Simulating ocean acidification and CO2 leakages from carbon capture and storage to assess the effects of pH reduction on cladoceran Moina mongolica Daday and its progeny

Chemosphere 155 (2016) 621e629

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Simulating ocean acidification and CO2 leakages from carbon capture and storage to assess the effects of pH reduction on cladoceran Moina mongolica Daday and its progeny Zaosheng Wang a, *, Youshao Wang b, Changzhou Yan a a

Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, 1799 Jimei Boulevard, Xiamen, 361021, People's Republic of China State Key Laboratory of Tropical Oceanography, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, People's Republic of China

b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Effects of pH reduction were assessed by simulation of OA and CCS-leakage.
Moina mongolica showed moderate sensitivity to pH reduction among phyla or classes.
Longevity in 21-day test was not significantly affected but at a cost of reproduction.
The decline of rm and energy content had profound implications on acidified oceans.
Both “quantity” and “quality” were negatively affected across pH reduction gradients.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 January 2016 Received in revised form 22 March 2016 Accepted 22 April 2016

In order to evaluate the effects of pH reduction in seawater as a result of increasing levels of atmospheric CO2, laboratory-scale experiments simulating the scenarios of ocean acidification (OA) and CO2 leakages of carbon capture and storage (CCS) were performed using the model organism Moina mongolica Daday. The LpH50s calculated in cladoceran toxicity tests showed that M. mongolica exhibited intermediate sensitivity to OA, which varied among species and with ontogeny, when compared with different phyla or classes of marine biota. Survival, reproduction and fecundity of parthenogenetic females were evaluated after 21-day exposures. Results showed that increased acidity significantly reduced the rate of reproduction of M. mongolica resulting in a decreased intrinsic rate of natural increase (rm) across the gradients of pH reduction. The analysis of macromolecule contents in neonates suggested that nutritional status in progeny from all broods were significantly reduced as seawater pH decreased, with increasing magnitude in latter broods, except the contents of protein from two former broods and lipids from the first brood. Our findings clearly showed that for this ecologically and economically important fish species, the negative effects of pH reduction on both “quantity” and “quality” of progeny may have farreaching implications, providing direct evidence that OA could influence the energetic transfer of marine food web and ecosystem functions in acidified oceans in the future. © 2016 Elsevier Ltd. All rights reserved.

Handling Editor: Caroline Gaus Keywords: CO2-driven acidification Rank species sensitivity Reproduction Intrinsic rate of natural increase Progeny Macromolecule contents

* Corresponding author. E-mail address: [email protected] (Z. Wang). http://dx.doi.org/10.1016/j.chemosphere.2016.04.086 0045-6535/© 2016 Elsevier Ltd. All rights reserved.

