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Effects of elevated pCO2 on reproductive properties of the benthic copepod Tigriopus japonicus and gastropod Babylonia japonica
Marine Pollution Bulletin 73 (2013) 402–408
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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Effects of elevated pCO2 on reproductive properties of the benthic copepod Tigriopus japonicus and gastropod Babylonia japonica Jun Kita a,⇑, Takashi Kikkawa b, Takamasa Asai c, Atsushi Ishimatsu c a
Demonstration Laboratory, Marine Ecology Research Institute, Arahama 4-7-17, Kashiwazaki-shi, Niigata 945-0077, Japan Central Laboratory, Marine Ecology Research Institute, Iwawada 300, Onjuku-machi, Isumi-gun, Chiba 299-5105, Japan c Institute for East China Sea Research, Nagasaki University, Taira-machi 1551-7, Nagasaki 851-2213, Japan b
a r t i c l e Keywords: CCS Elevated pCO2 Tigriopus japonicus Babylonia japonica Risk assessment Ocean acidification
i n f o
a b s t r a c t We investigated the effects of elevated pCO2 in seawater both on the acute mortality and the reproductive properties of the benthic copepod Tigriopus japonicus and gastropod Babylonia japonica with the purpose of accumulating basic data for assessing potential environmental impacts of sub-sea geological storage of anthropogenic CO2 in Japan. Acute tests showed that nauplii of T. japonicus have a high tolerance to elevated pCO2 environments. Full life cycle tests on T. japonicus indicated NOEC = 5800 latm and LOEC = 37,000 latm. Adult B. japonica showed remarkable resistance to elevated pCO2 in the acute tests. Embryonic development of B. japonica showed a NOEC = 1500 latm and LOEC = 5400 latm. T. japonicus showed high resistance to elevated pCO2 throughout the life cycle and B. japonica are rather sensitive during the veliger stage when they started to form their shells. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Offshore carbon dioxide capture and storage (CCS), i.e. geological storage of anthropogenic carbon dioxide under the seabed, is a prospective component of the emission mitigation strategy in some countries including Japan. However, it has been pointed out that there may be a potential risk of CO2 leakage into the marine environment, even if it is extremely unlikely (IPCC, 2005). Leaked CO2 may impact marine organisms near the leakage area, especially on benthic organisms. Recent attention on the issue of ocean acidification has led to an accumulation of data dealing with the effects of elevated pCO2 seawater on marine organisms (e.g., Royal Society, 2005; Orr et al., 2005; Kleypas et al., 2006; Gattuso et al., 2008). One of the ultimate objectives of these studies is to predict ecosystem level consequences resulting from elevated seawater CO2. We investigated the effects of elevated partial pressure of CO2 (pCO2) in seawater on the reproductive properties of the benthic copepod Tigriopus japonicus and gastropod Babylonia japonica with the purpose of accumulating basic data needed to consider ways to assess potential environmental impacts of offshore CCS in Japan. Experimental species were chosen because both species are common in coastal areas of the temperate zone of Japan and rearing and breeding techniques are well established for both species for ⇑ Corresponding author. Present address: CO2 Storage Research Group, Research Institute of Innovative Technology for the Earth, Kizugawadai 9-2, Kizu-shi, Kyoto 619-0292, Japan. Tel.: +81 774 75 2312; fax: +81 774 75 2313. E-mail address: [email protected] (J. Kita). 0025-326X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marpolbul.2013.06.026
the purpose of toxicological studies (e.g., Horiguchi et al., 1995; Raisuddin et al., 2007). T. japonicus is a copepod species which is commonly found in tide pools in the upper intertidal zone. Their developmental stages consist of six nauplius stages and five copepodite stages and then they reach the adult stage. They are an important grazer and omnivorous species, as well as a source of food for several marine and estuarine invertebrates and fish larvae. B. japonica is a gastropod species which inhabits sandy or muddy bottoms in shallow waters in the temperate zone of Japan. Their spawning season is in early summer and they lay successive egg capsules on solid substratum. Embryos develop in the capsule and veligers hatch from the capsule. B. japonica are scavengers in the inshore ecosystem, as well as an important local fisheries resource in Japan. 2. Materials and methods 2.1. Experiments on Tigriopus japonicus Continuously-cultivated T. japonicus at the Niigata laboratory of the Marine Ecology Research Institute (MERI) were used for the experiments. The cultivation containers were placed in an incubator which was set to 20 °C and 12 h of light and dark period. T. japonicus were fed 105 cells/mL of Tetraselmis tetrathela every 2 weeks. 2.1.1. Acute mortality tests on newly hatched nauplii of T. japonicus T. japonicus with eyed eggs, i.e. developmental stage just before hatching, were collected from the cultivation stocks. The egg
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capsule was separated from the body under a stereomicroscope and placed in a small Petri dish filled with filtered seawater of 20 °C. Thirty egg capsules were collected and the Petri dish was placed in an incubator set to 20 °C. Newly hatched nauplii within 4 h after hatching in the Petri dish were used for the tests. Exposure tanks of constant pCO2 seawater were placed in a constant temperature (20 °C) water tank. Seawater CO2 levels in the exposure tank were maintained by continuously bubbling the seawater with atmospheric air and mixture gases containing CO2 (five levels of predefined %), O2 (21%) and N2 (residual part) using gas mixture devices (KOFLOC GB-3C). Experimental nauplii were transferred to a glass vessel of 20 mL volume, which have been placed in the exposure tank, and the vessel was capped with a silicone rubber stopper. The vessel was then placed in the exposure tank. Twenty-five individuals of nauplii were introduced to a vessel. Five vessels containing nauplii and a small magnetic stirring bar were placed in each exposure tank. Three vessels were used for determination of nauplii activity and two vessels were used for measuring pH of the seawater in the vessels at the end of the exposure. After 24 h of exposure the nauplii in the vessel were transferred to a watch glass with a small amount of associated seawater and the number of mobile, immobile and dead individuals were counted using a stereomicroscope. Immobile state was determined as nauplii that could not move around even if it could move its appendages during 15 s after the seawater in a watch glass was gently stirred. Nauplii were assessed as dead if no movement was observed inside the body. Table 1 indicates experimental conditions.
2.1.2. Full life cycle tests on T. japonicus T. japonicus with eyed eggs were collected from the cultivation stocks. Thirty egg capsules were separated from the body and incubated in filtered seawater at 23 °C. Newly hatched nauplii within 24 h after hatching were used for the tests. The rearing container of the newly hatched nauplii was placed in an air tight box in which CO2 level was maintained by a continuous supply of atmospheric air or mixture gas (three levels of predefined CO2 %, as described above). To prevent evaporation of the rearing seawater, a beaker filled with seawater was placed in the air tight box and the mixture gas supplied to the air tight box was bubbled into the beaker (Fig. 1). The seawater in the beaker that reached a stable equilibrium with the mixture gas was used for exchange of the rearing seawater every 3 days. The air tight boxes were placed in an incubator set to 23 °C. Rearing seawater of 10 mL, mixture of 9 mL seawater and 1 mL seawater containing 106 cells of T. tetrathela, was filled into each well of a 6-well plate. The 6-well plate was then placed in the air tight box one day before the start of the experiment to facilitate the given pCO2 of the rearing seawater. On the starting day of the experiment, 20 newly hatched nauplii were introduced to each
Gas mixture device Air tight box pH electrode T. japonicus 6-well plate
Fig. 1. Schematic illustration of the experimental apparatus used for the full life cycle test on T. japonicus. Arrows indicate directions of gas flow.
well of the 6-well plates. Observations to assess survival, development and behavior under a stereomicroscope and pH measurement of rearing sweater were carried out daily. When a female with an egg capsule was appeared in the 6-well plate it was transferred individually to a well of a 12-well plate. A total of 10 incubating females were placed in the 12-well plate and reared until their eggs became eyed eggs. Then the egg capsule was separated from the body and placed in a well of another 12-well plate to prevent cannibalization of hatched nauplii by the adult. After removing 10 female, all the remaining individuals in the 6-well plate were grouped and reared in a Petri dish. The number of females and males of adults in the Petri dish were counted and the number of newly hatched nauplii and of undeveloped eggs in the 12-well plate was counted under a stereomicroscope after completion of the hatching. Table 2 indicates experimental conditions. Endpoints for the effect were determined as follows: First copepodid, i.e. days needed until first appearance of a copepodid after the exposure. First mating, i.e. days needed until first appearance of a mating pair after the exposure. First spawning, i.e. days needed until first appearance of an incubating female after the exposure. Hatching success, i.e. rate of hatching success of the first spawned egg capsule. Sex ratio, i.e. morphologically distinguished sex ratio at the end of the experiment. 2.2. Experiments on Babylonia japonica Freshly-caught B. japonica were purchased from fishermen’s cooperatives in Niigata near to the MERI laboratory. Males and females were kept in separate stock tanks until the start of the experiments. They were fed moist pellets, feed mixture for carnivorous aquacultured fish, once a day.
