 and 330 parts per million by

volume (ppmv) for 650,000 years prior to the Industrial Revolution (Siegenthaler et al.,

2005). Increased fossil-fuel burning associated with industrialization, as well as cement

production and land-use changes associated with agricultural activities, are causing

atmospheric CO2 concentrations to rise at increasing rates (rates of increase have risen

from 0.25% y-1 in the 1860s to 0.75% y-1 in recent years). Atmospheric CO2

concentration is expected to continue to rise by about 1% y-1 over the next few decades

(Houghton, et al., 2001). The rate of current and projected CO2 increase is about 100

times faster than what has occurred over the past 650,000 years, and the rise in

atmospheric CO2 levels is considered irreversible on human timescales (Dooney et al.,

2009). Even with immediate action, the reaction time of the ocean will result in a

declining pH in the oceans (Liu et al., 2010).

According to Steinacher et al. (2009), the issue of ocean acidification is linked with the

saturation state of aragonite, which controls the growth of corals. Steinacher et al. found

that the largest variation of pH occurs in the Arctic surface waters, where the hydrogen

ion has increased 185% (∆pH= -0.45) due to freshening and increased carbon uptake in

response to sea-ice retreat.

9

1.3.3.2 Implications for ocean acidification and carbon sequestration

It is predicted that, if the current rate of CO2 release into the atmosphere continues, by

the year 2100 the pH of the surface ocean will be lowered by 0.4 units compared to the

pH level at the start of the century, with a likely reduction of 0.7 units by the year 2250

(Caldeira and Wickett, 2003; IPCC 2013).

1.4 FORAMINIFERA

1.4.1 CLASSIFICATION OF FORAMINIFERA

Foraminifera are testate protists, i.e., single-celled organisms that produce a shell. The

name Foraminifera comes from “foramen”, which is the opening through the septum that

separates each chamber (University College London, 2002). This opening gives this

protozoan class its name.

Foraminifera have inhabited the oceans for more than 500 million years (Boudagher-

Fadel, 2008). Their shells maybe one chamber, though many have shells that are divided

into multiple chambers. These chambers are added during the growth of an individual

(Sen Gupta, 2003). Foraminifera have historically been classified based on their shell

structure. Three basic types of wall composition common amongst living foraminifera

are: a) the agglutinated shell, which can either be composed of very small particles

cemented together and have very even surface or be made up of larger particles and have

an uneven surface; b) the hyaline perforate shell, made of interlocking crystals of calcite

measured in micrometers, with glassy appearance and abundant pores; and c)

porcelaneous imperforate shell, made of microscopic randomly oriented rod-shaped

crystals of high magnesium calcite, resulting in a milky appearance (e.g., Loeblich and

Tappan, 1964; Wetmore, 1995; Sen Gupta, 2003). Due to the abundance of well-

preserved shells, foraminifera are very useful in analyzing recent and ancient marine

environments (Murray, 2002).

Two other features that are important when it comes to classification of the Foraminifera

are chamber arrangement and aperture style (University College London, 2002). The

common types of chamber arrangements are unilocular (shells with only a single

10

chamber) and multilocular (shells with several chambers) (Fig 1.5). Among the

multilocular arrangements there are the uniserial (the chambers are attached in a single

straight line); biserial (the chamber are attached in straight line but in pairs); triserial

(linear series in which chambers are attached in triplets); planispiral (the chambers take

up a coil shape); trochospiral (the coiling of chambers takes up the shape of a spire);

milioline (each chamber is added at an 180° angle from the previous chamber) (e.g.,

Loeblich and Tappan, 1964; Wetmore, 1995; Sen Gupta, 2003).

Figure 1.5 Principal types of chamber arrangement (University College London, 2002). 1, single chambered; 2, uniserial; 3, biserial; 4, triserial; 5, planispiral to biserial; 6, milioline; 7, planispiral evolute; 8, planspiral involute; 9, streptospiral; 10-11-12, trochospiral; 10, dorsal view; 11, edge view; 12, ventral view. (Loeblich and Tappan 1964).

11

Apertures link the external granuloreticulopodia (temporary projections of the cytoplasm

that are used for locomotion and ingesting food) with the internal endoplasm (Sharma,

2007). Apertures are typically found in the walls of the final chamber (Fig 1.6). Since

the aperture does not change position through ontogeny, each chamber is connected to

the next by a single (foramen) or several opening (foramina) that were originally

apertures (University College London, 2002).

Figure 1.6 Principal types of aperture (University College London, 2002). 1, open end of the tube;

2, terminal radiate; 3, terminal slit; 4, umbilical; 5, loop shaped; 6, interiomarginal; 7,

interiomarginal multiple; 8, areal crbrate; 9, with phialine lip; 10, with bifid tooth; 11, with umbilical

teeth; 12, with umbilical bulla. (Loeblich and Tappan 1964).

12

Fully grown individuals range from about 0.1 mm to almost 20 cm in size (Wetmore,

1999; Sen Gupta, 2003). A single individual may have one or many nuclei within its

cell. Many of the largest living species in the warm subtropical seas have symbiotic

relationships with algae. Most taxa, including those with algal symbionts, eat foods

ranging from dissolved organic molecules, bacteria, diatoms and other single-celled

phytoplankton, or small animals such as copepods (Wetmore, 1999).

Both planktonic and benthic species are sensitive to changes in food availability as well

as to physical and chemical environmental parameters, such as salinity, nutrient content

and temperature (Polyak et al., 2001). Because of this sensitivity, foraminifera are useful

indicators of environmental change, both on the local and global scales.

Foraminifera are well known for their excellent fossil record which is used in

biostratigraphic, paleoenvironmental, paleoceanographic and paleoclimatic

reconstructions (Pawlowski et al., 2003). In biostratigraphy, foraminiferal species or

assemblages are used to determine relative ages and to correlate sedimentary rock units

across regions or even globally. For paleoenvironmental interpretations, fossil

assemblages can indicate the environmental conditions under which sediments were

deposited. Past surface and bottom water temperatures have been mapped from the

measurements of stable oxygen isotopes in planktonic and benthic foraminiferal shells

(Wetmore, 1999). Together with foraminifera, other marine organisms such as diatoms

and testate amoebae can be used as sea level indicators (Allen, 2005). Gehrels (2001)

used a multi-proxy method to obtain more precise estimates of sea level change.

According to Lieberman (2000), ecological paleobiogeographic studies are an example

of an area of future growth for this field of study. Paleocology and paleobiogeography

can help the future scientists to understand how climate and ocean currents have changed

in the past and how they may change in the future.

This study will focus on Marginopora vertebralis (Fig 1.7). This large benthic

porcelaneous foraminiferal species lives in the shallowest ecological environment (i.e.,

the intertidal and shallow subtidal zone) and is an important producer of sediment in

Fijian beaches (Sharma, 2007); its demise could have serious consequences for beach

stability, especially under rising sea level.

13

The shells of foraminifera are preserved in the sediments after they die, allowing the

analysis of the conditions in which they lived. Marginopora vertebralis is a shallow-

water, tropical foraminifera species. Three stages of shell construction are evident in

adult shells (Fig 1.8): the embryonic, orbitoid and reproductive chamber stages (Ross,

1972). This arrangement of chambers reveal three bands of coloration: a narrow, light

yellowish-green band adjacent to the proloculus (initial chamber); a broader brownish-

yellowish green band that represents most of the orbitoid vegetative chambers, and, if

present, a cream-colored outer band of reproductive chambers (Ross, 1972). New

chambers are typically added every four to five days. At the third stage of growth, 4–58

days after formation of the last vegetative chamber (Rottger, 1974), reproductive

chambers are formed (Ross, 1972).

Domain : Eukaryota – Whittaker & Margulis,1978

Kingdom : Protozoa – (Goldfuss,1818) R.Owen,1858 – Protozoa

Subkingdom : Biciliata

Infrakingdom : Rhizaria – Cavalier-Smith,2002

Phylum: Foraminifera – (Eichwald,1830) Marguls,1974

Class: Foraminifera – Lee,1990

Order: Miliolida – Delage & Herouard,1896

Family: Soritidae – Ehrenberg,1839

Genus: Marginopora

Specific descriptor : vertebralis

� Scientific name: Marginopora vertebralis –

Authority: Quoy & Gaimard, 1830

Figure 1.7 Taxonomy of Marginopora vertebralis (www.zipcodezoo.com/Protozoa)

14

1.4.2 LIFE CYCLES OF FORAMINIFERA

Relatively little is known about how most species of Foraminifera live. The few species

that have been studied show a wide range of behavior, diet and life cycles. Individuals of

some species live for only a few weeks, while other species can live several years

(Goldstein, 1999). Some infaunal benthic species burrow actively into sediments at

speeds of up to 1 cm per hour, while some epifaunal species can attach themselves to the

surface of rocks and marine plants (Capriulo, 1990).

Of the approximately 4,000 living species of Foraminifera, the life cycle of only 20 or so

are known (University College London, 2002). However, the alternation of sexual and

asexual generations is basic to the phylum (Goldstein, 2003) and this feature

differentiates the Foraminifera from other members of the Class Granuloreticulosea

(Murray, 1973; University College London, 2002).

Sexual reproduction, in which gametes are produced, results in the diploid generation.

Because the gametes are very tiny, even when two fuse to produce a zygote, the resulting

proloculus tends to be very small. Hence such individuals are called microspherics

(University College London, 2002). When a diploid microspheric individual grows to

reproductive size, it will undergo asexual multiple fission. Whether the offspring are

haploid or diploid can vary by taxa and even environmental conditions (Leutenegger,

1977).

Figure 1.8: Growth stages of Marginopora vertebralis. A: embryonic, B: orbitoid and C: reproductive

0.7cm

15

The reproductive behavior of Foraminifera can involve several stages (Fig 1.9). The

common mode of asexual reproduction occurs when an individual undergoes multiple

fission, in which numerous offspring are produced. The offspring typically have a

relatively large initial chamber (proloculus) and are referred to as the megalospheric

generation. After reproducing, the parent shell becomes a dead sand particle. Whether

those megalospheric individuals reproduce asexually or sexually can also vary with

environmental conditions (Harney et al., 1998). Megalospheric individuals that

reproduce asexually are known as the schizont generations.

Figure 1.9 Life cycles of Foraminifera. Diagram showing a generalized foraminfera life cycle note

alteration between a haploid megalospheric from and a diploid microspheric form (Goldstein, 2003)

16

1.4.3 DISTRIBUTION AND ECOLOGY OF FORAMINIFERA

Foraminifera found in marine environments have either a planktonic or benthic mode of

life (Wetmore, 1999). According to Sen Gupta (2003), Foraminifera make up the most

diverse group of shelled microorganisms in modern oceans. Out of approximately 4,000

known living species of foraminifera, 40 species are planktonic and the remaining ones

are benthic (Wetmore, 1999). The shells of calcareous foraminifera act as a sink for

calcium carbonate (Langer, 2008).

Species of foraminifera are adapted to specific environmental conditions (Wetmore,

1999). Some are plentiful only in the deep ocean, some are found only on coral reefs,

and still other species live only in brackish estuaries or salt marshes along the shore

(Wetmore, 1999). Murray (2006) stated that the distribution of foraminifera is

determined by environmental variables, including depth, salinity, pH, sediment texture

(grain size), temperature, and oxygen availability. The majority of foraminifera are

classified as epifaunal, that is, are benthic and live at the surface of sediment or on algae

or other substrate that project above the seafloor. Some specimens of foraminifera are

infaunal and live below the surface, able to reach the deeper levels through bioturbation

by macrofauna (Saffert et al., 1998).

The ecology of modern foraminifera is also controlled by feeding mode and food

availability. Most foraminifera are heterotrophic and include micro-omnivores, which

feed on small bacteria, algae, protists, invertebrates and dead particles. Most

foraminifera feed by active searching or by suspension feeding in the case of sessile

taxa. Some taxa, especially those living in warm waters, host algal symbionts in a

relationship analogous to corals that host zooxanthellae. Many planktonic foraminifera

host either dinoflagellates or cryptophytes, while benthic taxa can host dinoflagellates,

diatoms, chlorophytes (green algae) or rhodophytes (red algae) (Hallock, 2003).

Predators of foraminifera include worms, crustaceans, echinoderms and fish (Rottger,

1983). Different foraminiferal species have different temperature requirements.

Moreover, the solubility of calcite is influenced by temperature (Erez, 2003).

17

1.4.4 CARBONATE PRODUCTION BY BENTHIC FORAMINIFERA

As noted in section 1.4.1, many foraminifera produce calcium carbonate shells, usually

calcitic, though a few produce aragonite shells. Moreover, many agglutinated taxa

produce calcite cements into which they incorporate sediment particles from the

environment. Among those that produce calcite shells, there are two major modes of

calcification, radial calcite and porcelaneous calcite (Sen Gupta, 1999; Erez, 2003).