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1. Introduction As the increasing levels of atmospheric CO2 diffuses passively into global ocean surface waters by air-sea fluxes, the marine environment will experience a gradual increase in the partial pressure of CO2 resulting in a decline in seawater pH and changes in carbonate chemistry. This phenomenon is commonly referred to as ocean acidification (OA). Predictably, the global surface ocean pH is projected to decrease by 0.3e0.5 units within this century and 0.7e0.8 units within the next 300 years together with the elevation of atmospheric CO2 to approximately 2000 ppm if no further measures are taken to control the current CO2 emission (Fitzer et al., 2012; Caldeira and Wickett, 2003). On the basis of this prediction, carbon capture and storage (CCS) is proposed as one of the better strategies to reduce CO2 emissions to alleviate future effects of global climate change (GCC) and OA (Rodriguez-Romero et al., 2014). Nevertheless, practical implementation of this technique could have a profound impact on marine ecosystems, because CO2 leaked deriving from the capture facilities, transport pipelines, and offshore installations would react with seawater and provoke more extreme acidification and abrupt changes in oceanic pH values. In vulnerable coastal areas, these changes are being exacerbated due to alterations in currents and upwelling, and coastal oceans are frequently becoming more acidic and consequently their pH variability is higher than that of open oceans (Feely et al., 2008; Li et al., 2014). In fact, the marine environment already faces the threat of unprecedented degrees of an alarming decrease in seawater pH from a number of sources, each of which differs in the severity of perturbation and the time scale over which it operates, potentially producing negative effects on biota and altering the balance of ecosystems (Wootton et al., 2008). Consequently, in order to achieve a complete and balanced assessment of the risks associated with CO2 changes in the Anthropocene, it is essential to explore the effects of CO2-induced acidification on keystone species. Several studies have been carried out to evaluate the effects of acidification on marine organisms (Azevedo et al., 2015; Li et al., 2013, 2014; Stumpp et al., 2014), while as yet, the effects of OA have mainly focused on marine calcifiers, which are believed to be particularly vulnerable to elevated seawater CO2 levels and the ensuing changes in carbonate chemistry because of the associated decline in the degree of saturation with respect to aragonite and calcite in seawater (Azevedo et al., 2015; Kurihara, 2008). However, recent studies have shown that not only calcification processes but also reproductive behavior can be negatively affected by OA, and sublethal reproductive processes are considered to be more sensitive (Hildebrandt et al., 2014). Further, the responses of marine species to OA are highly variable and species-specific. As a consequence, there is a lack of the broad-scale understanding to predict responses across taxa and appreciate the compromises and tradeoffs used by noncalcifying organisms to survive in an acidified ocean environment. Zooplankton crustaceans are a group of organisms that play an important biogeochemical role in ecosystems, because of their participation in energy transfer and nutrient regeneration as key herbivores in the food web (Halsband and Kurihara, 2013). In particular, the cladoceran Moina mongolica Daday, possessing a typical chitinous exoskeleton rather than an aragonite or calcite shell, is an indigenous and ecologically important species in the southeastern part of China, where it is extensively cultivated as major prey to predators such as fish and developing larvae of higher trophic levels, serving as crucial links and conspicuous components in the coastal food chain (Wang et al., 2009). Studies of zooplankton have become popular in OA research (Halsband and Kurihara, 2013). Characteristics such as an approximate size (~1.5 mm), easy to culture under laboratory conditions, a short but predictable

life cycle, and high fecundity make M. mongolica a good and costeffective indicator for studying the biological effects of climate change (Wang et al., 2007, 2009). To date, the observed responses of marine invertebrates such as copepods to elevated seawater pCO2 have predominantly been studied by assessing its effects on their reproduction after a shortor medium-term exposure (i.e., only a tiny fraction or a substantial part of the generation time, respectively), which limited our capacity to predict the likely long-term biological consequence and population variation of OA (Fitzer et al., 2012). In general, physiological adjustments to new environmental conditions and adaptive selection of resistant phenotypes take some time (Pedersen et al., 2014). In additional, exposure to adverse environmental conditions may have nutritional consequences as manifested in the progeny of subsequent generations, which are vital for zooplankton crustaceans providing essential energy in early life stage as the form of macromolecules such as proteins, lipids, carbohydrates or other cellular components and increasing their probabilities of survival under stressful conditions (Arzate-C ardenas and Martíneznimo, 2012). Therefore, additional studies on the quantification Jero of energy stores in offspring are required as a good indication of health status in organisms. In this study, the responses of M. mongolica to various OA scenarios were investigated based on 21-day sublethal tests, with particular emphasis on fecundity parameters calculated as the reproductive end points and nutritional conditions in progeny. Prior acute tests were conducted to determine acidities that resulted in 50% mortality to M. mongolica (LpH50) and rank species sensitivity were analyzed. A laboratory-scale controlled experiment was conducted using “seawater CO2 contact systems”. The experimental design involved the direct release of CO2 into filtered seawater (FSW) in nonpressurized chambers, mimicking both acidification due to the uptake of CO2 in a “business as usual scenario” for chronic and acute tests (pH- 7.3e7.8) and acidified seawater in possible scenarios of dramatic leakages from a CO2 storage site of physical sequestration scheme only for acute tests (pH- 6.0e6.9), where pH levels of 7.8 and 7.3 are generally similar to that predicted for the end of the century and the next 300 years by the Intergovernmental Panel on Climate Change (IPCC, 2007), and the lowest pH treatment of 6.0 is put forward as the local “worstcase” scenario (Wang et al., 2015). 2. Materials and methods 2.1. Study organisms M. mongolica was initially obtained from the culture collection in the laboratory at the Institute of Urban Environment and consecutively reared for approximately 1 year under controlled laboratory conditions. Briefly, the specimens were maintained in stock culture under semi-static conditions in a 2-L cultivation container filled with 0.22 mm filtered seawater (FSW, salinity 32.0 ± 0.3) at 20 ± 0.5  C and under a low light intensity of approximately 400 lux with a 16:8 h light:dark photoperiod in a programmable illuminated incubator (Boxun SPX-2501-G). Cladoceran cultures were fed daily with a mixture of two phytoplankton species: 1:1 ratio of Chlorella pyrenoidosa and Pavlova viridis in exponential growth phase at a constant rate of 8  105 cells mL1. For culturing of these two green microalgae, seawater-based f/2 medium was enriched with nutrients (Guillard and Ryther, 1962). Before using C. pyrenoidosa and P. viridis as food, it was centrifuged, washed, and resuspended in FSW to remove culture nutrients. Seawater was replaced every other day to avoid accumulation of metabolic wastes, and the seawater pH in the stock beakers was constant at 8.10 ± 0.05. All tests were conducted using