Table 1 Conditions of the test seawater for acute mortality test on T. japonicus. Exposure group
A (control) B C D E (control) F G
Measured
Calculated
Temp (°C)
Sal (psu)
pH
Alk (lmol/kg)
pCO2 (latm)
XCa
XAr
19.7 19.7 19.7 19.7 19.6 19.7 19.7
32.20
8.04 6.26 6.04 5.91 8.04 5.89 5.74
2158
550 39,000 65,000 88,000 560 92,000 130,000
3.17 0.06 0.04 0.03 3.16 0.03 0.02
2.05 0.04 0.02 0.02 2.04 0.02 0.01
33.00
2201
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Table 2 Conditions of the full life cycle tests on T. japonicus. Exposure group
A (control) B C D
Measured
Calculated
Temp (°C) Mean ± SD N=7
Sal (psu) Mean ± SD N=4
pH Mean ± SD N=7
Alk (lmol/kg) Mean ± SD N=4
pCO2 (latm)
XCa
XAr
22.9 ± 0.6 22.8 ± 0.4 22.8 ± 0.4 23.0 ± 0.5
33.05 ± 0.18
8.16 ± 0.13 7.11 ± 0.03 6.31 ± 0.04 5.85 ± 0.03
2251 ± 13
430 5800 37,000 110,000
4.43 0.48 0.08 0.03
2.89 0.32 0.05 0.02
2.2.1. Acute mortality tests on B. japonica Experimental setup was the same as used in the acute mortality tests on T. japonicus. Exposure tanks were set at 20 °C. Ten adult B. japonica were randomly selected from the stock tanks that were kept at 20 °C for more than 2 weeks and were placed in the exposure tank for 96 h. Seawater in each exposure tank was replaced daily with fresh seawater previously adjusted to the intended CO2 level. Conditions of B. japonica were checked twice a day. If there was no movement such as contraction of muscle of soft body when stimulated with tweezers, the specimen was defined as dead. Shell length and wet weight of test specimens were measured at the end of exposure or immediately after death. Mean wet weight (mean ± SD, and below) and mean shell height of experimental specimens were 30.4 ± 3.4 g and 58.3 ± 2.3 mm, respectively. Table 3 indicates experimental conditions.
Main tank (200 L)
13 pairs
Gas mixture device Seawater Sub tank (90 L) Pump
Cooling unit
Heater 2.2.2. Effects on reproductive properties of B. japonica 2.2.2.1. Effects on spawning properties. Experimental setup is shown in Fig. 2. Four sets of the apparatus were used, i.e. one for control and one each for the three different levels of elevated pCO2. Thirteen pairs of female and male B. japonica were randomly picked up from the stock tanks and placed in the main tank. Each pair was housed in a 15 cm3 cage in the main tank. Mean wet weight and mean shell height of the experimental specimens were 27.8 ± 4.1 g and 59.4 ± 3.7 mm respectively. To approximate the natural conditions at that time, temperature of the test seawater at the start of the experiment was set at 16 °C. The temperature was then gradually increased to 20 °C and kept at 20 °C for 1 month, subsequently increased to 22 °C and kept at 22 °C until end of the experiment. Experimental duration, from the start to the end of the experiment, was 80 days (Fig. 3). B. japonica were fed moist pellets twice a day. Spawning of individual B. japonica was recorded every day for number of spawned egg capsules and number of embryos in a capsule for 30 days after the first spawning. After the experiment, all the experimental specimens were boiled and the soft bodies were removed from the shell. These shells were dried and the shell surfaces were observed. Table 4 indicates experimental conditions. 2.2.2.2. Effects on embryonic development. Egg capsules spawned in the experiment above, which were spawned during 31–46 days after the first spawning and were within 12 h after the spawning,
Fig. 2. Schematic illustration of the experimental apparatus used for the reproductive properties test on B. japonica. Arrows indicate directions of water flow.
were used in this experiment. Egg capsules were carefully detached from their bonding surface and approximately 15 egg capsules were immediately placed in a Petri dish (9 cm in diameter and 6 cm in height) with seawater of 22 °C. The Petri dishes were placed in a water bath of 22 °C, i.e. natural seawater incubation group. Simultaneously, approximately 15 egg capsules were placed in a small cylindrical tube both sides capped with mesh fabrics and the tube was placed in the main tank in which the egg capsules were spawned, i.e. controlled CO2 level seawater incubation group. When putrid or dead embryos were found in an egg capsule, those egg capsules were removed from the Petri dish or the tube. The days needed for all veligers hatched out from the egg capsule was recorded. The number of undeveloped embryos was counted under a stereomicroscope after all the veligers had hatched out from the egg capsule. A part of the newly hatched veligers were fixed in 70% ethanol immediately after their hatching for observing their shell morphology. Fixed samples were then dehydrated by 99.5% ethanol and dried in a desiccator. They were coated with platinum using an auto fine coater (JEOL, JFC-1600) and observed using a scanning electron microscope (JEOL, JSM-6380LAK II). Rearing seawater of natural seawater incubation group was replaced every morning.
Table 3 Conditions of the test seawater for acute mortality test on B. japonica. Exposure group
A (control) B C D
Measured
Calculated
Temp (°C) Mean ± SD N=9
Sal (psu)
pH Mean ± SD N=9
Alk (lmol/kg)
pCO2 (latm)
XCa
XAr
19.8 ± 0.0 19.8 ± 0.0 19.8 ± 0.0 19.8 ± 0.1
33.58
8.09 ± 0.02 6.04 ± 0.07 5.89 ± 0.05 5.69 ± 0.03
2273
500 67,000 95,000 150,000
3.65 0.04 0.03 0.02
2.37 0.03 0.02 0.01
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(survival = 93%) was not significantly different from that of the control group (survival = 96%) (extended Fisher’s exact test, P < 0.05; Freeman and Halton, 1951).
8.50
8.00
3.2. Full life cycle tests on T. japonicus
pH
7.50
Delay of growth rates, those indicated by days needed for first appearance of a copepodid, a mating pair and incubating female after the exposure, were clearly indicated at pCO2 = 37,000 latm but at pCO2 = 5800 latm (Table 6). Hatching success of the first spawned egg at pCO2 = 37,000 latm decrease considerably and significantly different from that of the control group (Dunnett’s method, P < 0.05). Undeveloped eggs were frequently observed in the spawned egg capsule at pCO2 = 37,000 latm (Fig. 4). No growth and dead nauplii were observed at pCO2 = 110,000 latm. There were no significant difference in sex ratios at end of the exposure among control, pCO2 = 5800 latm and pCO2 = 37,000 latm groups (extended Fisher’s exact test, P < 0.05). These results indicated that NOEC (No Observed Effect Concentration) = 5800 latm and LOEC (Lowest Observed Effect Concentration) = 37,000 latm.
7.00
6.50
6.00 24.0 23.0
Temperature
22.0
Jun./2, First spawning
21.0 20.0 19.0 18.0 A (Control), 470 µatm B, 1,600 µatm C, 5,500 µatm D, 16,000 µatm
17.0 16.0 15.0 14.0 May/18
Jun./1
Jun./15
Jun./29
Jul./13
Jul./27
3.3. Acute mortality tests on B. japonica
Aug./10
Month / date Fig. 3. Temperature and pH changes during the exposure experiment on reproductive properties of B. japonica.
All adult B. japonica survived in the control (pCO2 = 500 latm), pCO2 = 67,000 latm and 95,000 latm, whereas all test specimens of pCO2 = 150,000 latm died before 72 h. Although the soft body of the test specimens at pCO2 = 95,000 latm was not completely covered by their shells and showed no spontaneous movement during the exposure, B. japonica showed normal movement and even feeding behavior if they were returned to normal seawater after the exposure.