The large benthic foraminifera that host with algal endo-symbionts are dynamic

producers of carbonates in the coral reef environments (Hallock, 1981; Fujita, 2008), as

they contribute an estimated 5% of the annual present-day carbonate production in the

world’s reefs, an approximately 2.5% of the CaCO3 produced in the oceans (Langer,

2008). The high carbonate productivity of the larger foraminifera is related to their

endosymbiosis with microalgae (Lee, 1998).

Due to the abundance of the larger benthic foraminifera in shallow shelf environments of

the West Pacific and East Indian Ocean (Hohenegger, 2006), they are major producers of

sediments deposited in lagoonal habitats and on beaches (Langer and Lipps, 2003).

Foraminifera contribute widely to the accumulation and stability of reefs and to the

building of reef barriers (Lee et al., 1994). According to Langer (2008), the larger

symbiont-bearing foraminifera are estimated to produce at least 130 million tonnes of

CaCO3/yr, whereas together with non-symbiont-bearing foraminifera, all benthic

foraminifera are estimated to produce 200 million tonnes of CaCO3/yr.

Based on population dynamics, calcium carbonate production by symbiont-bearing

foraminifera can be calculated (Muller, 1974; Hallock 1981). Hohenegger (2006) also

indicated that, besides the coastal erosion due to the natural forces like the abundant

tropical cyclones in the West Pacific, there were also three human induced interferences

that can destroy carbonate beaches. Firstly, the devastation of the production area (i.e.,

loss of habitat) by pollution or landfills; secondly, intensification of currents by coral

mining or deepening for boat passages; and thirdly, the separation of production area, the

reef crest, from the beach by breakwaters and groins that are inappropriate to stop the

erosion. Nukubuco Reef Flat sedimentology showed that carbonate bioclasts originate

18

mostly from coral debris (65%), molluscan shells (15%; bivalves and gastropods),

foraminifera (11%; mainly Marginopora vertebralis, some Amphistegina spp.) and in

minor amounts from calcareous green algae (Halimeda), crustaceans, bryozoans,

echinoderms and ostracods (Morris, 1998).

1.4.5 PREVIOUS STUDIES OF OCEAN ACIDIFICATION

1.4.5.1 Previous studies of responses of foraminifera

Benthic foraminifera have been the subject of many experimental ocean-acidification

studies compared to other calcifying marine protists (Bernhard et al., 2004; Dissard et

al., 2009; Kuroyanagi et al., 2009; Dias et al., 2010; Dissard et al., 2010a; Fujita et al.,

2011; Haynert et al., 2011; Glas et al., 2012; Macintyre-Wressnig et al., 2013; Knorr et

al., 2015). The response parameters taken into account for ocean acidification studies

(Table 1.1) were calcification, shell weight, size, growth, shell thickness, test diameter

and community shift. The responses varied from no pH effect (Vogel and Uthicke, 2012;

McIntyre-Wressnig, 2013) to decreases with lowered pH (Le Cadre et al., 2003; Russell

et al., 2004; Kuroyanagi et al., 2009; Allison et al., 2010; Dissard et al., 2010a; Fujita et

al., 2011; Sinutok et al., 2011; Uthicke and Fabricius, 2012; Haynert, 2013). An increase

in growth was found in M. vertebralis by Vogel and Uthicke (2012) and in Calcarina

gaudichaudii by Hikami et al. (2011). To date, no one has addressed the issue of ocean

acidification on benthic foraminifera in Fiji.

19

Table 1.1: Ocean acidification studies and the response of lower pH on benthic foraminifera.

RReference SSpecies RResponse parameter RResponse to low ppH/elevated ppCO2

Le Cadre et al., 2003 Ammonia beccarii calcification decrease

Russell et al., 2004 Marginopora kudakajimensis shell weight decrease

Kuroyanagi et al., 2009 Marginopora kudakajimensis size, shell weight, growth

decrease

Allison et al., 2010 Elphidium williamsoni shell thickness decrease

Cigliano et al., 2010 Foraminiferal assemblage community shift changes

Dissard et al., 2010a Ammonia tepida weight decrease

Haynert, 2011 Ammonia aomoriensis test diameter decrease

Fujita et al., 2011 Amphissorus hemprichii shell weight decrease

Hikami et al., 2011 Calcarina gaudichaudii calcification increase

Amphisorus kudakajimensis calcification decrease

Amphisorus hemprichii calcification no effect

Sinutok et al., 2011 Marginopora vertebralis calcification decrease

Uthicke and Fabricius, 2012

Marginopora vertebralis calcification decrease

Vogel and Uthicke, 2012 Amphistegina radiata growth no effect

Heterostegina depressa growth no effect

Marginopora vertebralis growth increase

McIntyre-Wressnig, 2013

Amphistegina gibbosa growth,fitness,survival no effect

Knorr et al., 2015 Archaias angulatus growth decrease

1.4.5.2 Culture setups for ocean acidification studies

A variety of culture methods are reported to have been used for experimental ocean

acidification studies by these different authors (Table 1.1).

20

Findlay et al. (2008) developed a flow-through tidal microcosm system that allows the

investigator to simultaneously manipulate temperature and CO2 to stimulate a realistic

intertidal scenario. The objective of the study was to investigate the survival, growth and

development of larvae, juveniles and adult intertidal organisms. The concentration of

CO2 flowing into the microcosms in that study was manipulated through an air to CO2

gas mixing system.

Several studies have used high precision pCO2 control systems to investigate the effect

of ongoing ocean acidification on the calcification of foraminifera. Living foraminifera

from three genera (i.e., Baculogypsina, Calcarina, and Amphisorus), were subjected to

seawater at five different pCO2 levels, within a range of 300-1000ppm (Hikami et al.,

2011). The pCO2 levels were adjusted and kept constant by bubbling CO2 gas with an

ALCAL system. During the culturing period of 12 weeks, Hikami et al. kept cultured

individuals under constant seawater temperatures, light intensity and photoperiod. Their

results suggested that ongoing ocean acidification will be favorable for some hyaline

taxa of symbiont-bearing reef foraminifera under intermediate levels of pCO2 (600 and

800ppm), but unfavorable for both hyaline and porcelaneous taxa at higher pCO2 levels

(>1000ppm).

Glas et al. (2012) added CO2 enriched air into a semi-closed circulation system of

filtered natural seawater, where the CO2 enriched air was humidified via a system of

Erlenmeyer flasks and bubbled into an aerated reservoir tank, connected to the

incubation chambers, which contained the organisms. Macintyre-Wressnig et al. (2013)

maintained elevated atmospheric pCO2 in incubators using a feedback-controlled

infrared CO2-sensor, together with a CO2 inlet tube that was inserted through a hole in

the incubators side wall to ensure fast mixing of gases. A recent study by Knorr et al.

(2015) tested the response of Archaias angulatus to simulated ocean acidification by

applying an automated pH controlled CO2 (g)-injection method.

21

For this study, methods were adapted from the culturing setup used by Widdicombe et

al. (2009), where the CO2 supplied to the seawater was monitored via a pH controller

and the water circulation was provided using a peristaltic pump.

1.4.5.3 Measuring growth (Weighing method and Calcein marking)

There are several ways to measure growth in foraminifera. A common method includes

weighing each specimen or a group of specimens at the start of an experiment, and at the

end of the treatment period (e.g., Hallock et al., 1986). Another useful method is to mark

or label the specimens prior to the experiment. For example, incorporation of the

fluorescent dye Calcein (Bis [N,Nbis(carboxymethyl)aminomethyl]-fluorescein) into the

skeleton, allows distinguishing pre-existing shell from chambers added during the

experimental treatment (Bernhard et al., 2004; De Nooijer et al., 2007; Dissard et al.,

2009) . The new chambers can then be analyzed for elemental concentrations and ratios

using EPMA and LA ICP-MS.

1.4.5.4 Elemental concentrations & ratios

Foraminiferal shells have been studied for records of Mg, Sr, Cd, Ba, F, Li, V, U and

other elements and their isotopes in a wide variety of environmental and

paleoceanographic studies (Hester and Boyle, 1982; Delaney and Boyle, 1986;

Rosenthal et al., 1997). Several trace elements in the benthic foraminiferal calcite have

been shown to be influenced by the carbonate ion concentration, either because of

dissolution or changing saturation (McCorkle et al., 1995; Elderfield et al., 1996;

Marchito et al., 2005).

Foraminifera have been cultured at different pH levels with constant temperature, where

together with Mg/Ca and Sr/Ca ratios, other ratios such as B/Ca, Cd/Ca, Ba/Ca, Nd/Ca

and U/Ca were also studied. The proxy using foraminiferal Cd/Ca is particularly

important. Cadmium concentrations in overlying waters are preserved in benthic

foraminifera (Hester and Boyle, 1982). According to Marechal-Abram et al. (2004); the

shell of a foraminifer can record the average dissolved cadmium concentration during

the whole lifetime of an individual. The boron isotopes in foraminiferal shells are known

to reflect seawater pH and for this reason they are used to reconstruct paleo-seawater pH

22

(Hönisch et al., 2009). Barium cycles like a nutrient but its regeneration occurs deeper

than the organic matter, which results in close correlation between organic matter and

alkalinity in today’s ocean (Lea, 1993). The Ba/Ca ratio in foraminifera has been used as

the paleo-tracer of alkalinity and water masses (Lea and Boyle, 1990b). The U/Ca ratio

of benthic foraminifera also links with the carbonate-system parameters such as pH and

CO32- (Russell et al., 2004) and is a possible alternative to the B/Ca ratio to reconstruct

seawater CO32-.

1.5 STUDY GOALS

As increasing amount of carbon dioxide in the atmosphere are taken up by the ocean,

calcification rates in large benthic foraminifera are likely to decrease, and such changes

will also affect trace element composition of resulting shells. Therefore, the main goals

of this study are to determine experimentally the effects of acidified seawater on

Marginopora vertebralis calcification and potential dissolution, as well as the effects on

their shell chemistry.

23

CHAPTER 2 GROWTH OF Marginopora vertebralis IN DIFFERENT PH TREATMENTS

2.1 INTRODUCTION

This chapter describes the design and implementation of a culture experiment with a

goal of determining growth rates of Marginopora vertebralis under three different pH

treatments. The results from the two methods of assessing growth are presented, as are

ancillary observations of behavior and visual characteristics of the experimental

specimens.

2.2 METHODOLOGY

2.2.1 SAMPLE COLLECTION

The samples of M. vertebralis were collected from Makuluva and Nukubuco reefs on the

Suva Barrier Reef (Fig 2.1). The location of the collection site was determined and

plotted using GPS data. Salinity and temperature of the sample collection area were

assessed using a YSI85 meter. The maximum diameter (d) of the specimens used in this

study ranged from 0.4 ≤ d≤ 1.4 cm.

The foraminifera and associated sediments were collected using hand scoops and

immediately placed in an aerated aquarium with filtered seawater (0.45µm), where they

were kept overnight. The next day, M. vertebralis specimens had crawled upward and

attached themselves to the sides of the aquarium and therefore could be easily identified

and removed, based on the methods of Sharma (2007).

24

Site of collection of M.vertebralis

Marginopora vertebralis

Figure 2.1: (A) Map of Laucala Bay ; (B) The mapping of the site of collection of Marginopora vertebralis ; (C) Algal flat habitat between Sandbank Island and Nukubuco Reef; and (D) M. vertebralis attached to algae and coral (arrow).Scale bars (A) 5km; (B) 0.06km (Source: SOPAC, 2012; Sharma, 2007); (D) 1 cm.

Research site (A)

(B)

(C)

(D)

25

2.2.2 EXPERIMENTAL SET-UP

Natural seawater (salinity 33.0 psu), contained in large (35 L) reservoir tanks, was

acidified by means of a bubbling system supplying CO2 gas. The gas, saturated with

water vapor (to limit evaporation) was injected through the water as very fine bubbles

allowing the gas to rapidly dissolve.

The seawater pH in the reservoirs was monitored using a flat-surface, combination pH

electrode (YSI Environmental pH 100). Once the pH reached the required level, the

supply of CO2 was halted via an automated feedback relay system (Accu-Max pH

controller). As the acidified water was pumped from the reservoir to supply the

experimental tanks, the overflow water was returned to the reservoir. Whenever the pH

increased, CO2 bubbling was triggered to ensure that the desired pH level was

maintained. Each reservoir contained a CO2 Reactor, an air pump and a water pump.

The reservoirs were refilled with natural seawater (pH=8.1) every 10 days from a

separate 300 m3 seawater tank, causing the pH in the reservoir tank to increase. This

increase triggered the supply of CO2 to be restarted and CO2 continued to bubble through

the water until the pH was reduced to the required level. Using this method it was

possible to supply large quantities of CO2 acidified seawater with a consistent pH.

