Effects of reduced pH on the growth and survivalof postlarvae of the donkey’s ear abalone,Haliotis asinina (L.)

Abduraji S. Tahil • Danilo T. Dy

Received: 2 February 2014 / Accepted: 4 June 2014� Springer International Publishing Switzerland 2014

Abstract High atmospheric CO2 levels primarily from burning fossil fuels have

increased CO2 concentration in the surface water of the ocean, this in turn has resulted in a

drop in seawater pH known as ‘‘ocean acidification’’ (OA) and presents a potential threat to

calcifying marine organisms. Up to the present, however, the impacts of OA on the early

developmental stages of tropical abalone were not known. For the first time in the Phil-

ippines, we investigated the direct effects of reduced pH on the growth and survival of the

postlarvae of the donkey’s ear abalone, Haliotis asinina L. H. asinina is the only com-

mercial species of haliotid in the country. We reduced the ambient pH (7.99) of experi-

mental seawater by bubbling food-grade CO2 to obtain the desired pH levels as expected

by the year 2100. Survival of H. asinina postlarvae was negatively affected by reduced pH.

Mean survival of postlarvae significantly decreased from 86.3 % at ambient conditions to

47.2 and 18.3 % at pH 7.62 and 7.42, respectively, after 20 days (P \ 0.001 for both).

Survival at pH 7.81 (69.9 %) was comparable with ambient conditions (P [ 0.05). Mean

shell lengths of postlarvae also differed significantly among treatments and across exposure

period (P = 0.000 for both). Postlarvae reared at pH 7.62 and pH 7.42 exhibited smaller

shell size (138 and 124 lm, respectively) compared to those at ambient conditions

(175 lm). Postlarvae reared at pH 7.81 showed larger shell length (162 lm) compared to

the postlarvae reared at pH 7.42 (124 lm). Across time, significant increase in shell size

from day 5 to day 20 was observed only in postlarvae reared at ambient conditions

(P = 0.000) and in the pH 7.81 treatment (P = 0.006). Daily growth rate decreased sig-

nificantly from 2.83 lm day-1 (ambient) to 0.95 and 0.30 lm day-1 at pH 7.62 and 7.42,

respectively. Our results suggest that growth and survival of postlarval H. asinina was

found to be sensitive to reduced pH of seawater. This implies that future decrease in pH

unit of the world’s oceans will most likely threaten the natural population of this eco-

nomically important species.

A. S. Tahil � D. T. DyDepartment of Biology, University of San Carlos Talamban, Nasipit, Cebu City, Philippines

A. S. Tahil (&)USC Marine Research Station, 6015 Maribago, Lapu-Lapu City, Cebu, Philippinese-mail: rajstahil@yahoo.com

123

Aquacult IntDOI 10.1007/s10499-014-9804-4

Keywords Abalone seed production � Daily growth rates �Mortality � Ocean acidification

Introduction

Concern over the negative effects of ocean acidification (OA) on marine ecosystem par-

ticularly on calcifying organisms is gaining more attention all over the world, especially in

the temperate region. Increasing concentration of CO2 in the world’s ocean due to high

anthropogenic CO2 emissions into the atmosphere could result in a decrease in the

availability of carbonate ions and a drop in seawater pH (Crim et al. 2011). Depending on

geographic region and season, the pH of the open sea (upper 100 m) varies between 7.9

and 8.3 (Andersen et al. 2013). This variation may be more pronounced in coastal waters

where relatively rapid changes in pH or pCO2 can occur (Kerrison et al. 2011). In the last

200 years, the oceans have already absorbed approximately 50 % of the atmospheric CO2,

which decreased surface ocean pH by an average of 0.1 units (Royal Society 2005; Orr

et al. 2005). This current drop in seawater pH units will continue to decline at a slower rate

to 0.3–0.5 units by 2100 (Caldeira and Wickett 2003). Recognizing the potential impact of

OA on the marine ecosystem, ecologically relevant CO2 perturbation experiments have

been conducted worldwide, revealing substantial adverse effects of OA on a wide range of

marine organisms (reviewed in Fabry et al. 2008).

Several studies have reported negative effects of OA on many early developmental

stages of calcifying invertebrates including fertilization, larval growth and survival,

shell calcification and dissolution, and larval duration and settlement (e.g., Albright

et al. 2012; Amaral et al. 2012; Andersen et al. 2013; Jansson et al. 2013; Kim et al.