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neonates (12e24 h) of the third brood of several females from parental continuous bulk cultures which were derived from a single individual to avoid variations due to culturing conditions. 2.2. Preparation of CO2-treated seawater The seawater used in the experiments was pumped by an inshore subsea pipeline system from the East Sea of Xiamen during high tide, continuously filtered by a high-power external filter containing nitrate removal stones before transported to the laboratory, and then poured to 200-L tanks. The experiment for assessing the effects of CO2-driven acidification on cladoceran species used seawaters of different pH and pCO2 levels, referred to as CO2-treated seawaters, which were prepared using the automatic nonpressurized laboratory-scale CO2 Venturi injector and manipulation system (AT Control System from Aqua Medic of North America, CO, USA). Briefly, the system connected exposure apparatus to a supply of CO2-enriched gas via mass flow controllers (MFCs) and solenoid valves injecting the gas to simulate CO2-induced acidification and set a desirable pH level or target pCO2 at each exposure chamber via an automated feedback relay system, which monitored and controlled the pH by opening the valves and adjusting the CO2 bubbling. Before injection, the exposure chambers were carefully filled with FSW. Then, CO2-enriched air was injected as bubbles through a ceramic bubble diffuser placed in the center of each chamber until equilibrium. Before toxicity tests, the pH of the treatment seawater was decreased slowly (~0.5 units/day) to reach the desired pH value in order to avoid sudden change of pH in the experimental vessels representing a worst-case exposure scenario (Wang et al., 2015). Furthermore, the pH values in the exposure chamber recorded by the CO2 manipulation system were regularly verified by a portable pH meter (model: Orion 520; Boston, MA, USA) to be accurately 0.01 pH units, calibrated using pH buffer solutions of 4.00 and 7.00 (NBS scale). Control seawater was prepared by aerating FSW (CO2 concentration: 385 ppm). 2.3. Seawater analysis For analysis of total alkalinity (TA), seawater samples were collected and preserved in darkness to avoid any head-space. Aliquots of 50 mL were taken each week throughout the experiments for TA measurements, and the values were obtained by an automatic titrator (Mettler Toledo, T50) using a combined glass electrode calibrated on the NBS scale. The TA, expressed in mM, and CO2 system pH were used to determine the seawater carbonate system speciation. Carbonic chemistry parameters such as total inorganic carbon 2 (TIC), bicarbonate (HCO 3 ), carbonate (CO3 ), partial pressure of CO2 (pCO2), and saturation states of aragonite and calcite were calculated using the CO2SYS program (Pierrot et al., 2006) based on the dissociation constants from Mehrbach et al. (1973) with a refit by Dickson and Millero (1987) and of KHSO4 from Dickson (1990). 2.4. Cladoceran toxicity tests A series of cladoceran toxicity tests using the species M. monogolica were conducted in exposure chambers according to the American Society for Testing Materials (ASTM) guidelines and previously established methods (Wang et al., 2009), with the modification that the exposure chamber was covered with a ribbed watch glass to prevent the water from exposure to the turbulent gas and the zooplankton from being trapped at the gas water interface and to maintain a stable pH. In advance, it had been confirmed that the exposure chamber was sufficiently large for the zooplankton to