Two seawater acidification reservoir tanks (pH 7.8, pH 7.5) and one control (ambient pH

8.1) were maintained. From each tank, water was supplied to the corresponding three

small tanks (25 x 10 x 15 cm) where the M. vertebralis specimens were cultured.

The experimental setup model in Figure 2.2 shows how the connections were made from

the reservoirs to the connecting tanks and vice versa. The CO2 system is plugged with

pH controllers (adjusted at pH 7.8 and 7.5). The CO2 is released with the help of the

bubble counter, check valve and CO2 injector. The pH controller regulates and stabilizes

the pH in the reservoirs and the peristaltic pump helps in the circulation of seawater,

creating an ocean-like environment.

Nine plastic tanks (3 treatments with 3 replicate each) were placed in a room (ambient

temperature ~ 27.5º C) and connected to a pH controller system (Fig 2.2). Each tank was

individually supplied with seawater at a rate of approximately 3.3 ml min-1 using a

26

peristaltic pump. The M. vertebralis specimens were randomly assigned to one of the

three pH treatment levels (8.1 [ambient seawater], 7.8, and 7.5). The treatments were

maintained under a 12-hour light and 12-hour dark cycle using a T5 Aquarium lamp

with a light intensity of 42.5 watts m-2.

Acidification of the seawater did not begin until 24 hours after the M. vertebralis

specimens were placed in their treatment tanks. Seawater pH was reduced gradually over

a period of two days and the experiment started when the final water chemistry for each

treatment was reached.

The experiment ran for 11 weeks, during which time, the pH of the water supplied to the

tanks was monitored daily via a pH controller (Accu Max I). Samples for Total

Alkalinity were taken at the beginning of the experiment and then once per week

throughout the duration of the experiment. The values given by the pH electrodes in the

reservoir tanks were cross-checked every week against values measured by a regularly

calibrated pH meter (InLab 413SG, Mettler-Teledo).

27

Fig

ure

2.2

Exp

erim

enta

l set

-up

mod

el

28

2.2.3 MEASURING GROWTH

2.2.3.1 Fluorescent Marker

Calcein (-bis [N, N-bis (carboxymethyl) aminomethyl]-fluorescein) was used to

fluorescently label existing chambers in M. vertebralis. Calcein makes newly formed

chambers distinguishable when the specimens are viewed under a confocal microscope

(Dissard et al., 2009).

Marginopora vertebralis in each treatment and replicates were placed in a solution at 40

�M of Calcein for approximately 48 hours to allow the incorporation of the green

fluorescent cell marker (Fig 2.3). Once Calcein is incorporated inside the cells, Calcein

AM (a nonfluorescent molecule) is hydrolyzed by endogeous esterase into the highly

negatively charged green fluorescent Calcein. The fluorescent Calcein is retained in the

cytoplasm in living cells but is not incorporated into the subsequently added chambers

(Papadopoulous et al., 1994). At the end of the experiment, this technique helps to

distinguish between the pre-existing and the newly grown calcite (Bernhard et al., 2004;

Dissard et al., 2009).

A confocal microscope (Nikon ECLIPSE TE2000-U) was used to create sharp images of

specimens that would otherwise appear blurred when viewed with a conventional

Figure 2.3 M.vertebralis treated in 40 μmol/L of Calcein

29

microscope. The software used was EZC1 3.91. Imaging is achieved by excluding most

of the light from the specimen that is not from the microscope’s focal plane. The image

has less haze and better contrast than that of a conventional fluorescent microscope and

shows a thin cross-section of the specimen. Thus, in addition to allowing better

observation of fine details, it is possible to build three-dimensional (3D) reconstructions

of a volume of the specimen by assembling a series of thin slices taken along the vertical

axis.

Figure 2.4: Basic setup of a confocal microscope. Light from the laser is scanned across the specimen by the scanning mirrors. Optical sectioning occurs as the light passes through a pinhole on its way to the detector.

In confocal microscopy, there is never a complete image of the specimen because at any

instant only one point is observed. The Calcein in the specimen is excited by the laser

light and fluoresces. The green fluorescent light is de-scanned by the same mirrors that

are used to scan the blue excitation light from the laser and then passes through the

dichroic mirror. Thereafter, it is focused onto the pinhole. The light that makes it through

the pinhole is measured by a detector such as a photomultiplier tube (Fig 2.4).

Growth of the selected specimens was assessed using confocal imagery. The thickness of

the calcite shell added after Calcein incubation revealed growth that occurred during

30

experimental treatments. The selected specimen was moved around under the confocal

microscope and the edges which showed growth was marked. Using the ruler property

from the EZC1 3.91 software, the increase in radius was noted in µm. The mean

increases in radius at each treatment for each replicate was recorded.

2.2.3.2 Weighing Method

The 25 M. vertebralis specimens in each treatment were weighed once each week as a

group using an analytical balance (Libror-Aex 200g). The specimens were collected

using hand scoops, pat dried using tissue, and wet weights of the group of specimens

from each replicate of each treatment were determined.

31

2.3 RESULTS

We cultured 225 M. vertebralis individuals in all, at three different pH values: pH 7.5

(n=75), 7.8 (n=75), and control (pH ~8.1; n=75). The M. vertebralis cultured at pH 7.5

showed a 429 µm mean increase in radius and at pH treatment of 7.8 the mean increase

in radius was 441 µm. The increase in radius was nearly the same for pH treatment of

7.5 and 7.8. The M. vertebralis cultured at pH 8.1 showed 1024 µm mean increase in

radius, which was more than doubled the increase noted for pH 7.5 and 7.8. The mean

increases in radius (n=25) at the end of each treatment for each replicate are presented in

Table 2.1. Examples of measurement for each treatment are shown in Figures 2.5 to 2.7.

Table 2.1: Mean increase in radius of shells of specimens in each replicate of each treatment after 11 weeks in culture.

TTrreatment RReplicate MMean increase in

radius (μm) SD n

7.5 R1 471 102 25

R2 489 76.8 25

R3 326 228 25

7.8 R1 476 427 25

R2 480 312 25

R3 367 38.2 25

8.1 R1 1019 346 25

R2 785 107 25

R3 1267 306 25

32

Figure 2.5. Confocal microscopy image (A1) of a specimen from pH treatment 7.5. The

growth measured for A1 was 561 μm and 724 μm.

Figure 2.6. Confocal microscopy image (B1) of a specimen from pH treatment 7.8. The

growth measured for B1 was 746 μm and 1000 μm.

A1

B1

33

Figure 2.7 Confocal microscopy images (C1) of specimens from two replicate tanks of pH treatment 8.1. The growth measured for C1 was 1400 μm and 934μm.

C1

34

Weekly weights of the 25 M. vertebralis specimens from each replicate of each

treatment are plotted in Figure 2.8. Linear regression parameter for each dataset for each

replicate is provided in Table 2.2, along with the total change in weight.

The M. vertebralis cultured at pH 7.5 showed very little growth, averaging only 0.0227

g/week and pH treatment 7.8 showed only slightly more growth, 0.0334 g/week. The

specimens cultured at pH 8.1 grew an average of 0.0658 g/week (Fig 2.8).

4

4.5

5

5.5

6

6.5

7

1 2 3 4 5 6 7 8 9 10 11

Tota

l wei

ght (

g) o

f 25

spec

imen

s of

M. v

ertb

ralis

Time (weeks)

pH7.5R1

pH7.5R2

pH7.5R3

pH7.8R1

pH7.8R2

pH7.8R3

pH8.1R1

pH8.1R2

pH8.1R3

Figure 2. 8 Weight increase (grams) of 25 specimens of M. vertebralis in each replicate for each pH treatment.

35

Table 2.2: Linear regression parameters for the increase in wet weight (g) of each treatment and replicate during the 11 week experiment.

TTreatment RReplicate sslope iintercept nn RR22 Change in weight pper individual (g)

7.5 R1 0.0195 5.04 11 0.879 0.0061

R2 0.0371 5.88 11 0.751 0.0123

R3 0.0405 6.25 11 0.825 0.0123

7.8 R1 0.0608 6.45 11 0.840 0.0187

R2 0.0414 6.22 11 0.846 0.0131

R3 0.0251 5.59 11 0.874 0.0117

8.1 R1 0.0623 5.84 11 0.951 0.0243

R2 0.0806 5.88 11 0.951 0.0273

R3 0.109 4.08 11 0.904 0.0354

The relative weight gain (%) of 25 individuals of M. vertebralis at each pH treatment for

a period of 11 weeks was calculated and graphed (Fig 2.9). Individuals treated at pH 7.5

had an average percentage growth of 3.24 % whereas individuals treated at pH 7.8 had a

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7 8 9 10 11

Tota

l gro

wth

of 2

5 sp

ecim

ens

of M

. ver

tebr

alis

(%)

Time (weeks)

pH7.5R1

pH7.5R2

pH7.5R3

pH7.8R1

pH7.8R2

pH7.8R3

pH8.1R1

pH8.1R2

pH8.1R3

Figure 2. 9 Relative weight gain (%) by 25 individuals of M. vertebralis at each pH treatment level

36

y = 6.3153e0.0015x R² = 0.7553

5

10

15

20

25

30

35

40

0 200 400 600 800 1000 1200 1400

Shel

l wei

ght (

mg)

per

indi

vidu

al

Mean shell radius per individual (μm)

percentage growth of 4.24 %. At pH 8.1; the highest percentage growth of 8.39 % was

noted.

Table 2.3 The mean increase in shell radius and the mean increase in shell weight, both measured after 11 weeks in culture.

Treatment Replicate Mean increase in radius (μm)

per individual Mean increase in weight (mg)

per individual

7.5 R1 471 6.05

R2 489 12.3

R3 326 12.3

Mean ± SD 429 ± 89.3 10.2 ± 3.60

7.8 R1 476 8.71

R2 480 13.1

R3 367 11.7

Mean ± SD 441 ± 64.1 14.5 ± 3.71

8.1 R1 1019 24.3

R2 785 27.3

R3 1267 35.4

Mean ± SD 1024 ± 241 30.0 ± 5.72

The mean shell radius of cultured individuals measured after 11 weeks of culture and the

mean shell weight measured every week of all cultured specimens of M. vertebralis are

presented in Table 2.3. The result in Figure 2.10 indicates that 1) shell size increased

Figure 2.10 Relationship between increase in shell radius and in average shell weight of Marginopora vertebralis measured after 11 weeks in culture (red - pH 7.5, green - pH 7.8, and blue - pH 8.1).

37

with time in all pH treatments and 2) pH of the medium significantly influenced the

increase in shell radius through a culture period (Table 2.3). The results in Table 2.3,

revealed notably less growth at the lowest pH condition both with respect to shell radius

and shell weight (Fig 2.10).

Table 2.4 Mean growth rates for group of specimens at each pH treatment and their replicates. (SD-Standard Deviation)

ppH Treatment Growth rate (mmg day-1)

Growth rate (mmg month-1)

Growth rate (gg month-1)

7.5 R1 1.96 58.8 0.0588

R2 3.98 119 0.119

R3 4 120 0.12

Mean ± SD 3.31 ± 1.17 99.4 ± 35.2 0.0994 ± 0.035

7.8 R1 6.08 182 0.182

R2 4.25 128 0.128

R3 3.8 114 0.114

Mean ± SD 4.71 ± 1.21 141 ± 36.2 0.141 ± 0.036

8.1 R1 7.88 236 0.236

R2 8.38 251 0.251

R3 11.5 344 0.344

Mean ± SD 9.25 ± 1.95 277 ± 58.5 0.277 ± 0.058

For comparison with previous studies, growth rates were calculated per day and per

month (Table 2.4). The 25 individuals of M. vertebralis together grew an average of

0.0994 g/month at pH 7.5, grew an average of 0.141 g/month at pH 7.8 and an average

of 0.277 g/month at pH 8.1.

38

2.3.1 OTHER OBSERVATIONS

At the end of the experiment, most specimens contained pale pink cytoplasm, which

indicated that they were alive throughout the entire experimental period.

Marginopora vertebralis has a disc shape. Individuals can roll on their edge and climb

the tank wall using their reticulopodia (Fig 2.11). It was observed that they disperse and

move more towards light.

Several specimens were observed to reproduce asexually. When an individual is ready

for reproduction, it changes its color to white with a pale purple ring in the middle.

During reproduction (Fig 2.12), the parent shell becomes white as the endoplasm

containing the dinoflagellate symbionts is incorporated into the megalospheric offspring.

All the replicate tanks had a mixture of adult and juvenile M. vertebralis but

reproduction was only noted at the pH treatment of 8.1.