2013; Kroeker et al. 2013; O’Donnell et al. 2013). It was shown that even minor

changes in pH can significantly impair larval development, survival and growth in

marine calcifying species (Dupont et al. 2008; Crim et al. 2011). This negative effect

may have severe consequences for commercially important species (i.e., abalone)

whose population hinges on larval recruitment, growth and survival of postlarvae in

their respective habitats. With all these observations, OA has now been recognized as

affecting key life history stages and transitions (including larval development, growth

and survival) of marine invertebrates. To date, however, the deleterious effect of OA

has not been investigated for tropical abalones or any high-value species of molluscs in

tropical areas.

Haliotis asinina is the only commercial species of abalone in the Philippines. In

1996, the country’s harvest of wild H. asinina was recorded at 448 metric tons with

China as the largest market for dried abalone meat, and the shells are also sold as

buttons, jewelry and inlay work (Fermin 2001). Juveniles of this species are produced

in the hatchery for grow-out culture and restocking in natural habitat. As an aquaculture

species, studying the postlarvae from the potential effects of reduced pH may help

enhance the sustainable seed production for the aquaculture industry. Here, we

investigated the effects of reduced seawater pH brought about by elevated CO2 con-

centration on the growth and survival of H. asinina postlarvae. The results presented

here provide a better understanding of the negative impact of low pH and the predicted

effects of OA on an ecologically and economically important gastropod species in a

tropical region, such as the Philippines.

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Materials and methods

Experimental design

The experiment was conducted at the Tawi–Tawi Multi-Species Hatchery in Latu–Latu,

Bongao, Tawi–Tawi, Philippines, from August–November 2013 using a randomized

complete block design (RCBD). An epoxy-coated wooden tank with eight compartments

(4 9 2) and aeration system was used for the experiment. Each compartment has a

dimension of 60 9 60 9 60 cm. Each tank compartment was provided with UV-treated

and filtered (sand-filter and 5 lm mesh filter bag) seawater pumped from the subtidal area

of Tawi–Tawi Bay. The experiment was conducted in four successive trials with two

replicates for each treatment per trial, and a new group of one-month-old postlarvae was

used during each trial. Part of the water quality management was a daily siphoning of fecal

material and dirt from the bottom of each tank compartment. The volume of water taken

out was approximately 5 L and replaced with an equal volume of UV-treated and filtered

seawater via the flow-through system. A flow-through, non-recirculating system was done

daily for 8 h (2 min L-1) to allow exchange with new filtered (sand-filter and 5-lm mesh

filter bag) seawater in the experimental units.

Manipulation and maintenance of pH

Immediately after stocking of postlarvae, the pH levels were adjusted daily and maintained

through manual addition of a food-grade CO2 delivered via thin-walled silicon tubings.

Based on the preliminary simulation to determine the amount of CO2 needed to maintain

the pH within the range, CO2 was added every 2.5–3 h for an average duration of 7–50 s

depending on the desired pH level for each treatment. Based on the predicted decrease of

0.3–0.5 units in the ocean pH by 2100 (Caldeira and Wickett 2003, 2005), four pH levels

each ranging from 7.95 to 8.02 (mean ± SD; 7.99 ± 0.01—Ambient), 7.78–7.83

(7.81 ± 0.02—Treatment I), 7.59–7.65 (7.62 ± 0.02—Treatment II) and 7.39–7.45

(7.42 ± 0.02—Treatment III) were used in this experiment. The pH and temperature of the

water were measured with a calibrated pH meter.

Total alkalinity (TA) was measured every 5 days throughout the experiment using an

alkalinity titration kit (Precision: ± 0.05 lmol kg-1). Concentrations of CO2, carbonate

(CO3-2) and bicarbonate (HCO3

-); partial pressure of CO2 (pCO2); saturation state of

calcite (Xcalc) and aragonite (Xarag) were then calculated from measured TA and pH using

the software CO2SYS (Pierrot et al. 2006) and by using the dissociation constants of

carbonic acid from Mehrbach et al. (1973) refitted by Dickson and Millero (1987) (Moulin

et al. 2011). Salinity and dissolved oxygen was measured with a refractometer and DO

meter, respectively.