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survive normally during the duration of tests. For acute tests, triplicates of 10 randomly juveniles selected (12e24 h) were assigned to control and various resultant CO2treated seawaters with pH values of 7.8, 7.6, 7.3, 6.9, 6.5 and 6.0. Test organisms were not fed during the test periods. Observations were made after 24 and 48 h of exposure, and organisms immobile for 15 s after gentle shaking and showing a change to milky coloration were classified as dead. On the basis of acute toxicity results, 21-day chronic toxicity tests were performed, and 10 replicates of individual juveniles (12e24 h; one neonate per exposure chamber) were exposed to a control and CO2-treated seawater with pHs 7.8, 7.6 and 7.3. Test organisms were fed a concentrated suspension of the mixture of green alga C. pyrenoidosa and P. viridis in the exponential growth phase. The seawater in each exposure chamber was renewed daily with fresh seawater previously adjusted to the desired pH levels and every time neonates were released to restore the algal concentrations to the predetermined level (5  105 cells mL1). This sequence of exposure and renewal was repeated until the end of the 21st-day of the test. During the experimental period, a number of juveniles and parent mortality were recorded each day, and the offspring produced were collected to determine nutrition levels. Dead animals were removed at each observation time. At the end of the 21-day exposure period, mortality and the number of juveniles were recorded for the last time. All toxicity tests were also conducted at 20 ± 0.5  C with a 16:8h light:dark photoperiod and maintained at the low intensity of 400 lux during the 16-h light period. The exposure chambers were gently agitated daily by a slow-rotating shaker apparatus to mimic the natural environment during the experimental period. The oxygen saturation was recorded using a YSI 52 dissolved oxygen meter (accuracy ± 0.5% of the measured value), and salinity was measured with Leici DDSJ-308A electronic salinity instruments (accuracy ± 1% of the measured value). 2.5. Determination of nutritional contents After collecting the progeny produced, the specimens of neonates were homogenized using a glass tube with a Teflon pestle in 1 mL of 0.9% physiological saline solution to quantify protein and carbohydrates. Lipids were extracted with a chloroform:methanol:water (2:1:1) solution using the same new neonates quantity. The organic phase was dried and resuspended in water for analysis. The total protein contents were determined by measuring absorbance at 595 nm following the method of Bradford (1976) using bovine serum albumin (BSA) as the standard. Total carbohydrates were quantified by the phenol sulfuric acid method (Dubois et al., 1979) measuring the absorbance at 492 nm, based on the formation of a yellow orange complex due to the reaction of carbonates with phenol in a strongly acidic medium. Total lipids were measured spectrophotometrically at 530 nm, using cholesterol as a standard according to the sulfophosphovanillin method (Zǒlner and Kirsch, 1962). All measurements were carried out in triplicate for each sample and results were expressed in micrograms (protein/carbohydrates/lipids) per individual. The caloric content of the specimens was calculated by multiplying each macromolecule content by its combustion factor: 4.10 cal mg1 for carbohydrates, 5.55 cal mg1 for proteins and 9.45 cal mg1 for lipids (Mann and Gallager, 1985), and the results were expressed in microcalories per individual. 2.6. Data treatment and statistical analysis Cumulative mortality in acute tests was determined by