(A) (B)

Figure 2.11 Net like structure of pseudopodia. (A) Movement of M. vertebralis during the culturing period;

(B) Pseudopodial projections of Marginopora vertebralis (white arrow)

39

Figure 2.12 Reproduction of Marginopora vertebralis at pH treatment 8.1 (black arrow)

40

2.4 DISCUSSION

The microscopic observations of Marginopora vertebralis specimens documented that

the precipitation of calcite did not occur evenly over the organic membrane of the

developing chamber during their calcification process. Calcification began at many small

sites on the membrane rather than in a uniform manner. After a few hours, the patchy

calcite interconnects and then thickens the chamber wall.

Erez (2003) stated that reticulopodia in foraminifera are important for food gathering,

movement, shell building, respiration and extraction of waste materials. Thus, the

reticulopodia in foraminifera enable them to interact with their surrounding (Goldstein,

2003). The shells of foraminifera are structured in such a way as to allow for effective

communication of cytoplasm between the intra-shell endoplasm and the outside

environment (Funnel et al., 1986; Hottinger et al., 1993 and Erez, 2003).

Sharma (2007) collected M. vertebralis from Laucala Bay and cultured them at ambient

pH of seawater (pH 8.1). The results revealed that a group of M. vertebralis larger than

or equal to a diameter of 1 cm grew at 0.13 g/month while M. vertebralis specimen

larger than 0.5cm and smaller than or equal to a diameter of 0.7 cm grew at 0.0063

g/month. In this study smaller and larger individuals were mixed (Table 2.4), 25

individuals of M. vertebralis together grew an average of 0.0994 g/month at pH 7.5, an

average of 0.141 g/month at pH 7.8 and an average of 0.277 g/month at pH 8.1. Based

on the reports of Hallock (1981) for Amphistegina spp. and Calcarina spp. and Smith

and Wiebe (1977) for Marginopora vertebralis, foraminifera are probably the major

CaCO3 producers in the low calcifying zones, as both species can produce sediment at

rates of 0.5 kg CaCO3 m-2yr-1.

Seawater with lower pH levels had a significant effect on the shell growth of M.

vertebralis. Individuals treated at pH 7.5 grew an average of 3.2% whereas individuals

treated at pH 7.8 grew by 4.2 % (Fig 2. 9), well below the 8.4 % growth at pH 8.1.

The smaller individuals of M. vertebralis showed a growth rate of 7.7% of their initial

body weight while the larger organisms showed a growth rate of 1.9% of their initial

body weight (Sharma, 2007). Sharma (2007) concluded from her study that the

41

approximate rate of sediment production from the three M. vertebralis colonies from the

Sandbank Island was 36 kg of sediments each month and 431 kg of sediments each year,

at an average of 0.13 kg/m2/yr. If the ocean acidification continues at predicted rates, the

sediment production rates by Sharma (2007) could be well overstated.

The results from this study showed that the rate of increase in shell weight of M.

vertebralis is more sensitive to changing pH (Fig 2.8). Furthermore, the relationship

between shell radius and shell weight is different among the three pH conditions (Fig

2.10). Our results are in agreement with Sinutok et al. (2011) and Uthicke and Fabricius

(2012), who also reported a decrease in M. vertebralis calcification in response to lower

pH. Thus, in an increasingly acidified ocean, the organisms may need more energy to

produce the same amount of calcite, which in turn will reduce the gross calcite

production by calcifying foraminifera (de Nooijer et al., 2009; Knorr et al., 2015).

Significant reductions in the growth rates of M. vertebralis were recorded in response to

the decreases in the seawater pH. This sensitivity to ocean acidification indicates that M.

vertebralis might not cope well with further increases in atmospheric and oceanic pCO2.

42

CHAPTER 3 CARBONATE CHEMISTRY OF CULTURE MEDIUM

3.1 INTRODUCTION

3.1.1 PURPOSE

The purpose of Chapter 3 of this thesis is to present the methods used to measure and

calculate carbonate species, and to report the resulting data on water chemistries to

which Marginopora vertebralis experienced during the experiment presented in Chapter

2.

3.1.2 INORGANIC CARBON IN SEAWATER

The ocean processes CO2 in multifaceted ways. Three inorganic forms of dissolved

carbon dioxide occur in seawater: free aqueous carbon dioxide (CO2 (aq)), bicarbonate

(HCO3-) and carbonate (CO3

2-) ions (Zeebe, 2011). Due to the influx of anthropogenic

CO2, the carbonate system of the world’s ocean is changing rapidly (Orr et al., 2005;

Guinotte and Fabry, 2008).

The inorganic carbon dioxide system in the oceans can be characterized using any two of

the four measurable parameters, pH, total alkalinity (TA), fugacity of carbon dioxide

(fCO2) and the total inorganic carbon dioxide (TCO2), given accompanying

measurements of temperature (ºC), salinity (psu) and pressure (atm). Dissociation

constants of carbonic acid are also needed to calculate the components of the CO2

system from these measurements.

The equilibrium of gaseous and aqueous CO2 when air-sea gas exchange occurs can be

represented as:

CO2 (g) CO2 (aq) (3.1)

Carbon dioxide in the atmosphere mixes with seawater to produce carbonic acid

(H2CO3), which readily dissociates into hydrogen (H+) and bicarbonate (HCO3-) ions:

CO2 (aq) + H2O H2CO3 HCO3- + H+ (3.2)

43

The bicarbonate ions can further dissociate, resulting in a hydrogen ion and a carbonate

ion:

HCO3- H+ + CO3

2- (3.3)

Foraminifera require both Ca2+ and CO32- ions to produce calcium carbonate shells

(CaCO3(s)):

Ca2+ + CO32- CaCO3(s) (3.4)

When dissolution occurs, the calcium carbonate dissolves into calcium ions and

carbonate ions:

CaCO3 (s) Ca2+ (aq) + CO3

2- (aq) (3.5)

The rate of dissolution depends on pH, temperature, salinity, pressure and the

concentration of carbonate ions in seawater. Hence, the addition of carbon dioxide to

seawater increases the proportions of hydrogen ions, carbonic acid, and bicarbonate and

lowers the proportion of carbonate ions. The generation of protons [H+] during the

carbon dioxide dissociation steps increases seawater acidity and causes a decrease of the

oceanic pH (Cao and Caldeira, 2008, Doney et al., 2009 ; Doney and Feely, 2011; Neuer

et al., 2014).

The decline in oceanic pH affects the proportions of the three inorganic carbon species:

CO2, HCO3-, and CO3

2-, which are in equilibrium with each other. The sum of all

dissolved inorganic carbon (CT):

CT = [CO2] + [HCO3-] + [CO3

2-] (3.6)

According to Riebesell et al. (2010), Total Alkalinity (AT) can be represented as:

AT = [HCO3-] + 2 [CO3

2-] + [B(OH)4-] + [OH-] –[H+] + minor components

Total Alkalinity can be determined by the titration of seawater with a strong acid. The

major component of this buffering system is the carbonate alkalinity (AC):

AC = [HCO3-] + 2 [CO3

2-] (3.7)

The dissociation constants K1 and K2 are defined as:

K1= [H+] [HCO3-] / [CO2] (3.8)

K2= [H+] [CO32-] / [HCO3

-] (3.9)

44

where the square brackets are used to denote the concentration in mol kg-1 of the species

in seawater.

The International Panel on Climate Change (IPCC) predicted future scenarios, indicating

that the atmospheric CO2 will increase to concentrations as much as 970 ppm by the year

2100 (IPCC, 2013). An increasing number of field and laboratory studies have indicated

negative impacts of increased seawater carbon dioxide and related changes in the

carbonate chemistry on marine calcifying organisms, such as corals and foraminifera

(Kleypas et al., 1999; Riebesell et al., 2000).

45

The 12 major constituents of seawater are listed in Table 3.0. These major components

have relatively long residence times (ocean mixing time of ̴ 1000 years) in the ocean

(Chester and Jickells, 2012).

Table 3.0 Major Seawater Constituents (Pilson,2013; Munsel, 2014)

CComponent AAverage conc [μM]]

H2O 54,880,000

Cl- 559,000

Na+ 480,000

Mg+2 54,100

SO4-2 28,900

K+ 10,500

Ca+2 10,500

HCO3- 1,890

Br- 863

CO32- 189

Sr+2 84

B(OH)4- 70

Trace elements are minor components of the seawater (less than 1 µmol/L) with shorter

residence times in the ocean than the major constituents (Chester and Jickells, 2012).

46

3.1.3 BENTHIC FORAMINIFERA UNDER OCEAN ACIDIFICATION

Based on the absence of calcareous benthic foraminifera in acidic seawater associated

with natural seafloor venting of carbon dioxide in a variety of shallow-water locations,

Uthicke et al. (2013) concluded that, if ocean acidification continues at predicted rates,

many benthic foraminifera will be extinct by 2100. However, Pettit et al. (2013) did not

find a significant response in the benthic foraminiferal assemblages to reduced pCO2 in

deeper shelf and slope settings.

Similarly, laboratory experiments examining responses of calcareous foraminifera to

lower pH and elevated pCO2 have shown significant differences among different taxa

and even within the same species (Doo et al., 2014). The majority of studies have shown

that pH lower than 7.8 can have deleterious effects on calcareous benthic foraminifera

(Kuroyanagi et al., 2009; Dias et al., 2010; Fujita et al., 2011; Vogel & Uthicke, 2012;

McIntyre-Wressnig et al., 2013; Knorr et al., 2015). These effects include reduced

calcification (Kuroyanagi et al., 2009; Moy et al., 2009; Haynert et al., 2011) and

decreased growth rates (Manno et al., 2012; Reymond et al., 2013), indicating the

decline in carbonate sediment production by foraminifera as ocean acidification

proceeds (Dias et al., 2010; Knorr et al., 2015). On the other hand, Fujita et al. (2011)

and Vogel & Uthicke (2012) reported that Marginopora vertebralis, in laboratory

experiments, responded to increased pCO2 with increased rates of calcification.

47

3.2 METHODOLOGY

3.2.1 DETERMINATION OF TOTAL ALKALINITY IN SEAWATER

To determine the carbonate chemistry in the culture treatments described in Chapter 2,

the following procedures and calculations were carried out based on best practices.

Borosilicate bottles, used to collect the water samples for alkalinity measurements, were

rinsed with concentrated HCl, followed by at least five rinses with deionised water.

Seawater samples were collected from the experimental aquaria. Borosilicate bottles

were fully filled and tightly capped. Analysis was carried out within 6 hours of sample

collection.

To standardize acid used in titrations, 10 ml aliquot of 0.01M sodium tetraborate was

placed into a 250 ml conical flask and a few drops of mixed indicator were added. The

aliquot was titrated with 0.01 M HCl until the end point was reached (color changed

from green to purple). The titre was recorded for the calculation of HCl concentration:

Conc of HCl = Vol. of Borax x Mass of Borax x 1000 x 2 Vol. of titre x 381.37 x 100

To determine the total alkalinity (AT) of a sample, 50 ml of the water sample were

pipetted into a 250ml Erlenmeyer flask and few drops of phenolphthalein indicator were

added. Once the solution color turned pink, it was titrated with 0.01M HCl until the

color disappeared and the volume of added acid was recorded. Then 2-3 drops of mixed

bromocrescol green/methyl red indicator were added to the same solution and titrated

with standardized 0.01M HCl to a pink color. Then AT was calculated using this

formula:

Total Alkalinity, AT (mg/L) = A x M x 50,000 Sample volume Where A = Volume of standard HCl used (ml) M = Molarity of HCl

48

The pH of the two acidification treatments described in Chapter 2 were adjusted and

maintained at 7.5 and 7.8 using a pH controller system w/electrode (Accu-Max, Ultralife

Reef Products). Since pH 8.1 was the pH of ambient seawater, carbon dioxide was not

injected into the reservoir or the replicate tanks. The pH was cross checked using a pH

meter (YSI Environmental pH 100).

3.2.2 ANALYSIS USING CO2SYS

The MATLAB program CO2SYS (single-input mode directly adapted from Lewis and

Wallace, 1998) was used to calculate and present seawater parameters where the salinity

was constant at 35 psu and temperature was constant at 27.5ºC. The input temperature

and pressure were from measurements performed in the laboratory. There are four

measurable parameters of the aquatic carbon dioxide system: pH, pCO2, total dissolved

inorganic carbon (DIC) and total alkalinity (TA). The measured pH and alkalinity values

were entered into the program. The equilibrium constants from Mehrbach et al. (1973)

as refit by Lueker et al. (2000) on a total scale were used. The measurements made by

Mehrbach et al. (1973) were made on real seawater.

Input variables (input conditions):

� Salinity (35 psu), Temperature (27.5ºC) and Pressure (1 atm).