Preparation of diatom slurry and plate substrates

Before the main experiment, several hatching protocols were done to ensure adequate

supply of one-month-old abalone postlarvae. A pure stock culture of benthic diatoms

(Amphora sp. and Navicula ramosissima) was acquired and cultured at the larval food

laboratory of the Tawi–Tawi Multi-Species Hatchery. The diatoms were cultured from the

(starter) stock to a larger culture vessel of 500 L using the multi-step batch culture method

(De la Pena et al. 2010). Diatom species were harvested during their exponential growth

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phase before transferring them to a larger culture vessel. Initially, 150 mL of diatom starter

was scaled up into 3 L of seawater in the laboratory using F/2 medium (Guillard and

Ryther 1962). After 3 days, this was scaled up into a 30-L culture using a white plastic

basin with TMRL (The Media Research Laboratory) medium (prepared from technical-

grade reagents composed of silicate, sodium phosphate, ferric chloride and sodium nitrate).

After another 3 days, the 30-L culture was inoculated into a 500-L concrete tank filled

with 300 L of seawater pumped from the subtidal area that passed through a sand filter and

a 5-lm mesh filter bag. The culture was vigorously aerated from the bottom of the tank and

enriched using the modified Commercial II medium (Renaud et al. 1991) composed of

technical- and agricultural-grade reagents (ammonium sulfate 21-0-0, 150.0 mg L-1; urea

46-0-0, 7.5 mg L-1; super phosphate 16-20-0, 25 mg L-1; FeCl3�6H2O, 5.0 mg L-1;

Na2EDTA, 5.0 mg L-1; and sodium silicate 15.0 mg L-1) (de la Pena et al. 2010). After

3 days of culture, additional seawater and a proportional amount of fertilizer were added to

fill-up the tank to full capacity (500 L).

A separate concrete tank was fitted with 100 pieces of 45 cm 9 40 cm corrugated

plastic sheet with crustose coralline algae (CCA) and arranged vertically on PVC pipes to

induce the growth of benthic diatoms on the plates. The plates with CCA were old plates

which have been placed in the sea for at least 3 months to allow the settlement of CCA

identified as Mesophyllum sp. on the top and Hydrolliton samoense at the bottom of the

pink crust (de la Pena et al. 2010). After 3–4 days in the tank, the corrugated plastic sheets

were examined as to the presence of diatom films growing on their surfaces. This hatchery

procedure was done to induce the abalone planktonic trochophores to settle onto the plates

with CCA and diatoms.

Stocking and rearing of abalone postlarvae

One-month-old postlarvae were obtained from the larval rearing facilities of the abalone

hatchery. Three replicate plates with CCA and diatom films were taken from the concrete

tank containing 100 plates and placed in each experimental unit during each trial as

substrate for the postlarvae. Each plate contained 20 postlarvae that have already settled

and developed for a month on the same plate. Plates with\20 postlarvae were augmented

by carefully detaching postlarvae from other plates using a soft paint brush or small plastic

spatula and placed on those plates containing \20 postlarvae. The postlarvae were reared

for 20 days in each of the four treatments. To sustain the food supply of the postlarvae

during the culture period, diatom slurry was added to each tank compartment every 3 days

for a period of 20 days.

To minimize counting error by avoiding repetitive counting of the same individuals, a

transparent plastic/acetate tape with grids of 2 cm2 was laid on the plate during counting of

surviving postlarvae.

Monitoring of growth and survival rates

The initial shell length (in lm) of the postlarvae was recorded at the time of stocking to the

experimental units using a micrometer scale inserted in the eyepiece of a monocular 409

microscope. Shell length was measured from the posterior end to the anterior end of the shell.

Subsequent measurement of shell length and monitoring of surviving postlarvae was done

every 5 days over a period of 20 days. For each sampling period, a total of 15 postlarvae were

randomly selected and dislodged from the three plates using a soft paint brush or small

spatula. All plates were numbered from which postlarval samples were collected using

Aquacult Int

123

random numbers. Increase in shell size of postlarvae in each treatment was calculated during

each sampling period, and the daily growth rate (in lm day-1) was expressed as the differ-

ence between the final and initial shell lengths divided by the number of days during which

measurements were taken. Postlarval survival for each treatment was determined by census of

individual postlarva during each sampling period and at the end of the 20-day rearing period

per trial. This was expressed in % and calculated from the accumulated mortality divided by

the initial number of postlarvae 9 100.