Table 5 indicates experimental conditions. 3.4. Effects on reproductive properties of B. japonica 2.3. Test seawater quality Temperature and pH of the test seawater was measured daily using a conventional pH meter (METTLER TOLEDO, SG8-ELK with pH probe InLab413SG). Salinity and alkalinity were measured weekly using an inductively coupled salinometer (YEO-KAL, MODEL 601MK1V) and by titration method respectively for the supplied seawater to the tests. The test seawater pCO2 and CaCO3 saturation state, calcite (XCa) and aragonite (XAr), were calculated from the measured values of temperature, pH, salinity and alkalinity using the program CO2SYS (Pierrot et al., 2006) and by using the dissociation constants from Mehrbach et al. (1973) refitted by Dickson and Millero (1987) and for K2SO4 from Dickson (1990). 3. Results 3.1. Acute mortality tests on newly hatched nauplii of T. japonicus Newly hatched nauplii of T. japonicus showed remarkably high tolerance to elevated pCO2 for 24 h up to 130,000 latm (pH = 5.74). The survival of the nauplii or appearance ratio of the mobile nauplii at the highest exposure of pCO2 = 130,000 latm
3.4.1. Effects on spawning properties All the test specimens showed active feeding behavior and survived the exposure. When the temperature of the test seawater reached 20 °C, spawning started in all experimental groups and continued until the end of the experiment, i.e. 65 days after the start of spawning (Fig. 3). Results of spawning during 30 days after the first spawning are indicated in Table 7. There were no significant differences between all exposure groups for all spawning properties (extended Fisher’s exact test on spawning events and egg capsules and Dunnett’s method, P < 0.05, on spawned times and embryos). These results indicated that the elevated pCO2 up to 16,000 latm did not affect the spawning of B. japonica. Appearances of shells after 80 days of the exposure are shown in Fig. 5. Small spirorbid specimens, specific name undetermined, were found only on the shells of control group. Appearances of shells of pCO2 = 1600 latm group were almost the same as control group but there were no spirorbid specimens. Parts of the periostracum peeled from the shells of pCO2 = 1600 latm group and the shells were severely affected in pCO2 = 16,000 group. These results indicated that longer exposure, i.e. 80 days in this case, to an elevated pCO2 of more than 1600 latm or CaCO3 saturation states
Table 4 Conditions of the test seawater for the spawning property test of B. japonica. Exposure group
A (control) B C D
Measured
Calculated
Temp (°C) Mean ± SD N = 119
Sal (psu) Mean ± SD N=9
pH Mean ± SD N = 119
Alk (lmol/kg) Mean ± SD N=9
pCO2 (latm)
XCa
XAr
20.0 ± 1.5 20.0 ± 1.8 20.0 ± 1.5 19.9 ± 1.4
33.44 ± 0.35
8.11 ± 0.04 7.64 ± 0.07 7.12 ± 0.03 6.66 ± 0.05
2261 ± 23
470 1600 5500 16,000
3.79 1.45 0.46 0.16
2.46 0.94 0.30 0.10
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Table 5 Conditions of the test seawater for the embryonic development test of B. japonica. Exposure group
Natural SW A (control) B C D
Measured
Calculated
Temp (°C) Mean±SD N = 48
Sal (psu) Mean±SD N=5
pH Mean±SD N = 48
Alk (lmol/kg) Mean±SD N=5
pCO2 (latm)
XCa
XAr
22.1 ± 0.1 21.9 ± 0.2 22.1 ± .01 21.9 ± 0.1 21.7 ± 0.1
33.15 ± 0.36 33.22 ± 0.40
7.98 ± 0.06 8.08 ± 0.02 7.66 ± 0.04 7.13 ± 0.03 6.69 ± 0.03
2232 ± 17 2241 ± 24
670 520 1500 5400 15,000
3.07 3.73 1.59 0.49 0.18
2.00 2.43 1.04 0.32 0.12
Table 6 Results of the full life cycle tests on T. japonicus. Exposure Group (pCO2)
First copepodid (days)a
First mating (days)b
First spawning (days)c
A (control, 430 latm) B (5800 latm) C (37,000 latm) D (110,000 latm)
6 12 14 6 12 14 9 15 20 No specimens molted to nauplius stage II (Dahms et al., 2007) Dead specimens were observed after 6 days of the exposure All specimens died after 21 days of the exposure
Hatching success (%)d Mean ± SD N = 10
Sex ratio (male %)e
99.2 ± 1.3 97.2 ± 6.2 11.3 ± 21.7*
0.66 0.75 0.75
a
Days needed for first appearance of a copepodid after the exposure. Days needed for first appearance of a mating pair after the exposure. c Days needed for first appearance of an incubating female after the exposure. d Rate of hatching success of the first spawned egg capsule. e Morphologically distinguished sex ratio at end of the exposure. Significantly different from the control, Dunnett’s method, P < 0.05. b
*
Control, 430µatm
37,000µatm
Fig. 4. Egg capsule of T. japonicus under control condition (left) and pCO2 = 37,000 latm (right). All eggs show normal development in the control while undeveloped eggs, i.e. transparent and green eggs, were observed at 37,000 latm.
Table 7 Results of the spawninga property test of B. japonica.
a b c d e
Exposure group (pCO2)
Spawning eventsb
Spawned timesc Mean ± SD
Egg capsulesd
Embryose Mean ± SD
A (control, 490 latm) B (1600 latm) C (5500 latm) D (16,000 latm)
39 38 56 44
3.0 ± 2.5 2.9 ± 1.9 4.3 ± 3.3 3.4 ± 2.4
1186 1146 1529 1074
32 ± 12 (N = 36) 25 ± 7 (N = 37) 30 ± 9 (N = 54) 24 ± 10 (N = 43)
(N = 10) (N = 10) (N = 10) (N = 10)
Observed for 30 days after the first spawning. Total number of spawning events. Spawning times per spawned females. Total number of spawned egg capsules. Number of embryos per egg capsules.
under ca. 1 (XCa < 1.45, XAr < 0.94) affected the maintenance of periostracum of B. japonica. Additionally, it was indicated that spiororbids are sensitive to elevated pCO2 and pCO2 = 1600 latm was critical for their survival. 3.4.2. Effects on embryonic development Results of the embryonic development in natural seawater and elevated pCO2 conditions are shown in Table 8. There were no significant differences (Dunnett’s method, P < 0.05) among the natural
seawater incubation groups for embryonic development properties, days from spawning to complete hatching of veligers and number of undeveloped embryos per capsule after completion of the hatching. This indicated that adult B. japonica could produce viable eggs under an elevated pCO2 up to 16,000 latm. In the controlled CO2 level incubation group, on the other hand, the number of undeveloped embryos in pCO2 = 5400 latm incubation group (XCa = 0.49 and XAr = 0.32) was significantly larger than that in control group (pCO2 = 520 latm) (Dunnett’s method, P < 0.05).
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Control, 470µatm
1,600µatm
5,500µatm
16,000µatm
Fig. 5. Shells of B. japonica after 80 days of the exposure. Spirorbid (small inserted picture, upper left) were only found on the shells of the control group.
Table 8 Results of the embryonic development test of B. japonica.
*
Exposure group (pCO2)
Incubation (pCO2)
A (control, 470 latm) B (1600 latm) C (5500 latm) D (16,000 latm)
Natural Natural Natural Natural
A (control, 470 latm) B (1600 latm) C (5500 latm) D (16,000 latm)
A (control, 520 latm) B(1500 latm) C (5400 latm) D (15,000 latm)
SW SW SW SW
(670 (670 (670 (670
latm) latm) latm) latm)
Days from spawning to complete hatching
Undeveloped embryos/capsule Mean ± SD
19, 19, 19, 19,
1.47 ± 0.40 1.40 ± 0.45 1.67 ± 0.25 1.17 ± 0.66
20, 20, 19, 20,
20 20 19 20
18, 19, 20, 20 20, 20, 21, 22 19, 20, 20, 22 No hatching
(N = 36) (N = 50) (N = 40) (N = 35)
0.65 ± 0.26 (N = 52) 1.38 ± 0.70 (N = 58) 2.93 ± 1.44 (N = 63)*
Significantly different from the control, Dunnett’s method, P < 0.05.