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comparing the number of individuals dead at the end of the exposure (24 and 48 h) with the number of individuals at the beginning of the exposure. These data were used to calculate the median lethal pH levels (LpH50) of toxic parameter, which was defined as the pH that caused lethal effects in 50% of the exposed population. These parameters and their significance (95% confidence intervals using bootstrapping) were estimated using the pH that provokes mortality in 50% of the population exposed to the different pH treatments and were calculated using interpolation of data fitted to the logistic sigmoid function for lethal toxicity. Then, acute tests results obtained were compared with those for species from different phyla or other taxonomic groups to evaluate the relative species sensitivity of M. mongolica. The following demographic parameters of the criteria used to quantify the effects of acidification on reproduction of test organisms were recorded daily: age-specific survival (survival rate of females at the age of producing neonates) and age at release of neonates, which included age at first reproduction and daily reproduction rates (number of neonates produced per surviving female per day). The tests were terminated at day 21, after which the fecundity parameters of intrinsic rate of natural increase (rm), net reproductive rate (R0), and mean generation time (T) were determined as (Lotka, 1913; Walthall and Stark, 1997) 21 X

lx mx erm x ¼ 1;

x¼0

P21 T¼

R0 ¼

21 X

lx mx

; and

x¼0

xlx mx ; respectively; R0

x¼0

where lx is the proportion of females surviving to age x (days) and mx is age-specific fecundity (number of juveniles produced per surviving female at age x). Because of the high importance of early reproduction, the rm calculated for M. mongolica organisms after 21 days was indistinguishable from the rm estimated for the entire life span. Consequently, all calculations were based on 21-day experiments. The longevity was defined as the average time that M. mongolica survived in the 21-day exposure. Macromolecule levels and caloric contents in progeny were normalized and then compared to those of controls statistically using two-sided Student's t-tests. The differences were classified according to their statistical significances as follows: *p < 0.05 and **p < 0.01. Reproduction and fecundity results in 21-day bioassays were analyzed by one-way analysis of variance (ANOVA), followed by comparing the means to detect the significant differences (p < 0.05) between the pH-treatment groups and the control using Student Newman Keuls multiple-range tests. Homogeneity of variance was tested using Bartlett's test and normality was measured when necessary. Statistical analysis was performed using the Statistical Analysis System (SAS 8.2; SAS Institute, North Carolina State University, Raleigh, NC, USA).

saturation (UAragonite and UCalcite) became lower than the corresponding values of control seawater. Measurements of seawater parameters revealed that dissolved oxygen concentrations were always >80% of air saturation and the salinity ranged from 31.8 to 32.3, respectively. 3.2. LpH50s and rank species sensitivity The results of acute toxicity tests are shown in Fig. S1. As can be seen, cumulative mortality of M. mongolica increased with the decline of pH levels. The median lethal pH levels (LpH50) for 24 and 48 h were calculated as 7.09 (7.06e7.11) and 7.37 (7.33e7.43), respectively, indicating that the LpH50 value increased with exposure time. At the end of testing period, no mortality was observed in the controls. For a comprehensive look into the sensitivity distribution among species of a taxon, LpH50s obtained in this study can be compared with the distribution of species sensitivity data determined from an analysis of existing toxicity data for various genera of different taxonomic groups (Fig. 1). The ranked acid sensitivities of the taxonomic groups were determined as follows. In total, species mean acute values (SMAVs) across 23 saltwater species included those for Arthropoda, Cnidaria, Echinodermata, Foraminifera, Mollusca and Rhodophyta, ranging from 7.87 of LpH50 for Echinodermata, Pseudechinus huttoni (being the most sensitive), to 6.81 for Cnidaria, Acropora digitifera (the most resistant) (listed in Table S2). Broadly, organism sensitivity to CO2-driven decreased pH levels appeared to be pH-dependent as shown in the regression equation, which was classified by two orders of magnitude (SMAVs of LpH50, <7.3 to >7.3) based on the projected tipping points of climate change (Fig. 1). Despite calcifying or noncalcifying organisms of all taxa, the rank of M. mongolica for species sensitivity was in the range of 20 and 10 for 24- and 48-h LpH50 values, respectively (Fig. 1), indicating that M. mongolica is moderately sensitive to acidification and vulnerable to climate change stressor compared with different phyla or different orders of the same phylum (Arthropoda) and class (Crustacea).

