Effects of CO2-driven ocean acidification on the embryonic and early larval development of

Epinephelus suillus

I. Introduction

A. Background of the Study

At present, approximately one-third of CO2 has already been absorbed by the earths

atmosphere that has accumulated for the past 100 years. This has led to the increase in the partial

pressure of carbon dioxide in the oceans and decrease in pH levels (Caldeira & Wickett 2003,

Royal Society, 2005, and German Advisory Council on Global Change, 2006). Moreover, this

level has been the highest CO2 concentration than any point in the past 800,000 years (Lthi D,

et al. 2008) and has the fastest rate experienced in the last 300 million years (Hnisch B, et al.,

2012).

It is known that ocean acidification affects a wide diversity of marine organisms. Most of

the research conducted on CO2-driven ocean acidification concerns on corals that focuses on

calcification and effects on other calcifying organisms. However, studies on the impact on fishes

are more limited both in understanding and in a variety of species. The early life stages of other

marine organisms particularly on fishes are also likely to be sensitive to these changes for they

are essential for replenishment and connectivity (Portner et al. 2004). Increasing attention has

been given to the impacts of ocean acidification on the early development stages of marine

species, but experiments in relation to these scenarios have provided mixed results (Munday, et

al., 2011 and Nilsson GE, et al., 2012). The early development stage is a vital phase in marine

fishes as this is the life stage which is more susceptible to mortality (Houde E., 1997). Therefore,

it is important to study the effects of ocean acidification on the early life stages of fish in

understanding the cumulative effect on fish populations.

 The species that will be used in the study, Epinephelus suillus, has a fast growth rate and

abundant supply compared to the other species in the genera (Kohno, 1987). In addition, it is one

of the most widely distributed tropical species with significant ecological and economic value.

Its life history and morphology are similar with other high-value, tropical fishes and thus their

use in these experiments provides a useful perspective of possible ocean acidification impacts on

other pelagic species of high ecological and economic value.

B. Objectives

This study aims to examine the development of the eggs and larvae of E. suillus, and their

rate of growth and survival when subjected to elevated CO2 levels (380, 600, 750 and 1000

ppm).

C. Hypothesis

CO2-driven ocean acidification will have damaging effects on the morphology of the

eggs, larvae, and will lower their survival rate.

II. Review of Related Literature

A. Introduction to ocean acidification

Ocean acidification is the process of lowering of the pH of the oceans waters by

increasing absorption of atmospheric CO2. The pre-industrial CO2 concentration in the

atmosphere at 278 ppm has risen to approximately 390 ppm in 2010 which causes significant

consequences on marine life.

When CO2 is absorbed in seawater, it reacts with H2O to form H2CO3 (carbonic acid).

In turn, the carbonic acid will break into H+ ions and HCO3- ions (bicarbonate ions). Carbonic
acid will break down into H ions and HCO3- (bicarbonate ions). Increase in H+ ions will lower

the pH thereby increases the acidity. The CO32- (carbonate ions) react with these H+ ions to

form more bicarbonate ions. Carbonate ion concentrations decreases while H+, H2CO3 and

HCO3- concentrations increase. When carbonate ions are less available, calcium carbonate

(CaCO) dissolution is more likely to occur, and consequently its formation is less likely to occur

(ASOC, 2010).

B. Impacts on vertebrates

The impacts of ocean acidification affects all marine life. However, studies on the effects

on vertebrates has been very limited but there are indication of detrimental effects on fishes

although these impacts are still unknown. It has been found out that the short-term effects of

ocean acidification on fishes includes acidbase status, respiration, blood circulation, and

nervous system functions, while long-term effects include reduced growth rate and reproduction

(Guinotte, JM and Fabry, VJ, 2008). Also, acidification of body fluids of marine animals

resulting from increasing hypercapnia occurs rapidly, in a matter of hours (Royal Society, 2005).

It is also found out that hypercapnia lowers respiratory rates and protein synthesis rates, and

maybe other physiological processes (Royal Society, 2005).

III. Materials and Methods

The experiment will be conducted at the Marine Pollution and Ecotoxicology Laboratory

on October 21-25, 2013.

A. Egg Collection
 E. suillus eggs will be supplied by the aquaculture department of SEAFDEC. Throughout

the experiment, the larvae will be fed with rotifers.

B. Experimental Design

A flow-through system will be used in the setup and will be equilibrated with different

CO2 concentrations. The aquaria will be placed in a water bath to minimize water temperature

fluctuations. The containers will be covered tightly with lids and a single hole will be cut for

seawater and gas tubing to fit inside. The seawater will be pre-bubbled with CO2 until it reaches

the current level of atmospheric CO2 which is approximately 380 ppm and three values projected

to occur in the future within the current century which is 600, 750 and 1000 ppm as

recommended by EPOCA protocol (Barry, JP, et al. 2010). There will be three replicates for

each treatment. CO2 concentration will be measured in ppm units using a CO2 gas probe. The

pH of the water will be monitored throughout the experiment using a pH meter. Total alkalinity

and dissolved organic carbon will be measured on subsamples and the carbonate system will be

calculated from it based on CO2SYS (Lewis and Wallace, 1998).