Embryos in pCO2 = 15,000 latm incubation group did not show normal development and the embryos could not reach veliger stage within 23 days after spawning. These results indicated that development of embryos and/or shell formation of veligers became diminished under the elevated pCO2 of more than pCO2 = 1500 latm or CaCO3 saturation states under ca. 1 (XCa < 1.59, XAr < 1.04), and halted under more than pCO2 = 15,000 latm. There were no marked differences among shell appearances of newly hatched veligers of elevated pCO2 incubation group (control, pCO2 = 1500 latm, pCO2 = 5400 latm) under observation by scanning electron microscope. It indicated that if veligers successfully started shell formation, they could form normal shells as same as the control group. From the above, it was indicated that for embryonic development of B. japonica NOEC = 1500 latm and LOEC = 5400 latm. 4. Discussion Acute mortality tests on T. japonicus indicated that newly hatched nauplii were tolerant to pCO2 = 130,000 latm for 24 h. Harpacticoida, in which T. japonicus is included, inhabit fresh, brackish and seawater and many of them live in seawater habitats. Range of distribution of harpacticoids in the sea is diverse, i.e. from sandy beaches and tide pools to benthic environments of the deep sea of thousand meters depth. Many of these habitats show severe and unique environmental fluctuations. Thus tolerances to elevated pCO2 of harpacticoids differ from species to species (Pascal et al., 2010; Thistle et al., 2006). High tolerance to elevated pCO2 described for newly hatched nauplii of T. japonicus may be explained by their adaptability to their habitats such as tide pool and sea bottom where CO2 concentration often becomes high due to respiration of organisms. For the planktonic copepod, it was reported that LC50 of 24 h for elevated pCO2 seawater ranges
between c.a. 20,000 and 110,000 ppm for epipelagic species at subarctic, epipelagic and mesopelagic species at subarctic and Paracalanus parvus, Tisbe gracilis and Stephos sp. (Watanabe et al., 2006). It can be concluded that nauplii of T. japnicus have a higher acute tolerance to elevated pCO2 compared to that of planktonic copepods. Full life cycle tests indicated that growth rate and hatching success of T. japonicus were not affected at pCO2 = 5800 latm but at pCO2 = 37,000 latm. It was reported for the planktonic copepod Acartia steueri that there were no significant differences in survival rate among test groups incubated for 8 days under + 2000 latm (pH = 7.40–7.55), +10,000 latm and control (pH = 8.14–8.17, pCO2 = equilibrated with atmospheric air) while their egg production was significantly lower for the +10,000 latm groups compared to the control group (Kurihara et al., 2004). Another planktonic copepod, Acartia tsuensis, was reported that there were no significant difference in egg production and hatching success between elevated pCO2 environment of pCO2 = 2380 latm and control of pCO2 = 380 latm when they were incubated for three generations (Kurihara and Ishimatsu, 2008). It can be concluded that although T. japonicus might be more tolerant to elevated pCO2 environment for longer exposures compared to planktonic copepods, both benthic and planktonic copepods would be tolerant to the pCO2 of several tens of thousands latm. Saturation state of CaCO3 at pCO2 = 5800 latm and pCO2 = 37,000 latm , NOEC and LOEC of T. japonicus respectively, were under ca. 0.5 and 0.01 respectively (Table 2). This suggests that under saturation of CaCO3 did not have a strong impact on T. japonicus compared to that of B. japonica described below. Acute mortality tests on B. japonica indicated that adults were tolerant to pCO2 = 95,000 latm for 96 h. B. japonica is a bottom dweller and a scavenger. They sometimes penetrate into the body of dead animals where O2 concentration would be at a minimum level and CO2 concentrations are expected to be high. Their habitat
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and feeding habits thus might select for tolerance to elevated pCO2 environments. Although shells of adult B. japonica were damaged under elevated pCO2 of more than 1600 latm or less than CaCO3 saturations of ca. 1 for 80 days exposure (Fig. 5), their damage would be more severe for longer exposure and thus their growth and survival might be affected. Gazeau et al. (2007) reported for bivalves that in Mytilus edulis shell dissolution was observed at pCO2 P 1800 ppm and the calcification rate showed a 25% decrease at pCO2 = 740 ppm and in Crassostrea gigas calcification rate showed a 10% decrease at pCO2 = 740 ppm. Kimura et al. (2011) reported for the gastropod in Haliotis discus that there were no effects on early development under exposure to pCO2 < 1100 latm but increasing deteriorations of early developments were observed with increased exposure to pCO2 = 1650 and 2150 latm. Shells of newly hatched veligers of B. japonica showed a normal appearance even at pCO2 = 5400 latm. It can be concluded that shell formation rates and dissolution rates under elevated pCO2 environment are quite different among species. However, mollusks with calcium carbonate shells such as B. japonica might be more sensitive to elevated pCO2 environment compared to marine organisms without shells (Fabry et al., 2008). It is expected that CaCO3 saturation state of less than 1 would be critical for calcification of many mollusks with calcium carbonate shells. Data on tolerances to elevated pCO2 of marine organisms have been accumulated because of the urgent need for research on ocean acidification due to anthropogenic CO2 (e.g., Gooding et al., 2009; Ries et al., 2009). Those studies showed that many species were susceptible to elevated pCO2, but some species such as T. japnicus and B. japonica were significantly tolerant than others. The differences in tolerance among species seem to involve their different physiological compensation systems against elevated pCO2 those arose during evolution. As a result, prediction of ecosystem fate under an elevated pCO2 resulting both from potential risks of CCS and ocean acidification needs further tolerance data of each species and reliable ecosystem model. However, ocean acidification is an issue of global scale while likely impact of CCS leakage will be limited to local. It would be useful and realistic to determine safety threshold of pCO2 for a local ecosystem using most susceptible data of local species when assessing potential impact of CO2 leakage by a CCS. Acknowledgement This work was supported by the project of Ministry of Environment, Japan, entitled ‘‘Investigating the Environmental Management System for Offshore CCS, Development of EIA Methodology’’ and was written based on the author’s independent consideration. References Dahms, H.U., Chullasorn, S., Kangtia, P., Ferrari, F.D., Hwang, J.-S., 2007. Naupliar development of Tigriopus japonicus Mori, 1932 (Copepoda: Harpacticidae). Zool. Stud. 46, 746–759.
Dickson, A.G., 1990. Thermodynamics of the dissociation of boric acid in potassium chloride solutions from 273.15 to 318.15 K. J. Chem. Eng. Data 35, 253–257. Dickson, A.G., Millero, F.J., 1987. A comparison of the equilibrium constants for the dissociation of carbonic acid in seawater media. Deep-Sea Res. 34, 1733–1743. Fabry, V.J., Seibel, B.A., Feely, R.A., Orr, J.C., 2008. Impacts of ocean acidification on marine fauna and ecosystem processes. ICES J. Mar. Sci. 65, 414–432. Freeman, G.H., Halton, J.H., 1951. Note on an exact treatment of contingency, goodness of fit and other problems of significance. Biometrika 38, 141–149. Gattuso, J.-P., Orr, J., Pantoja, S., Pörtner, H.-O., Riebesell, U., Trull, T. (Eds.), 2008. Special Issue: The ocean in the high-CO2 world II. Biogeosciences, www.biogeosciences.net/special_issue44.html. Gazeau, F., Quiblier, C., Jansen, J.M., Gattuso, J.-P., Middelburg, J.J., Heip, C.H.R., 2007. Impact of elevated CO2 on shellfish calcification. Geophys. Res. Lett. 34, L07603. http://dx.doi.org/10.1029/2006GL028554. Gooding, R.A., Harley, C.D.G., Tang, E., 2009. Elevated water temperature and carbon dioxide concentration increase the growth of keystone echinoderm. Proc. Natl. Acad. Sci. USA 106, 9316–9321. Horiguchi, T., Shiraishi, H., Shimizu, M., Yamazaki, S., Morita, M., 1995. Imposex in Japanese gastropods (Neogastropoda and Mesogastropoda): effects of tributyltin and triphenyltin from antifouling paints. Mar. Pollut. Bull. 31, 402– 405. IPCC, 2005. IPCC special report on carbon dioxide capture and storage. In: Metz, B., de Coninck, H.C., Davidson, O., Meyer, L.A., Loos, M. (Eds.), Prepared by Working Group III of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge, United Kingdom and New York. Kimura, R., Takami, H., Ono, T., Onitsuka, T., Nojiri, Y., 2011. Effects of elevated pCO2 on the early development of the commercially important gastropod, Ezo abalone Haliotis discus hannai. Fish. Oceanogr. 