3. Results 3.1. Seawater chemistry The values of seawater chemistry parameters observed are summarized in Table S1, where the CO2 manipulation system kept the pH within a moderate stable range in acidification apparatus over the entire experimental period. The seawater pH in the control chamber was maintained at 8.10 ± 0.01 through bubbling of ambient air. Acidified seawater had higher concentrations of pCO2, totally dissolved inorganic carbon (TDIC), and bicarbonate ion (HCO 3 ); on the contrary, the levels of pH, carbonate ion concentration (CO2 3 ), and calcium carbonate

Fig. 1. Distribution of acute toxicity data from various saltwater organisms of different phyla or class. The arrows are schematic representations illustrating how 24- and 48-h LpH50s of M. mongolica Daday exposed to CO2-treated seawaters compare and rank with these data. The error bars are the standard deviations of mean LpH50s for specific organisms.

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3.3. Reproductive effects and fecundity In order to further elucidate the effects of acidification on the reproductive process of M. mongolica, 21-day chronic toxicity tests were conducted at different pH values of seawater driven by CO2. At the end of the tests, mortality in the controls did not exceed 10%. As shown in Figs. 2e4, a decrease of pH levels had a negative impact on the reproductive variables. Except longevity, all other parameters examined were significantly affected by elevated pCO2 of sublethal acidification exposure, indicating that M. mongolica could be significantly affected by OA in the near future if atmospheric CO2 levels continue to increase as predicted. Although not statistically significant, acidification exposure still appeared to produce a slight decrease in reducing the life expectancy along the pH gradients from 8.1 to 7.3 (Fig. 2A). Furthermore, there was a delay in the time to first brood (i.e., by lengthening the time of maturation) (Fig. 2B), and a gradual decrease in the number of first brood (Fig. 2C), total reproduction (Fig. 3A), and mean generation time (T) (Fig. 3B) as the reduction of pH levels over the duration of the experiments. The decrease was even more evident in the lower pH levels. A significant difference (p < 0.05) between treated and control groups occurred when the pH was less than 7.6. The first significant difference in brood size (Fig. 3C) and net reproductive rate (R0) (Fig. 3D) occurred at pH 7.8 of near-future OA

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and continued in a context-dependent manner across the pH gradients. The reduction of pH levels also appeared to elicit a contextdependent decline in the number of broods (Fig. 2D), although the significant difference between the control and treated groups was only observed at pH 7.6. As for the intrinsic rate of natural increase (rm), low pH levels dramatically decreased this important fecundity parameter after a 21-day exposure (Fig. 4). With the reduction of pH from 8.1 to 7.3, the rm declined from 0.38 to 0.28. Furthermore, it is noteworthy that the rm was found to significantly vary at all pH values, illustrating that it is extremely sensitive as an examined fecundity parameter. 3.4. Nutritional status in progeny Fig. 5 illustrated the amounts of protein, carbohydrate and lipid as well as caloric contents in M. mongolica neonates of four broods produced by healthy (control) females and those under pH gradients, showing data from exposed groups converted to fractions of the absolute value obtained from controls, which were assigned a value of 1. Neonates produced by females under acidic conditions were expressed with lower nutritional levels than those born from control females. This is particularly evident for total carbohydrate levels in progeny, which were reduced significantly except for the neonates from first and third broods of females at pH 7.8. Compared

Fig. 2. (A) Longevity, (B) time to first brood, (C) number of first brood, and (D) number of broods of M. mongolica Daday after exposure to four pH conditions in 21-day chronic toxicity tests. Data are presented as mean ± SD (N ¼ 10) and analyzed by one-way ANOVA. Small letters above the gray bars indicate significant difference among treatments (StudenteNewmaneKeuls multiple-range tests, p < 0.05).