Statistical analysis

Results of the two-way ANOVA (factors pH and trials) on survival and shell size data did

not show significant differences among trials (survival: P = 0.254; shell size: P = 0.104 at

5 % level of significance). With these observations and since the postlarvae used for each

trial were obtained from the same group of broodstock and the conditions during the

experiment were similar, the data from the four trials were pooled for the statistical

analyses. Data on shell lengths were analyzed using repeated measures ANOVA to

determine the levels of differences in mean values among treatments and the effect of time

(day) on the growth and survival of postlarvae. Homoscedasticity of the shell length data

was done using Levene’s test. Differences between means were tested for significance

(P \ 0.05) using the post hoc Tukey HSD test. Differences in the daily growth rate and

survival of postlarvae at the end of the 20-day rearing period were determined using a

nonparametric Kruskal–Wallis ANOVA and multiple comparisons of mean ranks for all

treatment groups.

Results

Diffusion of CO2 into the experimental seawater resulted in reduced pH from ambient

conditions by 0.18, 0.37 and 0.56 units for treatment 1 (T-I), treatment 2 (T-II) and treatment

3 (T-III), respectively. The mean seawater pH was consistent within a desired level for each

treatment. At ambient conditions, the mean pH for the whole duration of the experiment was

7.99 ± 0.01. In CO2-treated waters, the mean pH levels were 7.81 ± 0.02 (T-I),

7.62 ± 0.02 (T-II) and 7.42 ± 0.02 (T-III) (Table 1). For all cases, the mean temperature

and salinity showed minimal variations across treatments which ranged from 26.8 ± 0.6 to

27.3 ± 0.5 �C and 34.1 ± 0.3 to 34.2 ± 0.4 psu, respectively (Table 1). The same variation

was recorded for the TA which ranged from 2,092.2 to 2,194.2 lmol kg-1 across treatments.

Minimal variations in O2 concentration were also observed among treatments (7.9 ± 0.6 to

8.2 ± 0.3 mg L-1), suggesting that the amount of CO2 addition did not significantly affect

the oxygen concentration of the seawater.

The carbonate chemistry of the experimental seawater is also shown in Table 1. Car-

bonate ion concentration was lowest in waters with high CO2 or lowest pH

(63.7 ± 2.4 lmol kg-1) and highest at ambient conditions (210.9 lmol kg-1) (Table 1).

The partial pressure of CO2 (pCO2) in the experimental seawater increased with increasing

CO2 concentration and was highest (pCO2 = 1,886.4 ± 73.6 latm) in T-III

(CO2 = 51.3 ± 1.8 lmol kg-1) compared to ambient conditions (CO2 = 11.8 ± 0.4

lmol kg-1) with a pCO2 of 440.6 ± 19.1 latm. Saturation states of aragonite (Xarag) and

calcite (Xcalc) decreased with increasing CO2 concentration or reduced pH level. In T-III

(pH = 7.42; CO2 = 51.3 lmol kg-1), Xarag and Xcalc of the experimental seawater were

only 1.0 ± 0.0 and 1.5 ± 0.1, respectively, compared to the ambient conditions

Aquacult Int

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(Xarag = 3.4 ± 0.1 and Xcalc = 5.1 ± 0.1) (Table 1). Calculations of these parameters

were based on the measured pH and TA as with other similar studies (i.e., Cunningham

2013; Jansson et al. 2013; Timmins-Schiffman et al. 2012).

Reduced pH negatively affected the survival of abalone postlarvae (Kruskal–Wallis

ANOVA; H (3, n = 32) = 29.19, P = 0.000) (Fig. 1). At the end of the rearing period,

mean survival of postlarvae in T-II (47.2 ± 2.2 %) and T-III (18.3 ± 16.0 %) was sig-

nificantly lower than that of ambient conditions (86.3 ± 2.0 %). Survival of postlarvae

was also significantly different between T-I (69.9 ± 2.4 %) and T-III but not in the fol-

lowing treatments: between ambient conditions and T-I; between T-I and T-II; and

between T-II and T-III.

Repeated measures ANOVA indicated significant differences in shell length of post-

larvae among pH treatments and across exposure period (P = 0.000 for both) (Fig. 2).