20 (5), 357–366. Kleypas, J.A., Feely, R.A., Fabry, V.J., Langdon, C., Sabine, C.L., Robbins, L.L., 2006. Impacts of ocean acidification on coral reefs and other marine calcifiers: a guide for future research, In: Report of a Workshop held 18–20 April 2005, St. Petersburg, FL, sponsored by NSF, NOAA, and the U.S. Geological Survey. Kurihara, H., Ishimatsu, A., 2008. Effects of high CO2 seawater on the copepod (Acartia tsuensis) through all life stages and subsequent generations. Mar. Pollut. Bull. 56 (6), 1086–1090. Kurihara, H., Shimode, S., Shirayama, Y., 2004. Sub-lethal effects of elevated concentration of CO2 on planktonic copepods and sea urchins. J. Oceanogr. 60, 743–750. Mehrbach, C., Culberson, C.H., Hawley, J.E., Pytkowicz, R.M., 1973. Measurement of the apparent dissociation constants of carbonic acid in seawater at atmospheric pressure. Limnol. Oceanogr. 18, 897–907. Orr, J.C., Pantoja, S., Pörtner, H.-O., 2005. Introduction to special section: the ocean in a high-CO2 world. J. Geophys. Res.-Oceans 110, C09S01. Pascal, P.-Y., Fleeger, J.W., Galvez, F., Carman, K.R., 2010. The toxicological interaction between ocean acidity and metals in coastal meiobenthic copepods. Mar. Pollut. Bull. 60, 2201–2208. Pierrot, D., Lewis E., Wallace D.W.R., 2006. MS Excel Program Developed for CO2 System Calculations. ORNL/CDIAC-105a. Carbon Dioxide Information Analysis Center, Oak Ridge National Laboratory, U.S. Department of Energy, Oak Ridge, Tennessee. http://dx.doi.org/10.3334/CDIAC/otg.CO2SYS_XLS_CDIAC105a. Raisuddin, S., Kwok, K.W.H., Leung, K.M.Y., Schlenk, D., Lee, J.-S., 2007. The copepod Tigriopus: a promising marine model organism for ecotoxicology and environmental genomics. Aquat. Toxicol. 83, 161–173. Ries, J.B., Cohen, A.L., McCorkle, D.C., 2009. Marine calcifiers exhibit mixed responses to CO2-induced ocean acidification. Geology 37, 1131–1134. Royal Society, 2005. Ocean acidification due to increasing atmospheric carbon dioxide. Policy Document 12/05. The Royal Society, London. Thistle, D., Sedlacek, L., Carman, K.R., Fleeger, J.W., Brewer, P.G., Barry, J.P., 2006. Simulated sequestration of industrial carbon dioxide at a deep-sea site: effects on species of harpacticoid copepods 330, 151–158. Watanabe, Y., Yamaguchi, A., Ishida, H., Harimoto, T., Suzuki, S., Sekido, Y., Ikeda, T., Shirayama, Y., Takahashi, M.M., Ohsumi, T., Ishizaka, J., 2006. Lethality of increasing CO2 levels on deep-sea copepods in the western North Pacific. J. Oceanogr. 62, 185–196.
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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Effects of elevated pCO2 on reproductive properties of the benthic copepod Tigriopus japonicus and gastropod Babylonia japonica Jun Kita a,⇑, Takashi Kikkawa b, Takamasa Asai c, Atsushi Ishimatsu c a
Demonstration Laboratory, Marine Ecology Research Institute, Arahama 4-7-17, Kashiwazaki-shi, Niigata 945-0077, Japan Central Laboratory, Marine Ecology Research Institute, Iwawada 300, Onjuku-machi, Isumi-gun, Chiba 299-5105, Japan c Institute for East China Sea Research, Nagasaki University, Taira-machi 1551-7, Nagasaki 851-2213, Japan b
a r t i c l e Keywords: CCS Elevated pCO2 Tigriopus japonicus Babylonia japonica Risk assessment Ocean acidification
i n f o
a b s t r a c t We investigated the effects of elevated pCO2 in seawater both on the acute mortality and the reproductive properties of the benthic copepod Tigriopus japonicus and gastropod Babylonia japonica with the purpose of accumulating basic data for assessing potential environmental impacts of sub-sea geological storage of anthropogenic CO2 in Japan. Acute tests showed that nauplii of T. japonicus have a high tolerance to elevated pCO2 environments. Full life cycle tests on T. japonicus indicated NOEC = 5800 latm and LOEC = 37,000 latm. Adult B. japonica showed remarkable resistance to elevated pCO2 in the acute tests. Embryonic development of B. japonica showed a NOEC = 1500 latm and LOEC = 5400 latm. T. japonicus showed high resistance to elevated pCO2 throughout the life cycle and B. japonica are rather sensitive during the veliger stage when they started to form their shells. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Offshore carbon dioxide capture and storage (CCS), i.e. geological storage of anthropogenic carbon dioxide under the seabed, is a prospective component of the emission mitigation strategy in some countries including Japan. However, it has been pointed out that there may be a potential risk of CO2 leakage into the marine environment, even if it is extremely unlikely (IPCC, 2005). Leaked CO2 may impact marine organisms near the leakage area, especially on benthic organisms. Recent attention on the issue of ocean acidification has led to an accumulation of data dealing with the effects of elevated pCO2 seawater on marine organisms (e.g., Royal Society, 2005; Orr et al., 2005; Kleypas et al., 2006; Gattuso et al., 2008). One of the ultimate objectives of these studies is to predict ecosystem level consequences resulting from elevated seawater CO2. We investigated the effects of elevated partial pressure of CO2 (pCO2) in seawater on the reproductive properties of the benthic copepod Tigriopus japonicus and gastropod Babylonia japonica with the purpose of accumulating basic data needed to consider ways to assess potential environmental impacts of offshore CCS in Japan. Experimental species were chosen because both species are common in coastal areas of the temperate zone of Japan and rearing and breeding techniques are well established for both species for ⇑ Corresponding author. Present address: CO2 Storage Research Group, Research Institute of Innovative Technology for the Earth, Kizugawadai 9-2, Kizu-shi, Kyoto 619-0292, Japan. Tel.: +81 774 75 2312; fax: +81 774 75 2313. E-mail address: [email protected] (J. Kita). 0025-326X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marpolbul.2013.06.026
the purpose of toxicological studies (e.g., Horiguchi et al., 1995; Raisuddin et al., 2007). T. japonicus is a copepod species which is commonly found in tide pools in the upper intertidal zone. Their developmental stages consist of six nauplius stages and five copepodite stages and then they reach the adult stage. They are an important grazer and omnivorous species, as well as a source of food for several marine and estuarine invertebrates and fish larvae. B. japonica is a gastropod species which inhabits sandy or muddy bottoms in shallow waters in the temperate zone of Japan. Their spawning season is in early summer and they lay successive egg capsules on solid substratum. Embryos develop in the capsule and veligers hatch from the capsule. B. japonica are scavengers in the inshore ecosystem, as well as an important local fisheries resource in Japan. 2. Materials and methods 2.1. Experiments on Tigriopus japonicus Continuously-cultivated T. japonicus at the Niigata laboratory of the Marine Ecology Research Institute (MERI) were used for the experiments. The cultivation containers were placed in an incubator which was set to 20 °C and 12 h of light and dark period. T. japonicus were fed 105 cells/mL of Tetraselmis tetrathela every 2 weeks. 2.1.1. Acute mortality tests on newly hatched nauplii of T. japonicus T. japonicus with eyed eggs, i.e. developmental stage just before hatching, were collected from the cultivation stocks. The egg
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capsule was separated from the body under a stereomicroscope and placed in a small Petri dish filled with filtered seawater of 20 °C. Thirty egg capsules were collected and the Petri dish was placed in an incubator set to 20 °C. Newly hatched nauplii within 4 h after hatching in the Petri dish were used for the tests. Exposure tanks of constant pCO2 seawater were placed in a constant temperature (20 °C) water tank. Seawater CO2 levels in the exposure tank were maintained by continuously bubbling the seawater with atmospheric air and mixture gases containing CO2 (five levels of predefined %), O2 (21%) and N2 (residual part) using gas mixture devices (KOFLOC GB-3C). Experimental nauplii were transferred to a glass vessel of 20 mL volume, which have been placed in the exposure tank, and the vessel was capped with a silicone rubber stopper. The vessel was then placed in the exposure tank. Twenty-five individuals of nauplii were introduced to a vessel. Five vessels containing nauplii and a small magnetic stirring bar were placed in each exposure tank. Three vessels were used for determination of nauplii activity and two vessels were used for measuring pH of the seawater in the vessels at the end of the exposure. After 24 h of exposure the nauplii in the vessel were transferred to a watch glass with a small amount of associated seawater and the number of mobile, immobile and dead individuals were counted using a stereomicroscope. Immobile state was determined as nauplii that could not move around even if it could move its appendages during 15 s after the seawater in a watch glass was gently stirred. Nauplii were assessed as dead if no movement was observed inside the body. Table 1 indicates experimental conditions.