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Fig. 3. (A) Total reproduction, (B) mean generation time (T), (C) brood size, and (D) net reproductive rate (R0) of M. mongolica Daday after exposure to four pH conditions in 21-day chronic toxicity tests. Data are presented as mean ± SD (N ¼ 10) and analyzed by one-way ANOVA. Small letters above the gray bars indicate significant difference among treatments (StudenteNewmaneKeuls multiple-range tests, p < 0.05).

females at pH 7.6 and from third and fourth broods at pH 7.3 decreased significantly, while, despite a reduction in this macromolecule content, all other groups did not differ statistically from controls. In the case of lipids, no significant differences were found among neonates from the first brood. However, total lipids decreased significantly in neonates from subsequent broods, with increasing magnitude, particularly for the fourth brood at pH 7.6 and 7.3. Variations in macromolecule contents directly affected the caloric content of offspring. The energy reserves of neonates were lower in offspring born from females under acidic conditions than in those born from control females, particularly those from third or fourth brood at lower pH levels. 4. Discussion

Fig. 4. Intrinsic rate of natural increase (rm) of M. mongolica Daday after exposure to four pH conditions in 21-day chronic toxicity tests. Data are presented as mean ± SD (N ¼ 10) and analyzed by one-way ANOVA. Small letters above the gray bars indicate significant difference among treatments (StudenteNewmaneKeuls multiple-range tests, p < 0.05).

with controls, protein contents in progeny from third brood of

Although previous studies have shown detrimental effects as a result of increasing OA on survival and reproductive process across a broad range of invertebrate taxa (Azevedo et al., 2015; Fitzer et al., 2012; Kurihara and Ishimatsu, 2008; Li et al., 2013, 2014; Stumpp et al., 2014), this study is, to our knowledge, the first to report the effects of varying pH levels and CO2 on an indigenous organism in China. OA is an issue of global scale, while impact of CCS leakage will likely be limited at specific locations, and for some species there are emerging patterns of change in ecosystem processes and

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Fig. 5. Macromolecules (AeC) and caloric contents (D) in neonates of M. mongolica produced by parthenogenetic females exposed to different pH-treatment seawaters. Columns show contents as a ratio of absolute control values. Asterisks indicate significant differences regarding proteins, carbohydrates, lipids, and caloric contents when compared with controls (*p < 0.05; **p < 0.01).

species occurrence (Nagelkerken and Connell, 2015). Although relating probability of survival in the laboratory by short-term acute tests to probability of presence in the field is limited, it would be useful and realistic to determine a safety threshold of OA such as LpH50s for a local ecosystem using the most susceptible data for an indigenous species (M. mongolica), which is of particular ecological importance when assessing potential outcomes of CO2 leakage by CCS. The lethal effects of OA on marine biota were species-specific and changed among phyla, taxa, species, and life history stages, and even varied within phyla and between closely related species (Azevedo et al., 2015; Dupont et al., 2010), while echinoderms and molluscs are more sensitive than Anthropoda crustaceans (Fig. 1). These results are consistent with the findings of Wittmann and Portner (2013), who reported that echinoderms and molluscs are vulnerable to ocean pCO2 values. In particular, marine crustaceans such as M. mongolica seem less sensitive to OA than echinoderms and bivalve molluscs. This might be attributed to their difference of physiological systems that the poor iono-and osmo-regulators in echinoderms and molluscs have limited abilities to compensate for acid-base disturbances in their body compartments by reduced pH levels or elevated pCO2 (Whiteley, 2011). Due to strong iono- and osmo-regulations, crustacean species possess compensatory mechanisms to respond to acid-base disruptions. On the contrary,