� Total Si (optional) and Total Phosphate (optional). If left empty, the total Si and

total P concentrations are assumed to be zero in the calculations.

� Two (2) known CO2 parameters (TA, pH).

Output (for both “input” and “output” conditions):

� The other CO2 parameters (pCO2, total dissolved inorganic carbon [DIC].

� Contributions to the alkalinity.

� Carbonate speciation

49

3.3 RESULTS

In each aquarium analyzed, 25 individual of M. vertebralis were placed as it is difficult

to assess the growth per individual in terms of shell weight and calcification. There were

little uncertainties which influence the calcification estimates of M. vertebralis, but it

does not affect the concluding result that calcification decreases as a function of

decreasing [CO32-] and the final weight observations.

Seawater chemistry contrasted strongly between the 2 treatments and control, with mean

values ranging from 8.1 to 7.5 pH units and 309 to 1717 matm pCO2, respectively. Mean

total alkalinity was 2277 µmol kg-1 (n=11, SD=101). The alkalinity values were between

2176 and 2408 µmol kg-1, a range we used together with the pH measurements from all

aquaria (treatments) to calculate pCO2 concentrations (Table 3.1).

Table 3.1 Carbonate system parameters pertinent to the experiment. The input parameters of temperature (T), salinity (S), total alkalinity (AT), and the total scale pH (pHT) were measured directly. The treatment and control values are mean of measurements (n=11) taken over the course of the experiment. The remaining values were calculated using Lewis and Wallace (1998). The equilibrium constants from Mehrbach et al. (1973) as refit by Lueker et al. (2000) on total scale were used. Temperature (T) is in °C, salinity (S) is in psu, partial pressure of CO2 in seawater (pCO2) is in milliatmospheres (matm); [OH-] is given in mmolkg-1, AT, [HCO3

-] and [CO32-] is given in mmolkg-1SW.

TT °C

SS

psu

pHT

AT

mmolkg-1SW

ppCO2

matm

[ OH- ]

mmolkg-1

[ HCO3- ]

mmolkg-1SW

[ CO32- ]

mmolkg-1

SW

8.1 control 27.5 35 8.1

(0.05)

2176 309.1 9.66 1577.3 240.6

7.8

treatment

27.5 35 7.8

(0.05)

2247 738.5 4.84 1891.4 144.6

7.5

treatment

27.5 35 7.5

(0.05)

2408 1717 4.8 2203.5 84.5

50

The pH measurements showed a good correlation to the long term averages of pCO2 for

the two treatments and control (Table 3.2, Fig 3.1, R2= 0.95).

Table 3.2 Mean pCO2 (matm) and mean [CO32-] (mmolkg-1 SW) calculated from pH and alkalinity

measurements and mean increase in weight (g) for 25 specimens. The treatment and control values are mean (n=11) of weekly measurements taken over the course of the experiment.

Treatment Replicate Mean pCO2

(matm)

Mean [CO3

2-] (mmolkg-1

SW)

Mean growth (g) n

7.5 R1 1717 84.5 0.1513 11

R2 1716.3 84.5 0.3063 11

R3 1716.5 84.5 0.3081 11

7.8 R1 738.5 144.6 0.4681 11

R2 738.4 144.6 0.3276 11

R3 738.6 144.6 0.2929 11

8.1 R1 308.3 240.6 0.6066 11

R2 309.1 240.6 0.6835 11

R3 308.6 240.6 0.8838 11

0200400600800

100012001400160018002000

7.4 7.5 7.6 7.7 7.8 7.9 8 8.1 8.2

pCO

2 (m

atm

)

pH

Year 2100

Figure 3.1: pCO2 in seawater of two pH treatments (7.5 and 7.8) and control (8.1).

51

The shell growth in M. vertebralis was negatively affected by elevated pCO2 (Fig 3.2) and was positively related to increased CO3

2- (Fig 3.3).

y = -0.0003x + 0.7194 R² = 0.6221

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 500 1000 1500 2000

Shel

l wei

ght (

g)

pCO2

y = 3.0769x - 34.251 R² = 0.8061

0

100

200

300

400

500

600

700

800

900

1000

0.0 50.0 100.0 150.0 200.0 250.0 300.0

Shel

l wei

ght (

mg)

CO32- (mmol kg-1 SW)

Figure 3.2: Mean increase in shell weight of 25 M. vertebralis, grouped by pH level (n=11) at three pCO2 levels.

Figure 3.3: Mean shell weight (mg) of 25 M. vertebralis, grouped by treatment (n=11) against CO3

2- (mmol kg-1 SW).

52

3.4 DISCUSSION

Every component in the carbonate system is interlinked; therefore, any changes in any

single component of the carbonate system will lead to changes in other components. The

burning of fossil fuels, deforestation, industrialization, and other land-use changes are

contributing to the increasing atmospheric CO2 levels which directly lead to decreasing

ocean pH through the air-sea gas exchange.

Foraminiferal species have specific ecological niches and populations respond quickly to

environmental changes (Hallock et al., 2003). Experimental evidence in the past two

decades has shown that foraminifera respond to the changes in seawater temperature and

chemistry (Erez and Luz 1982, 1983; Lea and Spero 1992, 1994; Russel et al., 1994; Lea

et al., 1999). The results of the laboratory work in this study indicate that, at the rate at

which ocean acidification is currently occurring, there will likely be intense biological

consequences for ocean ecosystems within the coming decades and centuries,

particularly those organisms that calcify. The results also are consistent with the

predicted decrease in calcification in foraminifera over a similar increase in pCO2

(Bijma et al., 1999; Le Cadre et al., 2003; Hikami et al., 2011; Sinutok et al., 2011;

Uthicke and Fabricius, 2012).

Calcification rates are largely related to the amount of available carbonate [CO32-] ions

in the water. A recent study by Dissard et al. (2010a) focused on shell weight and shell

size measurements of Ammonia tepida specimens, combined with the analysis of Mg/Ca

and Sr/Ca in shells, cultured under the different pCO2 conditions, temperatures and

salinities. The results indicated that the shell weight decreased with decreasing [CO32-].

The results obtained in this research for M. vertebralis from Laucala Bay are consistent

with those of Dissard et al. (2010a), and also can be compared to other studies of M.

vertebralis (Sinutok et al., 2011; Uthicke and Fabricius, 2012), which also found that

calcification of M. vertebralis decreased at higher pCO2 levels. Environmental records of past atmospheric CO2 levels and ocean pH imply that

projections of future atmospheric CO2 levels are higher than those of the past 45 million

years. In the very near future, the larger foraminifera, including M. vertebralis, will

53

therefore be exposed to very different ocean chemistry than has existed for millions of

years, and reductions in calcification rates are likely. If foraminifera must increase the

rates to active ion transport to maintain a fixed difference between seawater and their

calcifying vesicles, calcification at a lower pH will in turn decrease the carbonate

available for calcite precipitation (de Nooijer et al., 2009). The foraminifera will need to

spend more energy to produce the same amount of calcite and therefore, it would be less

likely that calcite precipitation will occur in an acidified ocean.

What remains to be determined is whether M. vertebralis exposed to elevated pCO2 for

an entire life cycle can acclimate or if long-term exposure will result in decreased

calcification rates, as has been observed for other benthic foraminifera (Dissard et al.,

2010a; Fujita et al., 2011; Haynert et al. ,2011; Knorr et al., 2015). Moreover, the

combined effects of temperature, nutrient availability and CO32- need to be studied in

order to estimate the impact of oceanic environmental changes on M. vertebralis calcite

production.

54

CHAPTER 4 ELEMENTAL RATIOS USING EPMA (ELECTRON PROBE MICRO-ANALYSER)

4.1 PURPOSE/INTRODUCTION

Magnesium/calcium ratios, strontium/calcium ratios and sulfur/calcium ratios were

determined in the shells of the benthic foraminifer, Marginopora vertebralis, which were

collected from Laucala Bay, Suva, Fiji and cultured in the laboratory under three

different pH levels. The goal of these analyses was to determine whether Mg, Sr and S in

the shells could be used as a proxy measurement for the pH of seawater in which the M.

vertebralis grew.

55

4.2 METHODOLOGY

4.2.1 EMBEDDING OF SPECIMENS

Specimens of M. vertebralis from culture experiments described in Chapters 2 and 3

were randomly selected from each treatment for analysis of magnesium/calcium ratios,

strontium/calcium ratios and other parameters using an electron probe micro-analyzer

(EPMA). These specimens were washed four times with double distilled water. For each

wash, the specimens were placed on a MRC lab rotator (model: 2100A) for five minutes.

Later the specimens were soaked overnight in sodium hypochlorite at a ratio of 1:10

(1ml of sodium hypochlorite to 10ml of double distilled water). The specimens were

again washed four times with double distilled water to remove traces of sodium

hypochlorite. Afterwards, the specimens were placed in petri dishes and dried for 90

minutes in an oven at 50ºC.

After removal from the oven, two specimens from each of the three pH treatments (7.5,

7.8, 8.1) were observed under a confocal microscope to mark the areas of growth (Fig

4.1), then one quarter of the specimen was cut as shown in Figure 4.2.

Each quarter specimen was embedded (Epoxy Resin 1: 0.7 Epoxy Hardener), using the

following protocol (Fig 4.3). First, Teflon tubes (1ʺ diameter) were greased and the

specimen area that was to be viewed was placed upside down in the tube. The epoxy

mixture was prepared by adding 2.6 ml of epoxy hardener to 4 ml of epoxy resin and

mixing slowly and gently until it turned from milky to clear. The epoxy mixture was

then poured into each Teflon tube, covering the specimens.

56

-1

pH 7.5 pH 7.8 pH 8.1

Figure 4.1 Growth was measured in RNAR2-5 (pH 7.5); RNBR3-2 (pH 7.8); RNCR1-1 (pH 8.1) using a confocal microscope (upper row of images). The growth areas were marked, cut, embedded with epoxy mixture, then imaged and analyzed with EPMA (lower row of images).

57

After one hour, the Teflon tubes containing the epoxy mixture were placed in a vacuum

jar until no bubbles were seen in the mixture. All samples were left in an open room for

12 hours to air dry and solidify. The embedded and cured specimens were then polished

on a series of diamond grit papers, starting from 60 µm followed by 30, 12, 9, 3 and 1

micron until the test was exposed. This critical step before analysis is important so that

surface imperfections would not interfere with electron beam-sample interactions. After

polishing, the embedded samples were placed in an ultrasonic cleaner for one minute.

Then they were coated with gold by means of evaporative deposition to prevent

electrical charging of the sample and observed under the EPMA.

Inner Layer

Middle Layer

Experimental Growth Layer

Figure 4.2 How the specimens were cut for embedding using epoxy mixture and then for viewing under EMPA; note that the outer layer in this case are the chambers added during the experiment.

58

The EPMA, informally called electron microprobe, is a microbeam instrument used

primarily for non-destructive chemical analysis of minute solid samples. The process is

fundamentally the same as SEM (Scanning Electron Microscopy), with the added

capability of chemical analysis (Bonetto et al. 2001). The EPMA bombards the

specimen with a beam of accelerated electrons, which are focused on the surface of a

specimen using a series of electromagnetic lenses, and these energetic electrons produce

characteristic X-rays within nine cubic microns of the specimen. The detection limits

differ for each element, and are affected by the overall composition of a sample and the

analytical conditions. For most elements, the detection limits for the wavelength

dispersive spectrometers (WDS) is between 50 and 500 parts per million (ppm). With an

added ability to detect elements down to the level of 1%, EPMA is well-suited to the

analysis of heterogeneous specimens. The instrument used in this study (Fig 4.4) was an

Electron Probe Micro-Analyzer (jeol, jxa -8600 superprobes).

B D

A C

Figure 4.3: Preparation of sample for EPMA: (A) The edges where growth has occurred were cut for analyses. (B) Epoxy mixture (Epoxy Resin1: 0.7 Epoxy Hardener) was prepared. (C) The specimens were embedded with epoxy mixture. (D) Epoxy blocks with embedded specimens were ground using a series of diamond paper to expose the shell of M. vertebralis for subsequent analysis.

59

Figure 4.4: A typical arrangement of a probe lab (Picture taken at the Freddy and Nadine Herrmann Institute of Earth Sciences, The Hebrew University of Jerusalem (Givat Ram/ Israel).

60

4.3 RESULTS

4.3.1 IMAGES FROM EPMA (ELECTRON PROBE MICRO ANALYZER)

Shell growth or dissolution was measured by observing the number of chambers formed

or dissolved after the specimens were marked using Calcien. In Figure 4.5, the areas

circled in red provide examples of calcification or dissolution of M. vertebralis

chambers. Images A1 & A2 are the EPMA images of specimens from the 7.5 pH

treatment, where there is little or no growth of new chambers, instead dissolution in

chambers is observed. Images B1 & B2 are of specimens from the 7.8 pH treatment,

again, where minimal growth of new chambers and some signs of dissolution are

observed. Images C1 & C2 are from the 8.1 pH treatment, where new growth of

chambers is seen; the newly grown chambers are very close together.