2.1.2. Full life cycle tests on T. japonicus T. japonicus with eyed eggs were collected from the cultivation stocks. Thirty egg capsules were separated from the body and incubated in filtered seawater at 23 °C. Newly hatched nauplii within 24 h after hatching were used for the tests. The rearing container of the newly hatched nauplii was placed in an air tight box in which CO2 level was maintained by a continuous supply of atmospheric air or mixture gas (three levels of predefined CO2 %, as described above). To prevent evaporation of the rearing seawater, a beaker filled with seawater was placed in the air tight box and the mixture gas supplied to the air tight box was bubbled into the beaker (Fig. 1). The seawater in the beaker that reached a stable equilibrium with the mixture gas was used for exchange of the rearing seawater every 3 days. The air tight boxes were placed in an incubator set to 23 °C. Rearing seawater of 10 mL, mixture of 9 mL seawater and 1 mL seawater containing 106 cells of T. tetrathela, was filled into each well of a 6-well plate. The 6-well plate was then placed in the air tight box one day before the start of the experiment to facilitate the given pCO2 of the rearing seawater. On the starting day of the experiment, 20 newly hatched nauplii were introduced to each
Gas mixture device Air tight box pH electrode T. japonicus 6-well plate
Fig. 1. Schematic illustration of the experimental apparatus used for the full life cycle test on T. japonicus. Arrows indicate directions of gas flow.
well of the 6-well plates. Observations to assess survival, development and behavior under a stereomicroscope and pH measurement of rearing sweater were carried out daily. When a female with an egg capsule was appeared in the 6-well plate it was transferred individually to a well of a 12-well plate. A total of 10 incubating females were placed in the 12-well plate and reared until their eggs became eyed eggs. Then the egg capsule was separated from the body and placed in a well of another 12-well plate to prevent cannibalization of hatched nauplii by the adult. After removing 10 female, all the remaining individuals in the 6-well plate were grouped and reared in a Petri dish. The number of females and males of adults in the Petri dish were counted and the number of newly hatched nauplii and of undeveloped eggs in the 12-well plate was counted under a stereomicroscope after completion of the hatching. Table 2 indicates experimental conditions. Endpoints for the effect were determined as follows: First copepodid, i.e. days needed until first appearance of a copepodid after the exposure. First mating, i.e. days needed until first appearance of a mating pair after the exposure. First spawning, i.e. days needed until first appearance of an incubating female after the exposure. Hatching success, i.e. rate of hatching success of the first spawned egg capsule. Sex ratio, i.e. morphologically distinguished sex ratio at the end of the experiment. 2.2. Experiments on Babylonia japonica Freshly-caught B. japonica were purchased from fishermen’s cooperatives in Niigata near to the MERI laboratory. Males and females were kept in separate stock tanks until the start of the experiments. They were fed moist pellets, feed mixture for carnivorous aquacultured fish, once a day.
Table 1 Conditions of the test seawater for acute mortality test on T. japonicus. Exposure group
A (control) B C D E (control) F G
Measured
Calculated
Temp (°C)
Sal (psu)
pH
Alk (lmol/kg)
pCO2 (latm)
XCa
XAr
19.7 19.7 19.7 19.7 19.6 19.7 19.7
32.20
8.04 6.26 6.04 5.91 8.04 5.89 5.74
2158
550 39,000 65,000 88,000 560 92,000 130,000
3.17 0.06 0.04 0.03 3.16 0.03 0.02
2.05 0.04 0.02 0.02 2.04 0.02 0.01
33.00
2201
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Table 2 Conditions of the full life cycle tests on T. japonicus. Exposure group
A (control) B C D
Measured
Calculated
Temp (°C) Mean ± SD N=7
Sal (psu) Mean ± SD N=4
pH Mean ± SD N=7
Alk (lmol/kg) Mean ± SD N=4
pCO2 (latm)
XCa
XAr
22.9 ± 0.6 22.8 ± 0.4 22.8 ± 0.4 23.0 ± 0.5
33.05 ± 0.18
8.16 ± 0.13 7.11 ± 0.03 6.31 ± 0.04 5.85 ± 0.03
2251 ± 13
430 5800 37,000 110,000
4.43 0.48 0.08 0.03
2.89 0.32 0.05 0.02
2.2.1. Acute mortality tests on B. japonica Experimental setup was the same as used in the acute mortality tests on T. japonicus. Exposure tanks were set at 20 °C. Ten adult B. japonica were randomly selected from the stock tanks that were kept at 20 °C for more than 2 weeks and were placed in the exposure tank for 96 h. Seawater in each exposure tank was replaced daily with fresh seawater previously adjusted to the intended CO2 level. Conditions of B. japonica were checked twice a day. If there was no movement such as contraction of muscle of soft body when stimulated with tweezers, the specimen was defined as dead. Shell length and wet weight of test specimens were measured at the end of exposure or immediately after death. Mean wet weight (mean ± SD, and below) and mean shell height of experimental specimens were 30.4 ± 3.4 g and 58.3 ± 2.3 mm, respectively. Table 3 indicates experimental conditions.
Main tank (200 L)
13 pairs
Gas mixture device Seawater Sub tank (90 L) Pump
Cooling unit
Heater 2.2.2. Effects on reproductive properties of B. japonica 2.2.2.1. Effects on spawning properties. Experimental setup is shown in Fig. 2. Four sets of the apparatus were used, i.e. one for control and one each for the three different levels of elevated pCO2. Thirteen pairs of female and male B. japonica were randomly picked up from the stock tanks and placed in the main tank. Each pair was housed in a 15 cm3 cage in the main tank. Mean wet weight and mean shell height of the experimental specimens were 27.8 ± 4.1 g and 59.4 ± 3.7 mm respectively. To approximate the natural conditions at that time, temperature of the test seawater at the start of the experiment was set at 16 °C. The temperature was then gradually increased to 20 °C and kept at 20 °C for 1 month, subsequently increased to 22 °C and kept at 22 °C until end of the experiment. Experimental duration, from the start to the end of the experiment, was 80 days (Fig. 3). B. japonica were fed moist pellets twice a day. Spawning of individual B. japonica was recorded every day for number of spawned egg capsules and number of embryos in a capsule for 30 days after the first spawning. After the experiment, all the experimental specimens were boiled and the soft bodies were removed from the shell. These shells were dried and the shell surfaces were observed. Table 4 indicates experimental conditions. 2.2.2.2. Effects on embryonic development. Egg capsules spawned in the experiment above, which were spawned during 31–46 days after the first spawning and were within 12 h after the spawning,
Fig. 2. Schematic illustration of the experimental apparatus used for the reproductive properties test on B. japonica. Arrows indicate directions of water flow.
were used in this experiment. Egg capsules were carefully detached from their bonding surface and approximately 15 egg capsules were immediately placed in a Petri dish (9 cm in diameter and 6 cm in height) with seawater of 22 °C. The Petri dishes were placed in a water bath of 22 °C, i.e. natural seawater incubation group. Simultaneously, approximately 15 egg capsules were placed in a small cylindrical tube both sides capped with mesh fabrics and the tube was placed in the main tank in which the egg capsules were spawned, i.e. controlled CO2 level seawater incubation group. When putrid or dead embryos were found in an egg capsule, those egg capsules were removed from the Petri dish or the tube. The days needed for all veligers hatched out from the egg capsule was recorded. The number of undeveloped embryos was counted under a stereomicroscope after all the veligers had hatched out from the egg capsule. A part of the newly hatched veligers were fixed in 70% ethanol immediately after their hatching for observing their shell morphology. Fixed samples were then dehydrated by 99.5% ethanol and dried in a desiccator. They were coated with platinum using an auto fine coater (JEOL, JFC-1600) and observed using a scanning electron microscope (JEOL, JSM-6380LAK II). Rearing seawater of natural seawater incubation group was replaced every morning.