Starting on day 10, postlarval growth was significantly different among treatments

(P \ 0.005). Mean shell lengths of postlarvae after 10 days (129.9 ± 19.3 lm) and

15 days (135.1 ± 20.5 lm) exposure to T-II and T-III (day 10 = 124.3 ± 19.8 lm; day

15 = 125.3 ± 20.8 lm) were significantly smaller than the ambient conditions (day

10 = 149.7 ± 12.6 lm; day 15 = 162.9 ± 14.0 lm). At the end of the 20-day rearing

period under reduced pH levels, postlarval shell lengths in T-II (138.2 ± 21.0 lm) and

T-III (124.7 ± 22.3 lm) were significantly smaller than the ambient conditions

(175.2 ± 14.0 lm) (Fig. 3). Significant difference in shell lengths was observed also

between T-I (161.6 ± 16.7 lm) and T-III at the end of the exposure period. Across time,

postlarvae displayed significant increase in size at ambient conditions (P = 0.000) and T-I

(P = 0.006) from day 5 to day 20 but did not significantly increase in size when exposed to

lower pH levels (i.e., T-II and T-III) during the same period.

Haliotis asinina postlarvae displayed a step-wise decrease in growth rate with reduction

in pH levels. The pH level had a significant effect on postlarval shell growth [Kruskal–

Wallis ANOVA: H (3, n = 31) = 22.93, P = 0.000; Fig. 3]. With decreasing levels of pH

Table 1 Carbonate chemistry of experimental seawater in ambient condition and at reduced pH levels

Parameter pH treatment

7.97–8.02(Ambient)

7.78–7.83(T-I)

7.59–7.65(T-II)

7.39–7.45(T-III)

Temperature (�C) 27.3 ± 0.5 27.3 ± 0.6 26.9 ± 0.5 26.8 ± 0.6

Salinity (psu) 34.2 ± 0.4 34.1 ± 0.5 34.2 ± 0.4 34.1 ± 0.3

Dissolved oxygen (mg L-1) 7.9 ± 0.6 8.0 ± 0.3 8.1 ± 0.4 8.2 ± 0.3

pH 7.99 ± 0.01 7.81 ± 0.02 7.62 ± 0.02 7.42 ± 0.02

Total alkalinity (lmol kg-1) 2,194.2 ± 23.2 2,124.2 ± 28.4 2,109.3 ± 25.2 2,092.2 ± 24.6

CO2 (lmol kg-1) 11.8 ± 0.4 19.1 ± 1.1 31.3 ± 2.0 51.3 ± 1.8

CO3-2 (lmol kg-1) 210.9 ± 4.9 144.7 ± 5.2 98.1 ± 5.5 63.7 ± 2.4

HCO3- (lmol kg-1) 1,682.3 ± 20.7 1,771.4 ± 31.2 1,870.1 ± 27.2 1,936.9 ± 22.7

pCO2 (latm) 440.6 ± 19.1 708.5 ± 39.1 1,153.9 ± 65.5 1,886.4 ± 73.6

Calcite saturation (Xcalc) 5.1 ± 0.1 3.5 ± 0.1 2.4 ± 0.1 1.5 ± 0.1

Aragonite saturation (Xarag) 3.4 ± 0.1 2.3 ± 0.1 1.6 ± 0.1 1.0 ± 0.0

Measured were: pH, temperature, salinity, dissolved oxygen, and TA. Calculated using the software CO2-

SYS were: CO2, carbonate (CO3-2) and bicarbonate (HCO3

-) concentrations, partial pressure of CO2

(pCO2), and saturation state of calcite and aragonite. Data are mean ± SD; n = 8

Aquacult Int

123

0

20

40

60

80

100

5 10 15 20

Ambient (pH 7.99)

pH 7.81

pH 7.62

pH 7.42

Days

% s

urv

ival

Fig. 1 Mean percent survival of H. asinina postlarvae exposed to reduced pH levels for 20 days. Survivaldiffered significantly across treatments [Nonparametric Kruskal–Wallis ANOVA test: H (3,n = 32) = 291.19, P = 0.000]. Error bars are SD, n = 8

110

120

130

140

150

160

170

180

0 5 10 15 20

Ambient (pH 7.99)

pH 7.81

pH 7.62

pH 7.42

Sh

ell l

eng

th (

µm

)