A1 A2

61

Accelerating Voltage: 15 KeV Magnification: 60

Resolution: 1024 x 1024 Pixel Size: 2.07603 microns

Figure 4.5: Growth or dissolution was determined by observing the number of chambers formed or dissolved. Areas marked with a red circle in A1, A2, B1, B2, C1, C2 display the calcification or dissolution of M. vertebralis chambers. Images A1& A2 are the EPMA images of specimens from the 7.5 pH treatment (sample name: RNAR3-1; RNAR2-5) where there was little or no growth of new chambers, instead dissolution in chambers can be observed. Images B1 & B2 are the EPMA images of specimens from the 7.8 pH treatment (sample name: RNBR2-1; RNBR3-2), again where little growth of new chambers and some signs of dissolution was observed. Image C1 &C2 are the EPMA images of specimens from the 8.1 pH treatment (sample name: RNCR1-1; RNCR3-1) where new growth of chambers can be seen; newly grown chambers are very close together.

B1 B2

C2 C1

62

4.3.2 EPMA DATA ANALYSIS

Two specimens from each pH treatment were imaged using EPMA and an outline of

inner, middle and outer areas was drawn to locate points for analysis (Fig. 4.6, 4.7.4.8).

Using the images of the two specimens from each pH treatment (Fig 4.5), areas for

analysis were marked for each (Fig 4.6–4.8). An outline of inner, middle and

experimental growth areas for analysis was drawn as indicated by the blue lines in each

figure, and the red triangles indicate the points analyzed. Spectral data were generated

from each point. Examples of spectral data from one analysis point for each treatment

are shown in Figure 4.9. Relative concentrations of Mg, S, Ca, and Sr were extracted

from each spectral analysis, and averaged for each layer for each specimen examined

(Table 4.1). From those data, Mg/Ca and S/Ca ratios were calculated; the Sr/Ca ratios

were not calculated because Sr was close to or below detection limits in most

foraminifer specimens.

63

Outline of RNAR2-5tested samplePoint of Analysis

Exp Growth Layer Middle Layer Inner Layer

Outline of RNAR3-1tested sample

Outline of RNAR3-1outer layer

Point of Analysis

Inner Layer Exp Growth Layer

Middle Layer

(a)

(b)

Figure 4.6: Outline of areas of each specimen cultured at pH 7.5 that was analyzed and points of analysis of RNAR3-1 (a) and RNAR2-5 (b)

64

Outline of RNBR3-2 tested sample

Point of Analysis

Exp Growth Layer

Middle Layer

Inner Layer

Figure 4.7: Outline of areas of each specimen cultured at pH 7.8 that was analyzed and points of analysis of RNBR2-1 (a) and RNBR3-2 (b)

(a)

Outline of RNBR2-1tested sample

Point of Analysis

Inner Layer Middle Layer Exp Growth Layer

(b)

65

Outline ofRNCR1-1 testedsample

Point ofAnalysis

Inner Layer

Middle Layer

Exp Growth Layer

Outline ofRNCR3-1tested sample

Point ofAnalysis

Exp Growth Layer Middle Layer

Inner Layer (a)

(b)

Figure 4.8: Outline of areas of each specimen cultured at pH 8.1 that was analyzed and points of analysis of RNCR3-1 (a) and RNCR1-1 (b)

66

((cc))

((b)

((aa))

Figure 4.9: Peaks generated by EPMA while analyzing M. vertebralis cultured at (a) pH 7.5; (b) pH 7.8; (c) pH 8.1. The accelerating voltage was 16KeV with a takeoff angle of 40˚. The analysis time for the generation of peaks was 100 seconds.

67

TTable 4.1 Relative concentrations of Mg, S, Ca, P, Na and Sr extracted from each spectral analysis, and averaged for each layer for each specimen examined. From these data, Mg/Ca and S/Ca ratios were calculated. TTreatment SSpecimen

## LLayer MMg SS CCa PP NNa SSr MMg/Ca SS/Ca

7.5 RNAR3-1 Inner 8.18 0.9 90.5 0.062 0.29 0.00 0.0903 0.00994 Middle 6.67 1.08 91.9 0.14 0.16 0.00 0.0725 0.0117 Outer 7.13 0.853 91.6 0.110 0.283 0.00 0.0779 0.00931 RNAR2-5 Inner 2.4 0.19 22.3 0.025 0.12 0.045 0.107 0.00852 Middle 2.65 0.195 27.2 0.065 0.08 0.00 0.0971 0.00716 Outer 2.07 0.21 27.6 0.0767 0.0833 0.03 0.075 0.00984

7.8 RNBR2-1 Inner 7.34 0.95 91.3 0.00 0.37 0.00 0.0804 0.0104 Middle 7.93 0.89 90.7 0.12 0.36 0.00 0.0874 0.00981 Outer 7.39 0.856 91.3 0.116 0.35 0.00 0.0809 0.00938 RNBR3-2 Inner 2.36 0.26 23.5 0.035 0.15 0.00 0.100 0.0111 Middle 2.84 0.3 29.2 0.1 0.18 0.00 0.0972 0.0103 Outer 2.72 0.247 29.5 0.0367 0.113 0.00 0.0921 0.00835

8.1 RNCR3-1 Inner 2.63 0.205 21.8 0.00 0.14 0.03 0.121 0.0094 Middle 2.66 0.2 22.5 0.03 0.1 0.01 0.118 0.0089 Outer 2.23 0.167 19.5 0.0367 0.153 0.0367 0.114 0.00856 RNCR1-1 Inner 3.48 0.24 30.9 0.045 0.125 0.00 0.113 0.00777 Middle 2.79 0.315 31.09 0.03 0.145 0.005 0.0896 0.0101 Outer 2.77 0.277 30.5 0.05 0.147 0.01 0.0908 0.00907

4.3.3 VARIATIONS IN ELEMENTAL RATIOS IN-RELATION TO [CO32-]

The Mg/Ca and S/Ca ratios from the points analyzed in the outer layer of each specimen (Table 4.1), which represented growth in culture, were compared with the carbonate concentrations in the culture media [CO3

2-] in Figure 4.9. The Mg/Ca ratios were directly correlated with [CO3

2-], while the S/Ca ratios were inversely correlated.

y = 0.1664x + 62.407 R² = 1

60

65

70

75

80

85

90

95

100

105

0 50 100 150 200 250 300

Mg/

Ca (m

mol

/mol

)

CO32- (mmol kg-1 SW) (a)

68

y = -0.0045x + 9.7851 R² = 0.6847

8.68.78.88.9

99.19.29.39.49.59.69.7

0 50 100 150 200 250 300

S/Ca

(mm

ol/m

ol)

CO32- (mmol kg-1 SW)

Figure 4.10 Mean Mg/Ca (mmol/mol) against CO32- (a) and mean S/Ca (mmol/mol) against CO3

2- (b). The Mg/Ca ratio (mmol/mol) increased with increasing CO3

2- with a R2 value of 1. The opposite trend was observed for S/Ca ratio (mmol/mol) against with a R2 value of 0.685.

(b)

69

4.4 DISCUSSION

Elemental ratios of foraminiferal shells have become a vital tool in estimating past

oceanic conditions (Martin et al., 1999; Russell et al., 2004; Dissard et al., 2009). The

magnesium/calcium (Mg/Ca) ratio in the shells of foraminifera is widely used as a paleo-

temperature proxy in paleoceanography because the magnesium content of calcite is a

function of its precipitation temperature (Elderfield et al., 2006). However, most studies

have considered responses of hyaline taxa, especially low Mg-calcite species, while

relatively few have looked at the high Mg-calcite porcelaneous taxa. Major differences

in Mg/Ca ratios among lineages can represent different biomineralization pathways that

developed during the evolution of the Foraminifera (Bentov and Erez, 2006).

In this study, micro-distributions of selected elements in the shells of the porcelaneous

species, Marginopora vertebralis, cultured at three pH (pCO2) levels, were examined to

determine if water chemistry influenced incorporation of those elements from seawater.

Prior to the experiment, M. vertebralis specimens were marked with Calcein, which

helped in distinguishing the unstained areas of growth using confocal microscopy.

According to Dissard et al. (2009), the fluorescent marker Calcein does not affect Mg

and Sr incorporation into foraminiferal calcite.

The elemental measurements were designed to examine three different stages of growth

of the specimens: inner (embryonic), middle (juvenile, pre-treatment) and experimental

growth. In theory, the inner and middle stages should depict the natural environment in

which the M. vertebralis were living before they were collected for the experiment. The

the unstained outer chambers were added during the experimental treatments. Indeed,

experimental results from this study showed that the Mg/Ca ratios in the outer layers

were lower in the lower seawater pH treatments (Table 4.1, Fig 4.10). Moreover, the

specimens cultured at pH 7.5 and 7.8 exhibited minimal growth (see Chapter 2) and even

some dissolution (Fig 4.5). Thus, the Mg/Ca differences across the layers may have been

diminished by dissolution. Engel (2010) found that Amphisorus hemprichii that were

partly dissolved by acidic waters exhibited a detectable decline in Mg/Ca ratios, which

she predicted because Mg is more soluble than Ca at reduced pH.

70

The results of the present study on the response of M. vertebralis in relation to

increasing ocean acidity are in contrast to a study by Lea et al. (1999) on two species of

planktonic foraminifera, Globigerina bulloides and Orbulina universa, which indicated

that seawater pH has secondary influence on the shell Mg/Ca ratio and that a decrease in

the Mg/Ca ratio was noted with an increase in pH.

Studies have proposed that benthic foraminiferal Sr/Ca ratio decreases with increasing

calcification rate (Elderfield et al., 1996), increasing post-depositional dissolution

(McCorkle et al., 1995), and increasing pressure (Rosenthal et al., 1997). Higher pH

appeared to increase shell Sr/Ca through the kinetic influence of calcification (Lea et al.,

1999). However, it was not possible to show the relationship of Sr to pH or [CO32-] as

the Sr concentrations in M. vertebralis were too low to be measured by the instrument

(Jeol, Jxa -8600 Superprobes) used in this study.

Erez (1994) cultured the benthic foraminifera Amphistegina lobifera at the salinity of

35‰ and seawater pH adjusted between 7.9 and 8.4, with results showing a linear

relationship between the sulfate content and pH, with higher S/Ca ratio at lower pH. The

cultured M. vertebralis from the present study support the expected inverse relationship

of foraminiferal S/Ca ratio with pH or [CO32-] shown by Erez (1994).

The results from this study revealed that porcelaneous high Mg-calcite foraminifera may

respond differently from the hyaline low-Mg calcite foraminifera (such as M.

vertebralis) with respect to environmental pH. The hyaline taxa actively concentrate

Ca2+ in vacuoles (Erez, 2003), and may actively remove Mg2+ (Mewes et al., 2014). The

porcelaneous taxa appear to concentrate seawater. Their increase in shell Mg/Ca ratios

with increased temperature is well-documented (Raja et al., 2005) and may be associated

with increased carbonate saturation, which would be consistent with the current results

that show Mg/Ca ratios declining as [CO32-] and pH declined.

71

CHAPTER 5 ELEMENTAL CONCENTRATIONS USING LA-ICPMS 5.1 INTRODUCTION

5.1.1 PURPOSE

The present study focused on changes in physical proxies as the seawater chemistry is

altered, to see the response of shell chemistry of Marginopora vertebralis to a decrease

in pH or [CO32-]. B/Ca, Mg/Ca, Sr/Ca ratios, 10B/11B (denoted δ11B) and 88Sr/86Sr

(denoted δ88/86Sr) from cultured specimens described in Chapters 2 and 3 were

examined. Although the dominant parameter that has been studied as the cause of

variation in Mg/Ca ratios in benthic foraminiferal shells is temperature (Rosenthal et al.,

1997; Barker et al., 2003; Russell et al., 2004), this study focused on the effects of lower

seawater pH on Mg/Ca ratios in M. vertebralis, while keeping the temperature constant.

5.1.2 BACKGROUND

Large climatic changes have occurred in the near geological past (Pleistocene, early

Holocene). The sea surface and deep-sea records of stable isotopes in foraminiferal

shells, as well as those of major and trace elements, provide proxy information for

paleotemperatures, palecirculation and paleochemistry of the oceans (Emiliani, 1955;

Broecker and Peng, 1989, 1982; Boyle, 1988; Bijma et al., 1999). Such

paleoceanographic information is essential for the tests and calibrations of global

circulation models trying to predict the response of the atmosphere-ocean system to

changes such as increase in atmospheric CO2 (Archer and Maier-Reimer, 1994).