Table 3 Conditions of the test seawater for acute mortality test on B. japonica. Exposure group
A (control) B C D
Measured
Calculated
Temp (°C) Mean ± SD N=9
Sal (psu)
pH Mean ± SD N=9
Alk (lmol/kg)
pCO2 (latm)
XCa
XAr
19.8 ± 0.0 19.8 ± 0.0 19.8 ± 0.0 19.8 ± 0.1
33.58
8.09 ± 0.02 6.04 ± 0.07 5.89 ± 0.05 5.69 ± 0.03
2273
500 67,000 95,000 150,000
3.65 0.04 0.03 0.02
2.37 0.03 0.02 0.01
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(survival = 93%) was not significantly different from that of the control group (survival = 96%) (extended Fisher’s exact test, P < 0.05; Freeman and Halton, 1951).
8.50
8.00
3.2. Full life cycle tests on T. japonicus
pH
7.50
Delay of growth rates, those indicated by days needed for first appearance of a copepodid, a mating pair and incubating female after the exposure, were clearly indicated at pCO2 = 37,000 latm but at pCO2 = 5800 latm (Table 6). Hatching success of the first spawned egg at pCO2 = 37,000 latm decrease considerably and significantly different from that of the control group (Dunnett’s method, P < 0.05). Undeveloped eggs were frequently observed in the spawned egg capsule at pCO2 = 37,000 latm (Fig. 4). No growth and dead nauplii were observed at pCO2 = 110,000 latm. There were no significant difference in sex ratios at end of the exposure among control, pCO2 = 5800 latm and pCO2 = 37,000 latm groups (extended Fisher’s exact test, P < 0.05). These results indicated that NOEC (No Observed Effect Concentration) = 5800 latm and LOEC (Lowest Observed Effect Concentration) = 37,000 latm.
7.00
6.50
6.00 24.0 23.0
Temperature
22.0
Jun./2, First spawning
21.0 20.0 19.0 18.0 A (Control), 470 µatm B, 1,600 µatm C, 5,500 µatm D, 16,000 µatm
17.0 16.0 15.0 14.0 May/18
Jun./1
Jun./15
Jun./29
Jul./13
Jul./27
3.3. Acute mortality tests on B. japonica
Aug./10
Month / date Fig. 3. Temperature and pH changes during the exposure experiment on reproductive properties of B. japonica.
All adult B. japonica survived in the control (pCO2 = 500 latm), pCO2 = 67,000 latm and 95,000 latm, whereas all test specimens of pCO2 = 150,000 latm died before 72 h. Although the soft body of the test specimens at pCO2 = 95,000 latm was not completely covered by their shells and showed no spontaneous movement during the exposure, B. japonica showed normal movement and even feeding behavior if they were returned to normal seawater after the exposure.
Table 5 indicates experimental conditions. 3.4. Effects on reproductive properties of B. japonica 2.3. Test seawater quality Temperature and pH of the test seawater was measured daily using a conventional pH meter (METTLER TOLEDO, SG8-ELK with pH probe InLab413SG). Salinity and alkalinity were measured weekly using an inductively coupled salinometer (YEO-KAL, MODEL 601MK1V) and by titration method respectively for the supplied seawater to the tests. The test seawater pCO2 and CaCO3 saturation state, calcite (XCa) and aragonite (XAr), were calculated from the measured values of temperature, pH, salinity and alkalinity using the program CO2SYS (Pierrot et al., 2006) and by using the dissociation constants from Mehrbach et al. (1973) refitted by Dickson and Millero (1987) and for K2SO4 from Dickson (1990). 3. Results 3.1. Acute mortality tests on newly hatched nauplii of T. japonicus Newly hatched nauplii of T. japonicus showed remarkably high tolerance to elevated pCO2 for 24 h up to 130,000 latm (pH = 5.74). The survival of the nauplii or appearance ratio of the mobile nauplii at the highest exposure of pCO2 = 130,000 latm
3.4.1. Effects on spawning properties All the test specimens showed active feeding behavior and survived the exposure. When the temperature of the test seawater reached 20 °C, spawning started in all experimental groups and continued until the end of the experiment, i.e. 65 days after the start of spawning (Fig. 3). Results of spawning during 30 days after the first spawning are indicated in Table 7. There were no significant differences between all exposure groups for all spawning properties (extended Fisher’s exact test on spawning events and egg capsules and Dunnett’s method, P < 0.05, on spawned times and embryos). These results indicated that the elevated pCO2 up to 16,000 latm did not affect the spawning of B. japonica. Appearances of shells after 80 days of the exposure are shown in Fig. 5. Small spirorbid specimens, specific name undetermined, were found only on the shells of control group. Appearances of shells of pCO2 = 1600 latm group were almost the same as control group but there were no spirorbid specimens. Parts of the periostracum peeled from the shells of pCO2 = 1600 latm group and the shells were severely affected in pCO2 = 16,000 group. These results indicated that longer exposure, i.e. 80 days in this case, to an elevated pCO2 of more than 1600 latm or CaCO3 saturation states
Table 4 Conditions of the test seawater for the spawning property test of B. japonica. Exposure group
A (control) B C D
Measured
Calculated
Temp (°C) Mean ± SD N = 119
Sal (psu) Mean ± SD N=9
pH Mean ± SD N = 119
Alk (lmol/kg) Mean ± SD N=9
pCO2 (latm)
XCa
XAr
20.0 ± 1.5 20.0 ± 1.8 20.0 ± 1.5 19.9 ± 1.4
33.44 ± 0.35
8.11 ± 0.04 7.64 ± 0.07 7.12 ± 0.03 6.66 ± 0.05
2261 ± 23
470 1600 5500 16,000
3.79 1.45 0.46 0.16
2.46 0.94 0.30 0.10
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Table 5 Conditions of the test seawater for the embryonic development test of B. japonica. Exposure group
Natural SW A (control) B C D
Measured
Calculated
Temp (°C) Mean±SD N = 48
Sal (psu) Mean±SD N=5
pH Mean±SD N = 48
Alk (lmol/kg) Mean±SD N=5
pCO2 (latm)
XCa
XAr
22.1 ± 0.1 21.9 ± 0.2 22.1 ± .01 21.9 ± 0.1 21.7 ± 0.1
33.15 ± 0.36 33.22 ± 0.40
7.98 ± 0.06 8.08 ± 0.02 7.66 ± 0.04 7.13 ± 0.03 6.69 ± 0.03
2232 ± 17 2241 ± 24
670 520 1500 5400 15,000
3.07 3.73 1.59 0.49 0.18
2.00 2.43 1.04 0.32 0.12
Table 6 Results of the full life cycle tests on T. japonicus. Exposure Group (pCO2)
First copepodid (days)a
First mating (days)b
First spawning (days)c
A (control, 430 latm) B (5800 latm) C (37,000 latm) D (110,000 latm)
6 12 14 6 12 14 9 15 20 No specimens molted to nauplius stage II (Dahms et al., 2007) Dead specimens were observed after 6 days of the exposure All specimens died after 21 days of the exposure
Hatching success (%)d Mean ± SD N = 10
Sex ratio (male %)e
99.2 ± 1.3 97.2 ± 6.2 11.3 ± 21.7*
0.66 0.75 0.75
a
Days needed for first appearance of a copepodid after the exposure. Days needed for first appearance of a mating pair after the exposure. c Days needed for first appearance of an incubating female after the exposure. d Rate of hatching success of the first spawned egg capsule. e Morphologically distinguished sex ratio at end of the exposure. Significantly different from the control, Dunnett’s method, P < 0.05. b
*
Control, 430µatm
37,000µatm
Fig. 4. Egg capsule of T. japonicus under control condition (left) and pCO2 = 37,000 latm (right). All eggs show normal development in the control while undeveloped eggs, i.e. transparent and green eggs, were observed at 37,000 latm.
Table 7 Results of the spawninga property test of B. japonica.
a b c d e
Exposure group (pCO2)
Spawning eventsb
Spawned timesc Mean ± SD
Egg capsulesd
Embryose Mean ± SD
A (control, 490 latm) B (1600 latm) C (5500 latm) D (16,000 latm)
39 38 56 44
3.0 ± 2.5 2.9 ± 1.9 4.3 ± 3.3 3.4 ± 2.4
1186 1146 1529 1074
32 ± 12 (N = 36) 25 ± 7 (N = 37) 30 ± 9 (N = 54) 24 ± 10 (N = 43)
(N = 10) (N = 10) (N = 10) (N = 10)
Observed for 30 days after the first spawning. Total number of spawning events. Spawning times per spawned females. Total number of spawned egg capsules. Number of embryos per egg capsules.
under ca. 1 (XCa < 1.45, XAr < 0.94) affected the maintenance of periostracum of B. japonica. Additionally, it was indicated that spiororbids are sensitive to elevated pCO2 and pCO2 = 1600 latm was critical for their survival. 3.4.2. Effects on embryonic development Results of the embryonic development in natural seawater and elevated pCO2 conditions are shown in Table 8. There were no significant differences (Dunnett’s method, P < 0.05) among the natural
seawater incubation groups for embryonic development properties, days from spawning to complete hatching of veligers and number of undeveloped embryos per capsule after completion of the hatching. This indicated that adult B. japonica could produce viable eggs under an elevated pCO2 up to 16,000 latm. In the controlled CO2 level incubation group, on the other hand, the number of undeveloped embryos in pCO2 = 5400 latm incubation group (XCa = 0.49 and XAr = 0.32) was significantly larger than that in control group (pCO2 = 520 latm) (Dunnett’s method, P < 0.05).