Days

a1

a1

b1 b

1

d1

f 12

d1

f1

a1

a1

b1

b1

c2

e3

i1

ef2

cd12

gh 2 3

hi 1 2

g3

Fig. 2 Mean shell length (lm) of postlarvae of H. asinina reared at different pH levels for 20 days.Different letters and numbers above the columns denote significant difference among treatments and acrossrearing period, respectively. Repeated measures ANOVA, P = 0.000. Error bars are SD, n = 8

a

a

b

b

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

7.99±0.01 7.81±0.02 7.62±0.02 7.42±0.02

Gro

wth

rat

e (µ

m d

ay -1

)

pH treatment

Fig. 3 Daily growth rate (lm day-1) in shell length of H. asinina postlarvae after 20 days of culture atdifferent pH levels. Different letters above the columns denote significant difference among treatments.Nonparametric Kruskal–Wallis ANOVA test: H (3, n = 31) = 22.93, P = 0.000. Error bars are SD, n = 8

Aquacult Int

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from 7.99 (ambient), 7.81, 7.62 to 7.42, daily growth rates of the postlarvae decreased from

2.83 ± 0.26, 2.18 ± 0.47, 0.95 ± 0.81, to 0.30 ± 0.42 lm day-1 at the end of the 20-day

exposure period (Fig. 3).

Discussion

The reduction in pH units (0.18–0.56) as a result of CO2 infusion into seawater was within

the ocean surface water pH reduction (0.3–0.5 units) expected by 2100 (Caldeira and

Wickett 2003). However, reducing the ambient pH by 0.56 units is relatively lower than

that resulting from the expected worst-case scenario (-0.5 units). We used this lowest pH

to forecast how future worst scenario may affect the postlarvae of this abalone species

should the emission of anthropogenic CO2 into the atmosphere continue with its current

trend of ‘‘business-as-usual’’ in the coming century. Here, we provide the first direct

experimental evidence that the expected pH reduction of the ocean’s surface waters will

negatively impact the tropical abalone species (H. asinina). We recorded an ambient pH

ranging from 7.97 to 8.02 (mean = 7.99 ± 0.01) for seawater pumped from the subtidal

area (2.5–4.5 m depth) of Tawi–Tawi Bay, which is partly surrounded by mangrove forest

and where pH levels are expected to be relatively low and variable compared to the open

ocean. The calculated pCO2 (441 latm) for ambient conditions was closely similar to the

reports of other workers studying molluscs from the Pacific area [e.g., 468 latm (Timmins-

Schiffman et al. 2012); 469 latm (Andersen et al. 2013)]. H. asinina is most common in

the shallow reef flat (ca. 0.5–4.5 m depth) around the bay where they may be exposed to

such variations in pH levels in nature. Thus, the use of reduced pH levels here revealed

how these variations in nature may affect the post-settlement stage and population integrity

of this abalone species. We calculated also low saturation states of calcium carbonate (1.5)

and aragonite (1.0) at the lowest pH (7.42) treatment, suggesting that shell dissolution of

postlarvae may be rapid at reduced pH levels. For shell-forming molluscs (i.e., abalone)

living in such conditions, high organism energy expenditure is expected to maintain a

normal shell structure and obtain better survival and growth (Jansson et al. 2013).

Survival

In the context of tropical conditions, our results on larval survival of H. asinina exposed to

reduced pH for 20 days indicated that OA has highly negative impact on the postlarval

stage of this tropical abalone. The observed reduction in % survival of postlarvae from

86.3 % at ambient conditions to 47.2 % at pH 7.62 and 18.3 % at pH 7.42 is a clear

evidence of such negative effects. These observations corroborated other studies for some

temperate abalone and shelled molluscs (Table 2). In the southern California red abalone

H. rufescens, only 40 % of the juveniles survived after 15 days of exposure to pH 7.50

(Kim et al. 2013). Significantly, higher mortalities were also reported for two species of

abalone from Australia after 68 days at pH 6.79 (for H. laevigata) and pH 7.76 and 6.79

(for H. rubra) (Harris et al. 1999). Considering that the pH of the open sea (upper 100 m)

varies between 7.9 and 8.3 (Andersen et al. 2013), the use of pH 6.79 (ca. -1.3 units

relative to the ambient value) for these two species is very much lower than that resulting

from the worst-case scenario (pH decrease of 0.5 units) expected by the end of the present

century. Such pH reduction (-1.3 units) can be considered highly hypothetical and most

likely far from reality even after the next century. In a short-term experiment conducted by

Crim et al. (2011), the survival of the northern abalone (H. kamtschatkana) larvae was