To interpret past environments through the use of proxies, it is necessary to associate

these proxies with specific environmental conditions. This is done by measuring these

proxies under different conditions in living specimens. To identify past oceanic

environments, analyses of the elemental compositions of foraminiferal shells are widely

used (Martin et al., 1999; Russel et al., 2004).

Nutrient proxies (Cd and Ba), physical proxies (Mg, Sr and 10B/11B) and chemical

proxies (Li, U, V, 87Sr/86 Sr and 142Nd/146Nd) are three groups of elemental proxies that

72

are expressed as a trace element/Ca ratio (Lea, 1999). Chemical proxies include those

elements whose variability in shells is primarily controlled by changes in the ocean

carbonate chemistry, while nutrient proxies provide information on seawater nutrients.

Physical proxies include those elements whose variability is primarily affected by

physical factors such as pressure, temperature, salinity and pH. Both Sr and B have

important isotope systems that can be assessed using foraminiferal shells (Lea, 2003).

Strontium has four isotopes, 84 (~0.56), 86 (~9.87), 87 (~7.04), and 88 (~82.53), having

the isotopes of 84, 86, 88 as stable isotopes, in contrast to 87 on geological time scales

(Veizer, 1989). Foraminifera actively precipitate their shells, affecting the chemistry and

structure of shell calcite. For example, calcification rate increases with rising pH, which

increases the incorporation of Sr into calcite (Morse and Bender, 1990). As described by

Pingitore (1986), there are two general mechanisms for trace element incorporation in

foraminiferal calcite: direct solid solution (e.g., trace element substitutes directly for

Ca2+ in the calcite structure) and trapping (e.g., trace elements occurring as adsorbed

ions). 5.2 METHODOLOGY

Experimental specimens of M. vertebralis were maintained under constant temperature,

pressure and salinity at three different pH levels (7.5, 7.8 and 8.1, as described in

Chapters 2 and 3). After Calcein marking, calcite deposited under experimental

conditions was easily identified using confocal microscopy (as described in Chapter 4)

and analyzed for isotopes of Ca, B, Mg, and Sr using Laser Ablation ICP-MS. Factors

that are known to influence life activities in foraminifera, such as light, temperature,

salinity and pH, were carefully controlled during culture experiments. Temperature and

pH were monitored every 2nd day to ensure stable conditions.

For analysis with LA-ICPMS, two specimens were examined per treatment. Twelve

points were ablated per specimen along the growth marked area (see Fig 5.1). To prepare

specimens for analysis, they were soaked in 3-7% NaOCl solution for 30 mins to remove

the organic matter (Dissard et al., 2010b). Specimens were removed from the NaOCl

solution as soon as they bleached completely. A stereomicroscope (low magnification)

73

was used to observe the specimens during the cleaning process. Immediately after

removal from NaOCl solution, the samples were rinsed with deionised water to ensure

complete elimination of reagent, as recommended by Dissard et al. (2010b).

The newly formed chambers of M. vertebralis (identified using confocal microscopy)

were ablated using a laser ablation system (Figs. 5.2, 5.3) inside an ablation chamber

flushed with helium (similar procedure as used by Dissard et al., 2010b).

Elemental concentrations were determined using the UP193-FX Excimer laser ablation

system (Fig 5.4) at the laboratory in Freddy and Nadine Herrmann Institute of Earth

Sciences, The Hebrew University of Jerusalem (Givat Ram/ Israel). Analyses were

calibrated against JCp-1 (coral), JCt-1 (clam) and Arag. AK Standards. Samples and

standard solutions were systematically adjusted to 100 ppm Ca through dilution to (1)

avoid dominant Ca signal increasing the salt deposition on cones and affecting the ICP-

MS stability, and (2) adjust the Ca concentrations being introduced in the ICP-QMS to

control Ca matrix effects. To compensate for signal derivation, standards (JCp-1, and

JCt-1 and Arag. AK) were run on every five and ten samples, respectively. Instrumental

calibration was achieved using standard solution for each element (Harding et al., 2006)

and by routinely measuring carbonate standards (JCp-1, JCt-1 and Arag. AK; Inoue et

al., 2004). The UP193 Laser Ablation System coupled effectively and transferred its

photon energy directly to the sample matrix for accurate and precise measurement of

trace elements and isotopic compositions (Table 5.1). The samples were bombarded via

(a) (b)

Figure 5.1 Example of M. vertebralis specimen (a) before (b) after analysis with LA-ICPMS. The arrows show the path of analysis.

74

UP193 Laser Ablation System for polishing and the distance cleaned was 600 microns.

Before ablating the sample, the background of each standard was measured, this took 40

seconds.

Table 5.1: Laser Ablation ICP-MS operating parameters

Standard Sample Repetition frequency 10 Hz Repetition frequency 30 Hz

Power/energy input 70% Power/energy input 80%

Ablation pattern: Line,50μm

spot

Ablation pattern: Line,150μm

spot

Scan speed 3 μm/sec Scan speed 6 μm/sec

Figure 5.2 Shows a laser – ablation inductively – coupled – plasma mass – spectrometry (LA–ICP–MS) Instrument Control setup (image captured from the computer screen while preparing ICP-MS for analyses of M. vertebralis.

75

Figure 5.3 Typical transient LA–ICP–MS signal from different elements while analyzing for elemental concentration in Marginopora vertebralis (in Freddy and Nadine Herrmann Institute of Earth Sciences, The Hebrew University of Jerusalem (Givat Ram/ Israel).

Figure 5.4 Shows UP193-FX Excimer laser ablation system in Freddy and Nadine Herrmann Institute of Earth Sciences, The Hebrew University of Jerusalem (Givat Ram/ Israel)

76

5.2.1 LA ICP MS ANALYSIS WITH SILLS

The Laser Ablation ICP-MS results were analyzed with SILLS (Signal Integration for

Laboratory Laser Systems) software version: V 1.1.0. SILLS is a data processing

package based on MATLAB (Figs. 5.5, 5.6).

Spike elimination (based on Grubbs test) was conducted by SILLS (Fig 5.7) where the

outliers were detected and replaced with calculated and suggested values obtained from

the analyses of M. vertebralis using ICP-MS.

Figure 5.5 shows a STANDARD window when the standards are loaded in SSILLS.

Figure 5.6 shows a SAMPLE (Unknown) window when the unknowns are loaded in SSILLS.

Figure 5.7 shows spike elimination window

77

After the standards and unknowns were loaded in SILLS, calculations were carried out

and calibration graphs were plotted (Fig 5.8). Then Element/Ca2+ ratios were calculated,

and means and standard errors were calculated and recorded.

Figure 5.8 Calibration Graphs window PPLOT 1: RELATIVE SENSITIVITY: The graph in the top-left compares the sensitivity of any two elements, i.e. plots cps/ppm for isotope A (eg 88Sr) vs. cps/ppm for isotope B (eg 43Ca). A separate line is shown for each standard measured. The relative sensitivity of different isotopes was compared by selecting them from the drop-down lists. PPLOT 2: DRIFT IN RELATIVE SENSITIVITY: The graph in the top-right shows the relative sensitivity of isotope A (eg 88Sr) and isotope B (eg43 Ca) as a function of time (as specified by the user). White dots represented the standards and yellow dots represented the unknowns. The slope is the least-squares regression line through the standard data, with each standard weighted equally. PPLOT 3: The table comprises of the standard names and the time each analysis. The values of relative sensitivities of element/Ca were calculated using SILLS. PPLOT 4: % DRIFT IN RELATIVE SENSITIVITY: The bar graph in the bottom-right shows the percentage drift in each isotope (based on the calculated drift between the first and last measured standard).

78

The 88Sr/86Sr and 11B/10B values were calculated for the M. vertebralis samples to study

how these stable isotope systems may be affected by increasing acidity of seawater. To

be consistent with notations for strontium isotopes, as reported by Fietzke and

Eisenhauer (2006) and Krabbenhöft et al. (2010), and boron isotopes (Sanyal et al.,

1995; Rollion-Bard and Erez, 2010), the results are expressed in terms of the δ-notation:

δ11/10B = {[(11B/10B) sample / (11B/10B) standard] –1}* 1000

and

δ88/86 Sr = {[(88Sr/86Sr) sample / (88Sr/86Sr) NBS987] *1000 – 1000}.

79

5.3 RESULTS

The mean values of the elemental composition for two specimens (12 ablation points per

specimen) of M. vertebralis, assessed in shell produced under each pH treatment, are

presented in Table 5.2, along with the ratios for standard associated with those analyses.

The resulting element/Ca ratios, calculated from the data in Table 5.2, are summarized in

Table 5.3. Because the standards are for aragonite and low-Mg calcite, their values for

Mg24/Ca43 and for Mg26/Ca43 are substantially lower than those for the experimental data,

across treatments.

Table 5.2 Mean values of isotopes of selected elements in M. vertebralis in shell produced under three different pH treatments, two specimens per treatment, 12 analyses per specimen. The units are in mmol, except for Ca, which are in mol. 10 B 11 B 24 Mg 26 Mg 43 Ca 46 Ca 86 Sr 88 Sr

pH 7.5 standard 126.7 299.2 254.17 275.84 664.18 985 478 46.7

sample 75 136.1 183.33 165.74 283.34 460.2 259 45.4

pH 7.8 standard 118.7 385 198.9 279.12 349.46 803.3 2123 62.6

sample 72.73 159.1 176.37 114.55 148.18 358.2 296 36.4

pH 8.1 (control) standard 73.42 311.4 120.25 154.43 98.735 260.8 624 32.9

sample 65.09 223.6 114.15 121.7 99.057 218.9 539 25.5

80

Table 5.3. Mean values of Element/Ca ratios of M. vertebralis in shell produced under each pH treatment. The values are in mmol mol-1.

Treatment 10B /43Ca 11B/43Ca 24Mg/43Ca 26Mg/43Ca 86Sr/43Ca 88Sr/43Ca

pH 7.5 ratio mean 0.000259 0.00156 21.03 3.06 0.641 5.51

standard error 0.000026 0.0000548 0.0523 0.00821 0.00179 0.0117

pH 7.8 ratio mean 0.000272 0.00149 22.6 3.29 0.586 5.002

standard error 0.000012 0.0000257 0.0769 0.0109 0.00193 0.0166

pH 8.1 (control)

ratio mean 0.000377 0.00217 24.4 3.52 0.532 4.46

standard error 0.000009 0.0000265 0.0502 0.00751 0.00125 0.0105

The mean B10/Ca43 and B11/Ca43 ratios for shell produced under each pH treatment are

presented in Figure 5.8, revealing linear increases in ratios with increasing pH. The

slopes for the two regressions indicate a roughly 5 times faster increase in the B11/Ca43

ratios as compared with the B10/Ca43 ratio. The Mg24/Ca43 and Mg26/Ca43 ratios similarly

revealed increases with increasing pH (Fig 5.9), with the ratio for Mg24/Ca43 increasing

roughly 7 times faster than that for Mg26/Ca43. The Sr/Ca ratios (Fig 5.10) in the shells

produced in the experimental treatments revealed negative correlations with pH, with the

ratio for the heavier isotope (Sr88/Ca43) declining nearly 10 times faster than the ratio for

the lighter isotope (Sr86/Ca43). Given these fractionation differences between isotopes of

B, Mg and Sr (Figs 5.8–5.10), changes in isotopic ratios with pH were calculated for B

(δ 10/11B) and Sr (δ 88/86Sr); both decreased with increasing pH (Table 5.4, Figs 5.11 and

5.12)

.

81

y = 0.0002x - 0.0012 R² = 0.836

y = 0.001x - 0.0063 R² = 0.6704

0

0.0005

0.001

0.0015

0.002

0.0025

7.4 7.5 7.6 7.7 7.8 7.9 8 8.1 8.2

B/Ca

in c

ultu

red

M.v

erte

bral

is

(mm

olm

ol-1

)

pH total scale

10B/43Ca 11B/43Ca

y = 5.6147x - 21.109 R² = 0.999

y = 0.7652x - 2.67 R² = 1

0

5

10

15

20

25

30

7.4 7.5 7.6 7.7 7.8 7.9 8 8.1 8.2

Mg/

Ca in

cul

ture

d M

. ver

tebr

alis

(m

mol

mol

-1)

pH total scale

24Mg/43Ca 26Mg/43Ca

Fig 5.9: Mean Mg/Ca plotted against pH of culture media. Mg/Ca data represent means of all measurement for specimens from that treatment.

Fig 5.8: Mean B/Ca plotted against pH of culture media. B/Ca data represent means of all measurement for specimens from that treatment.