Three hundred eggs will be randomly assigned to each treatment (100 eggs for each

replicate). Five randomly selected eggs per aquaria will be monitored every fifteen minutes one

hour after the fertilization for the duration of five hours, and every three hours for the next 24

hours for development stage.

Dead cells will be removed from the treatments for morphometric analysis. E. suillus will

be photographed and measured for each developmental stage and will be compared to the

controlled. Each of them will be dried to a constant mass to obtain the dry mass.

C. Computations
 Hatching rates will be determined using the formula:
.
=

.
=


= 100

D. Statistical Analyses

Differences in the mean number of E. suillus larvae completing metamorphosis among

the four pCO2 treatments will be tested with one-way ANOVA. Tukeys HSD multiple

comparison test will be used to determine differences in pCO2 levels. Any bias in these data will

be explored by residual analysis and if detected the data were transformed as noted in the

ANOVA tables. A contingency table will be used to test whether the number of larvae settling

was independent of the level of pCO2.

IV. Timeframe
J J A S O N D J F M A
Capsule proposal
Proposal first draft
Final proposal
Experiment proper
Thesis first draft
Thesis second draft
Final thesis

V. Budgetary requirements

Amount (Php)
Grouper eggs 100
Transportation 100
CO2 750
Others 1000
 TOTAL 1950

VI. Literature cited

Barry JP, Tyrell T, Hansson L, et al. (2010) Atmospheric CO2 targets for ocean acidification
perturbation experiments. In: Riebesell U, Fabry VJ, Hansson L, Gattuso JP (eds) Guide to
best practices for ocean acidification research and data reporting European project on ocean
acidification Luxembourg, pp 53-64
Bofill, A. (1992). Embryonic development and hatching rate of grouper (Epinephelus suillus)
eggs incubated at different stocking densities. Undergraduate thesis BS Biology, UP
Visayas, Iloilo. 30 p.
Caldeira K, Wickett ME (2003) Anthropogenic carbon and ocean pH. Nature 425:365
Frommel, AY., Stiebens V., Clemmesen C., Havenhand, J. (2010). Effect of ocean acidification
on marine fish sperm (Baltic cod: Gadus morhua). Biogeosciences, 7, 3915-3919, 2010.
http://www.biogeosciences.net/7/3915/2010/bg-7-3915-2010.pdf
German Advisory Council on Global Change (2006). The future oceans warming up, rising
high, turning sour. WBGU, Berlin. Available at: www.wbgu.de/wbgu_sn2006_en.html,
doi:10.1029/2006GL028554
Guinotte, JM and Fabry, VJ (2008). Ocean acidification and its potential effects on marine
ecosystems. Annals of the New York Academy of Sciences 1134: 320 342.
Hnisch B, et al. (2012). The geological record of ocean acidication. Science 335(6072):1058
1063.
Houde E (1997) Patterns and trends in larval-stage growth and mortality of teleost sh. J Fish
Biol 51(Supplement A):5283.
Kohno, H., Duray, M., and Suntayo S. (1990). Introduction to Lapu-lapu of the Philippines.
Asian Aquaculture, B(6), 9(1): B-11; 5,10 (part of series)
Lewis, E. and Wallace, DWR. (1998). Program developed for CO2 systems calculations,
ORNL/CDIAC-105, Carbon Dioxide Information Analysis Center, Oak Ridge National
Laboratory, US Department of Energy, Oak Ridge, Tennessee.
Lthi D, et al. (2008) High-resolution carbon dioxide concentration record 650,000-800,000
years before present. Nature 453(7193):379382. http://epic.awi.de/18281/1/Lth2008a.pdf
Munday PL, Gagliano M, Donelson JM, Dixson DL, Thorrold SR (2011) Ocean acidication
does not affect the early life history development of a tropical marinesh. Mar Ecol Prog Ser
423:211221.
Nilsson GE, et al. (2012) Near-future carbon dioxide levels alter sh behavior by interfering with
neurotransmitter function. Nat Clim Chang 2(3):201204
Portner HO., Langenbuch M., Reipschlager A. (2004). Biological impact of elevated ocean CO2
concentrations: Lessons from animal physiology and earth history. J Oceanogr 60:705-718
Royal Society (2005) Ocean acidification due to increasing atmospheric carbon dioxide. Policy
Document 12/05, The Royal Society, London. Available at:
www.royalsoc.ac.uk/displaypagedoc.asp?id=13539