Aquacult Int

123

Tab

le2

Eff

ects

of

redu

ced

pH

/ele

vat

edpC

O2

assi

ng

lest

ress

or

on

the

gro

wth

and

surv

ival

of

som

ete

mp

erat

eab

alo

ne

and

oth

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elle

dm

oll

usc

s

Sp

ecie

sS

tag

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fd

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op

men

tp

H/p

CO

2w

ith

neg

ativ

eef

fect

s;ex

po

sure

tim

eN

egat

ive

effe

cts

Ref

eren

ces

Red

abal

on

e,H

ali

oti

sru

fesc

ens

Juven

ile

stag

epH

7.5

0;

24

hre

pea

ted

twic

eover

15

day

sJu

ven

iles

exp

erie

nce

d6

0%

mort

alit

y;

gro

wth

rate

sw

ere

sign

ifica

ntl

ylo

wer

than

that

of

con

trols

Kim

etal

.(2

01

3)

Gre

enli

pab

alo

ne,

Ha

lio

tis

laev

iga

taJu

ven

ile

stag

ep

H7

.76

–6

.79

;6

8d

ays

Mo

rtal

ity

was

sig

nifi

can

tly

hig

her

atp

H6

.79;

gro

wth

was

sig

nifi

can

tly

red

uce

d—

no

shel

lg

row

that

pH

\6

.90

Har

ris

etal

.(1

99

9)

Bla

ckli

pab

alone,

Ha

lio

tis

rub

raJu

ven

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stag

ep

H7

.76

–6

.79

;6

8d

ays

Mo

rtal

ity

was

sig

nifi

can

tly

hig

her

atp

H7

.76

and

6.7

9;

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wth

Jan

sson

etal

.(2

01

3)

Aquacult Int

123

significantly reduced by 40 % after 8 days of exposure to elevated pCO2 of 800 ppm

(Table 2).

A correlation between larval survival and pH levels has also been reported for other

shell-forming molluscs. In the Pacific oyster Crassostrea gigas, a small reduction in larval

survival by 7.3 % was observed after 3 days of exposure to pH 7.66 (Timmins-Schiffman

et al. 2012). In our experiment, survival of H. asinina was reduced by 39 % when the

postlarvae were exposed to a similar pH level (7.62) relative to the ambient conditions (pH

7.99). Andersen et al. (2013) reported a significant reduction in larval survival of the Great

scallop (Pecten maximus) from 45 % to ca. 20 and 12 % on day 7 post fertilization when

the ambient pH (7.98) was reduced to pH 7.78 and pH 7.51, respectively (Table 2). For

H. asinina, we obtained 47.2 % postlarval survival at pH 7.62 and 18.3 % at pH 7.42 after

20 days of exposure. These variations in survival rates among shelled molluscs indicate

inter-species differences at certain life stages and exposure period, and some species may

be robust to the perturbations related to seawater chemistry changes.

Growth

Our results show that decreases in pH due to increased CO2 concentration negatively

affected the growth of H. asinina postlarvae. The shell growth of the postlarvae was

reduced by 28 % at pH 7.62 and 20 % at pH 7.42 relative to ambient conditions. In the

New Zealand black-foot abalone (H. iris), the relative growth of the postlarvae was 50 %

lower than that of ambient conditions after 100 days of exposure to pH 7.60 (Cunningham

2013) (Table 2). In the Green-lip abalone (H. laevigata) and Black-lip abalone (H. rubra)

from Australia, juvenile growth rates of both species were significantly depressed at lower

pH levels, and no shell growth was observed after 68 days of exposure to a very low pH of

\6.99 (Harris et al. 1999). In the red abalone (H. rufescens), juvenile shell growth was

significantly reduced when exposed to low pH (Kim et al. 2013). With these negative

effects on growth and in the context of natural scenario, a reduction in seawater pH can be

considered as an important factor influencing the maintenance of abalone populations. This

is so because reproductive success and susceptibility to predation of a species is influenced

by its growth (Rossetto et al. 2012).

Reduced shell growth under reduced pH conditions was observed also in some species

of shell-forming molluscs. The average shell growth of the Baltic Sea bivalve, Macoma

balthica, larvae was 25 % lower than that of the control treatment after 20 days at reduced

pH of 7.72 (Jansson et al. 2013). A mussel species, Mytilus galloprovincialis, displayed

20 % decrease in shell length of larvae when the ambient pH (8.21) was reduced to pH

7.43 over a period of 6 days (Kurihara et al. 2008). In the Pacific oyster C. gigas, the shell

length of larvae became smaller by 20 % after the 2-day exposure to pH 7.4 (Kurihara et al.