82

Table 5.4 Mean values of the Sr and B isotopic compositions of M. vertebralis cultured at three different pH treatments. The values are expressed as ‰. The number of analyses on M. vertebralis from each treatment was 12.

δ 11B δ 88/86Sr n

pH treatment 7.5 0.551 0.17 12

pH treatment 7.8 0.457 0.12 12

pH treatment 8.1(control) 0.291 0.05 12

y = -0.1816x + 2.0035 R² = 1

y = -1.7485x + 18.629 R² = 0.9996

0

1

2

3

4

5

6

7.4 7.5 7.6 7.7 7.8 7.9 8 8.1 8.2Sr/C

a in

cul

ture

d M

. ver

tebr

alis

(m

mol

mol

-1)

pH total scale

86Sr/43Ca 88Sr/43Ca

y = -0.4333x + 3.813 R² = 0.9751

0

0.1

0.2

0.3

0.4

0.5

0.6

7.4 7.5 7.6 7.7 7.8 7.9 8 8.1 8.2δ11B

(‰) o

f cul

ture

d M

. ver

tebr

alis

pH total scale

Fig 5.10: Mean Sr/Ca plotted against pH of culture media. Sr/Ca data represent means of all measurement for specimens from that treatment.

Fig 5.11: The δ 11B of M. vertebralis increases with increasing seawater acidity.

83

y = -0.2x + 1.6733 R² = 0.9908

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

7.4 7.5 7.6 7.7 7.8 7.9 8 8.1 8.2

δ88/8

6 Sr(

‰) o

f cul

ture

d M

. ver

teba

lis

pH total scale

Fig 5.12: The δ 88/86Sr of M. vertebralis increases with increasing seawater acidity.

84

5.4 DISCUSSION

Foraminifera are organisms that precipitate their shells by complex physiological

processes that are biologically controlled (Lowenstam and Weiner 1989). As a

consequence, most paleoceanographic proxies, such as isotopic and elemental ratios in

shells, are affected to some degree by these processes during the lives of the organisms

(e.g., foraminifera, coccolithophores, and corals). Such influences are called “vital

effects” and are influenced by respiration, symbiont photosynthesis and

biomineralization processes (Dissard et al., 2010a). Moreover, as noted in Chapter 4,

major differences in Mg/Ca ratios between lineages can represent different

biomineralization pathways that developed during the evolution of the Foraminifera

(Bentov and Erez, 2006). The porcelaneous Miliolida produce Mg-calcite relatively

close to equilibrium with seawater, while many (but not all) hyaliine Rotalida and

Globigerinida produce low Mg-calcite of generally less than 4 micromol Mg/mol Ca.

The use of controlled laboratory cultures can provide insights into the controls on

foraminiferal shell chemistry (Filipsson, 2008). Differences in the rates of calcification

are often used to explain the vital effects on isotopes and trace elements (Boyle, 1995;

Elderfield et al., 1996), as the effects of these physiological processes are observed in

stable isotopes, as well as concentrations of major and trace elements (Erez, 1978; Erez

and Honjo, 1981; Boyle, 1995; Spero et al., 1997; Weiner and Dove, 2003). Trace

elements are incorporated directly from seawater during shell precipitation, reflecting

both seawater composition and the physical and the biological conditions present during

precipitation (Sen Gupta, 2003).

The Mg/Ca ratio varies also within single individual foraminiferal shells (Toyofuku and

Kitazato, 2005) and through chamber walls (Eggins et al., 2004; Sadekov et al., 2005;

Kunioka et al., 2006). Different parts of a shell also can have different concentrations of

trace elements (Eggins et al., 2003), reflecting physiological and environmental

differences that occur during the life of the specimen. This is particularly true in larger

benthic foraminifera such as M. vertebralis that can live for a year or more, so calcify

under a seasonal range of conditions. Moreover, even specimens grown under identical

85

conditions have revealed individual differences in both Mg and Sr composition (Reichart

el al., 2003; Dissard et al., 2010a, b). Thus, natural variability in Mg/Ca ratios can be

expected in the results such as those reported here.

Interest in using shell the Mg/Ca ratios as a proxy of sea surface temperature (SST)

intensified in the 1990’s. In benthic foraminifera, Mg/Ca ranges from 0.5 to more than

10 mmol/mol had been observed, with the highest values in shells from the shallowest

(warmest) sites (Rathburn and Decker, 1997; Rosenthal et al., 1997b). Moreover, it has

long been known that the porcelaneous foraminifera produce Mg-calcite, and can

incorporate more than 20 mmol Mg per mol Ca (Rathburn and Decker, 1997). In the

present study utilizing the porcelaneous, tropical benthic foraminifera, M. vertebralis,

mean Mg/Ca ranged from 21.0 to 24.4 mmol/mol (Table 5.2), with the highest values in

shells from the 8.1 pH treatment (control).

Elderfield et al. (2006) presented the carbonate ion hypothesis, which proposed that

lower [CO32-] results in lower Mg/Ca in benthic foraminifera. The results obtained in

this study for M. vertebralis are consistent with this hypothesis. Mean Mg/Ca ratios,

from multiple spot measurements on the experimental M. vertebralis, confirm that

seawater acidification (lower pH) is an important factor controlling Mg uptake into

porcelaneous foraminiferal shells (Fig 5.9). As noted in Chapter 4, the results for M.

vertebralis are in contrast to results reported by Lea et al. (1999) on two species of

planktonic foraminifera, Globigerina bulloides and Orbulina universa, who reported that

seawater pH had secondary influence on the shell Mg/Ca ratio and that a decrease in the

Mg/Ca ratio was noted with an increase in pH.

During the deposition of calcium carbonate, the amount of magnesium (Mg)

incorporated into the mineral matrix can also alter the properties of the shell (Lin and

Dexter, 1988; Kuffner et al., 2007; Engel et al., 2015; Knorr et al., 2015). Skeletons with

significant amounts of magnesium incorporated into the matrix (greater than 12%) are

more soluble, so the presence of this mineral can negatively impact shell durability

(Kuffner et al., 2007; Ferguson et al., 2008). As seawater pH declines in the future as

86

atmospheric and ocean pCO2 increase, the Mg/Ca ratios in calcite shells will decrease,

actually reducing solubility, and increasing strength of high Mg-calcite shells.

The Sr/Ca ratio is commonly measured along with Mg/Ca ratio in foraminifera. The

Sr/Ca ratios in M. vertebralis shells grown under controlled pH showed a decrease with

increasing pH (Fig 5.10).

An 80-million-year record of shell Sr concentrations has indicated that oceanic Sr

concentrations have changed significantly, recording processes such as tectonic uplift

and continental erosion (Delaney and Boyle, 1986). Moreover, studies have found that,

during glacial advances, benthic foraminiferal Sr/Ca ratios were elevated by 3-6% and

the changes varied among species and locations (Martin et al., 1999; Shen et al., 2001).

Because the seawater Sr/Ca ratio remained near constant on glacial-interglacial

timescales during the late Pleistocene, Yu et al. (2014) suggested that Sr/Ca in deep-sea

benthic foraminifera may be used as a secondary proxy for deep water changes in [CO32-

]. Studies on benthic foraminifera have demonstrated that there is a linear decrease in

benthic shell Sr with water depth suggesting that pressure is the most likely cause of

change with depth in the oceans (Elderfield et al., 1996; Rosenthal et al., 1997b).

The Sr/Ca ratio is also used as a proxy for sea-level fluctuations. Lower Sr

concentrations in seawater reflect high rates of deposition of aragonite on continental

shelves (Schlanger, 1988) since Sr preferentially substitutes into the aragonite crystal

lattices. The 87Sr/86Sr ratio of the oceans, as recorded in foraminifera shells and bulk

deep-sea carbonates, has risen from about 0.7082 (24 Mya) to 0.7091 at present (Lea,

2003). Raymo and Ruddiman (1992) hypothesized that the cause of this increase is due

to the changes in the isotopic composition and flux of Sr from rivers draining uplifted

continental areas, which includes rocks with high 87Sr/86Sr. The δ88/86Sr of water masses

can provide additional knowledge about ocean circulation and biomineralization

processes (Fietzke and Eisenhauer, 2006).

The two isotopes of strontium recorded in this study in cultured M. vertebralis shells

were 86Sr and 88Sr. Given the projected decline in ocean pH by 2100 (8.1 to 7.5), the 88Sr/86Sr ratio in M. vertebralis could increase from 0.05 to 0.17 (Table 5.4, Fig 5.12).

87

Another set of useful geochemical proxies in environmental reconstructions are the

boron isotopes, which are used to determine pH changes in the ocean water (Sanyal and

Bijma, 1999), using B as a pH proxy on the isotopic composition of skeletal carbonates

(Rollion-Bard and Erez, 2010). The ratio of 10B to 11B does not vary within the ocean

(Lea, 2003), but boron exists as a mixture of the two species, borate [B(OH) 4-] and boric

acid [B(OH)3], the proportions of which are pH dependent. Boron isotopic

measurements of marine biogenic carbonate provide a tool for estimating paleo-pH of

seawater because in modern oceans, borate is proportional to pH (Sanyal et al.; 2001; Yu

et al., 2007, Kaczmarek, 2015). Both the B10/Ca and B11/Ca ratios increased linearly

with increasing pH (Fig 5.8), though the B11/Ca ratio increased faster. As a consequence,

the boron isotope ratio, 10B/11B, in cultured specimens of M. vertebralis decreased with

increasing pH (Fig 5.11).

As noted in Chapter 4 and demonstrated again in this chapter, porcelaneous Mg-calcite

foraminifera may respond differently from the hyaline low-Mg calcite foraminifera with

respect to environmental pH. The hyaline taxa actively concentrate Ca2+ in calcifying

vacuoles (Erez, 2003), and may actively remove Mg2+ (Mewes et al., 2014). The

porcelaneous taxa do not, so their well-documented increases in shell Mg/Ca ratios with

increased temperature (Raja et al., 2005) may be associated with the increased carbonate

saturation, which would be consistent with the results from this study that show Mg/Ca

ratios declining as [CO32-] and pH declined.

88

CONCLUSIONS

The experimental results from the present study showed that calcification and growth

rate of Marginopora vertebralis decreased with decreasing pH, and the shell weight was

closely related to changing pH between 7.5 and 8.1 (NBS scale). This tendency of the

growth rate of M. vertebralis to decrease with decreasing pH is consistent with the

results reported for many other calcifying marine organisms. In addition, M. vertebralis

showed a decrease growth in response to a change in pH from 8.1 to 7.5, in par with

other organisms that also display a decrease in growth due to the elevated pCO2 at

around this pH range. The results also showed that the calcification and growth rates

were similar between pH 7.5 and 7.8, and substantially less than observed at pH

8.1(control). The laboratory experiment showed that calcification of M. vertebralis will likely

decrease in response to the projected (IPCC, 2013) future increases in atmospheric CO2.

This study also supports the observation that CO32- is the limiting factor in M. vertebralis

calcification in relation to increasing pCO2.

The calcification rate of M. vertebralis will decline substantially if pH drops to 7.5 or

lower; such conditions may not allow the survival of this species unless it has time to

adapt over several generations.

Marginopora vertebralis is a major contributor to sediment for Fijian beaches and its

diminished calcification rate could have serious consequence on the beach stability,

especially as sea level rises. When calcification rates of the major calcifers in the ocean,

including foraminifera, decline, the result is a small negative feedback mechanism to

global CO2 increase (Bentov et al., 2009).

EPMA-corrected and LA-ICP-MS data are used to compare the Mg/Ca, Sr/Ca, Sr and B

isotope ratios in M. vertebralis shells produced under three pH levels. Comparison of

Mg/Ca and Sr/Ca compositions obtained by both methods against pH suggests that

89

Mg/Ca increases and Sr/Ca decreases with increasing pH. These results clearly indicate

that if ocean acidification (lower pH and [CO32-]) continues, the Mg/Ca ratio will

decrease and Sr/Ca ratio will increase in M. vertebralis. Similarly, δ11B and δ88/86Sr

ratios increased with increasing ocean acidity.

Since it is possible to calibrate the shell composition against the controlling factors,

foraminiferal trace elements can provide researchers with vital and relevant proxies to

investigate the physical, biological and chemical changes in the ocean.

6.1 FUTURE RESEARCH TOPICS

� Future studies may seek to culture other important species of foraminifera around

Fiji and compare their response to increasing ocean acidity.

� Future studies could look at other parameters that may affect the inclusion of

major, minor and other trace elements in the M. vertebralis shells.

� Modeling and documentation of carbonate production differences in

Foraminifera in a historical context, along with present and future projections

will be vital to determine coastal protection and conservation strategies.

� Further developmental work in high purity sample preparation should be done to

improve the analytical reproducibility in the isotope analysis of benthic

foraminiferal shells, where each element can be examined in more detail.

90

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