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Control, 470µatm
1,600µatm
5,500µatm
16,000µatm
Fig. 5. Shells of B. japonica after 80 days of the exposure. Spirorbid (small inserted picture, upper left) were only found on the shells of the control group.
Table 8 Results of the embryonic development test of B. japonica.
*
Exposure group (pCO2)
Incubation (pCO2)
A (control, 470 latm) B (1600 latm) C (5500 latm) D (16,000 latm)
Natural Natural Natural Natural
A (control, 470 latm) B (1600 latm) C (5500 latm) D (16,000 latm)
A (control, 520 latm) B(1500 latm) C (5400 latm) D (15,000 latm)
SW SW SW SW
(670 (670 (670 (670
latm) latm) latm) latm)
Days from spawning to complete hatching
Undeveloped embryos/capsule Mean ± SD
19, 19, 19, 19,
1.47 ± 0.40 1.40 ± 0.45 1.67 ± 0.25 1.17 ± 0.66
20, 20, 19, 20,
20 20 19 20
18, 19, 20, 20 20, 20, 21, 22 19, 20, 20, 22 No hatching
(N = 36) (N = 50) (N = 40) (N = 35)
0.65 ± 0.26 (N = 52) 1.38 ± 0.70 (N = 58) 2.93 ± 1.44 (N = 63)*
Significantly different from the control, Dunnett’s method, P < 0.05.
Embryos in pCO2 = 15,000 latm incubation group did not show normal development and the embryos could not reach veliger stage within 23 days after spawning. These results indicated that development of embryos and/or shell formation of veligers became diminished under the elevated pCO2 of more than pCO2 = 1500 latm or CaCO3 saturation states under ca. 1 (XCa < 1.59, XAr < 1.04), and halted under more than pCO2 = 15,000 latm. There were no marked differences among shell appearances of newly hatched veligers of elevated pCO2 incubation group (control, pCO2 = 1500 latm, pCO2 = 5400 latm) under observation by scanning electron microscope. It indicated that if veligers successfully started shell formation, they could form normal shells as same as the control group. From the above, it was indicated that for embryonic development of B. japonica NOEC = 1500 latm and LOEC = 5400 latm. 4. Discussion Acute mortality tests on T. japonicus indicated that newly hatched nauplii were tolerant to pCO2 = 130,000 latm for 24 h. Harpacticoida, in which T. japonicus is included, inhabit fresh, brackish and seawater and many of them live in seawater habitats. Range of distribution of harpacticoids in the sea is diverse, i.e. from sandy beaches and tide pools to benthic environments of the deep sea of thousand meters depth. Many of these habitats show severe and unique environmental fluctuations. Thus tolerances to elevated pCO2 of harpacticoids differ from species to species (Pascal et al., 2010; Thistle et al., 2006). High tolerance to elevated pCO2 described for newly hatched nauplii of T. japonicus may be explained by their adaptability to their habitats such as tide pool and sea bottom where CO2 concentration often becomes high due to respiration of organisms. For the planktonic copepod, it was reported that LC50 of 24 h for elevated pCO2 seawater ranges
between c.a. 20,000 and 110,000 ppm for epipelagic species at subarctic, epipelagic and mesopelagic species at subarctic and Paracalanus parvus, Tisbe gracilis and Stephos sp. (Watanabe et al., 2006). It can be concluded that nauplii of T. japnicus have a higher acute tolerance to elevated pCO2 compared to that of planktonic copepods. Full life cycle tests indicated that growth rate and hatching success of T. japonicus were not affected at pCO2 = 5800 latm but at pCO2 = 37,000 latm. It was reported for the planktonic copepod Acartia steueri that there were no significant differences in survival rate among test groups incubated for 8 days under + 2000 latm (pH = 7.40–7.55), +10,000 latm and control (pH = 8.14–8.17, pCO2 = equilibrated with atmospheric air) while their egg production was significantly lower for the +10,000 latm groups compared to the control group (Kurihara et al., 2004). Another planktonic copepod, Acartia tsuensis, was reported that there were no significant difference in egg production and hatching success between elevated pCO2 environment of pCO2 = 2380 latm and control of pCO2 = 380 latm when they were incubated for three generations (Kurihara and Ishimatsu, 2008). It can be concluded that although T. japonicus might be more tolerant to elevated pCO2 environment for longer exposures compared to planktonic copepods, both benthic and planktonic copepods would be tolerant to the pCO2 of several tens of thousands latm. Saturation state of CaCO3 at pCO2 = 5800 latm and pCO2 = 37,000 latm , NOEC and LOEC of T. japonicus respectively, were under ca. 0.5 and 0.01 respectively (Table 2). This suggests that under saturation of CaCO3 did not have a strong impact on T. japonicus compared to that of B. japonica described below. Acute mortality tests on B. japonica indicated that adults were tolerant to pCO2 = 95,000 latm for 96 h. B. japonica is a bottom dweller and a scavenger. They sometimes penetrate into the body of dead animals where O2 concentration would be at a minimum level and CO2 concentrations are expected to be high. Their habitat
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and feeding habits thus might select for tolerance to elevated pCO2 environments. Although shells of adult B. japonica were damaged under elevated pCO2 of more than 1600 latm or less than CaCO3 saturations of ca. 1 for 80 days exposure (Fig. 5), their damage would be more severe for longer exposure and thus their growth and survival might be affected. Gazeau et al. (2007) reported for bivalves that in Mytilus edulis shell dissolution was observed at pCO2 P 1800 ppm and the calcification rate showed a 25% decrease at pCO2 = 740 ppm and in Crassostrea gigas calcification rate showed a 10% decrease at pCO2 = 740 ppm. Kimura et al. (2011) reported for the gastropod in Haliotis discus that there were no effects on early development under exposure to pCO2 < 1100 latm but increasing deteriorations of early developments were observed with increased exposure to pCO2 = 1650 and 2150 latm. Shells of newly hatched veligers of B. japonica showed a normal appearance even at pCO2 = 5400 latm. It can be concluded that shell formation rates and dissolution rates under elevated pCO2 environment are quite different among species. However, mollusks with calcium carbonate shells such as B. japonica might be more sensitive to elevated pCO2 environment compared to marine organisms without shells (Fabry et al., 2008). It is expected that CaCO3 saturation state of less than 1 would be critical for calcification of many mollusks with calcium carbonate shells. Data on tolerances to elevated pCO2 of marine organisms have been accumulated because of the urgent need for research on ocean acidification due to anthropogenic CO2 (e.g., Gooding et al., 2009; Ries et al., 2009). Those studies showed that many species were susceptible to elevated pCO2, but some species such as T. japnicus and B. japonica were significantly tolerant than others. The differences in tolerance among species seem to involve their different physiological compensation systems against elevated pCO2 those arose during evolution. As a result, prediction of ecosystem fate under an elevated pCO2 resulting both from potential risks of CCS and ocean acidification needs further tolerance data of each species and reliable ecosystem model. However, ocean acidification is an issue of global scale while likely impact of CCS leakage will be limited to local. It would be useful and realistic to determine safety threshold of pCO2 for a local ecosystem using most susceptible data of local species when assessing potential impact of CO2 leakage by a CCS. Acknowledgement This work was supported by the project of Ministry of Environment, Japan, entitled ‘‘Investigating the Environmental Management System for Offshore CCS, Development of EIA Methodology’’ and was written based on the author’s independent consideration. References Dahms, H.U., Chullasorn, S., Kangtia, P., Ferrari, F.D., Hwang, J.-S., 2007. Naupliar development of Tigriopus japonicus Mori, 1932 (Copepoda: Harpacticidae). Zool. Stud. 46, 746–759.
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