2007). These observations strongly corroborate our results under closely similar pH levels

and CO2 concentrations (Table 1; Fig. 2).

Slow growth of H. asinina postlarvae at reduced pH may be attributed to the effect of

pH stressor on the development of larval shell structure. Shell deformity and dissolution

was observed in postlarvae exposed to pH 7.42 during the experiment (Fig. 4). This

condition was observed also in H. rufescens larvae that showed decreased shell length after

exposure to the low pH treatment, suggesting that low pH may have caused the dissolution

of larval shells (Kim et al. 2013). The decrease in growth rates as manifested in the

abnormal shell development of larvae due to reduced pH had been observed also in the

great scallop, P. maximus, larvae (Andersen et al. 2013). Normally shaped shells decreased

from 64 % at ambient pH (7.98) to 28 % when the larvae were exposed to pH 7.51 for

Aquacult Int

123

1 week. In the present study, we observed severe dissolution of the prismatic (outer/

epidermal) layer of the postlarval shell exposed to pH 7.42; only the shell edge was visible

(Fig. 4). Although they were still alive, they appeared weak at the time of observation.

These naked larvae will eventually succumb to death over time. In nature, this morpho-

logical abnormality potentially threatens the survival of abalone populations because naked

larvae are most likely susceptible to predation. Hence, the negative effect of reduced pH on

larval survival and growth of H. asinina is alarming to the livelihood of the people

dependent on the haliotid fishery in southern Philippines. In our previous experiment,

larvae that experienced slow or reduced growth would most likely face the risk of delayed

settlement and high predation as they remain part of the plankton community over an

extended period.

In conclusion, our results provide concrete evidence that the severe effects of reduced

pH levels on abalone larvae are seemingly undisputable. For calcifying organisms, whose

populations were already decimated due to other factors (i.e., overfishing), changes in

Fig. 4 Morphological conditions of H. asinina postlarvae after the 20-day exposure to reduced pH levels.a Normal postlarva at pH 7.99 (Ambient). b Postlarva exposed to T-I (pH 7.81) with deformed shell shape.c Postlarva from T-II (7.62) with highly deformed shell, the prismatic (epidermal) layer started to dissolve.(D) Postlarva reared at T-III (pH 7.42) became shell less [prismatic (outer/epidermal) layer was totallydissolved), only the shell edge (se) is visible (the larva was still alive at the time of sampling). Scale bar100 lm

Aquacult Int

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ocean carbonate chemistry and in particular reduction in surface water pH may pose severe

impact on their existence. The reduced pH-mediated decline in larval survival and growth

that we observed in our study suggested that highly commercial and hence heavily

exploited species, such as abalone, may succumb to higher rates of natural mortality. More

investigations between larval growth and survival of abalones and seawater pH or CO2

concentration should be done across the region as there are possibly other haliotid species

which could possibly have higher tolerance to environmental stressors and could serve as

alternate candidate for aquaculture. Together with fishing mortality, it may further threaten

the existence of the natural populations. Since OA is seemingly irreversible and may

continue to pose detrimental conditions, long-term studies across biogeographical regions

are needed to elucidate the negative effects of OA on the early life history of both tem-

perate and tropical species of abalone. Such studies should involve investigation on the

additive effects of pH together with other environmental parameters (i.e., temperature,

salinity, dissolved oxygen) associated with OA.

Acknowledgments The authors thank the two anonymous reviewers for their critical review and helpfulsuggestions. We also thank the management of the Tawi–Tawi Multi-Species Hatchery, Latu–Latu, Bongao,Tawi–Tawi, Philippines, for allowing the authors to conduct the experiment using their facilities andmanpower. Due recognition and gratitude are afforded to Ms. Arlyn C. Carroz, Hatchery Manager; Ms.Juliet F. Francisco, Senior Hatchery Technician; and our Research Assistants, Mr. Japrin L. Hadji-Amin andMr. Abdurasul S. Sarail for their support and assistance during the study. This paper is a joint marine sciencecontribution of the University of San Carlos and the Mindanao State University Tawi–Tawi, Philippines.

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