most crustaceans are characterized by a chitinous exoskeleton, so they are not as vulnerable to calcium carbonate under saturation as other calcifying organisms. These physiological traits can also be used to explain species-related differences in sensitivity (Fig. 1), which, in turn, may be used to predict changes in individual performance and survival when facing a stress such as the CCSinduced acidification or OA scenarios, including the possibility of thresholds or tipping points (Kroeker et al., 2010). For instance, the echinoderms were the most sensitive species, with the highest LpH50 value, and may thus be vulnerable to CCS leakage events. Despite in fact, most species will be lethally affected by leaking CO2 decreasing chances of survival when the pH decreases beyond 6.0 (De Vries et al., 2013). Within the range of pH reduction tested, reproduction was affected more markedly than survival, as indicated by the absence of significant differences in the longevity occurred at pH levels of 7.8e7.3 (Fig. 2A), while all pH reductions had a negative impact on fecundity. To a certain extent, decreased fecundity was a predictable response as the energy primarily available to organisms is preferentially allocated to basal maintenance under acid conditions. Once these needs have been met, energy may be allocated to highly demanding processes such as reproduction, creating a tradeoff between two responses of highest biological import as survival and reproduction, both of them required for species maintenance

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rdenas and Martínez-Jero nimo, 2012). Thus, even if (Arzate-Ca some individuals would be able to survive the 21-day exposure of acidified conditions, the basal maintenance in life expectancy does not imply that there is no effect, likely at an energetic cost of a reduction in reproduction that will result in trade-offs. Overall, it appears that long-term exposure to pCO2 levels more representative of OA has the potential to adversely affect reproduction by diverting energy toward the maintenance of effective compensatory responses. Our results are consistent with the findings by Whiteley (2011) and Pedersen et al. (2014), who indicated that energy budget can be diverted away from key biological processes such as growth and reproduction toward compensatory responses. Little is known about the impact of OA on quality of progeny in marine organisms. In this study, macromolecule and caloric contents measured in neonates reflected the general health status of rdenas and Martínez-Jero  nimo, 2012; Stumpp progeny (Arzate-Ca et al., 2014). Compared with controls, the reduction of energy levels suggested that the neonates of stressed females were born at a disadvantaged status in terms of poor health and fewer available energy resources, which face potentially adverse conditions in acidified oceans. Under unfavorable OA conditions, the reductions of nutrition levels in neonates may be related to the complex mechanisms among energy reserve, resource reallocation and trade-off as stated above. Despite these conditions, because the nutrition qualities in zooplankton play a major role in the food web of marine ecosystems, including fatty acid composition of lipids, amino acid composition of proteins, and sugar composition of rdenas and Martínez-Jero  nimo, 2012; carbohydrates (Arzate-Ca Stumpp et al., 2014; Wang et al., 2007), the decline in macromolecule and caloric contents provided direct evidence that OA could affect the energetic transference in marine food chains. Overall, pH reduction caused by CO2 elicited a change in reproduction toward not only “quantity” (in terms of the numbers of offspring produced) but also “quality” (in terms of the normal energy contents for new individuals). Early life stages are considered to be more sensitive to environmental stress than maturity stages, and its effects have received much attention, with crucial implications for population abundances, species diversity and ecosystem functions (Kurihara and Ishimatsu, 2008). In this study, exposure to acidified seawaters at three stages from the neonate to the juvenile and then adult stages decreased the rate of reproduction and the negative impacts to population was additive, indicating a strong carry-over effect, resulting in more than a 26% decrease in rm for M. mongolica during the 21-day exposure (Fig. 4). When the predicted reduction in the offspring and the reproductive success of this species are applied to broodstocks of cultivated or natural populations of M. mongolica, such a decrease as a result of CO2-acidified seawater represents consequent effects on population dynamics and community structure throughout the species range. It is anticipated that the resulting variations may have a substantial economic impact on the population fitness of this important fish species and far-reaching ecological consequences on the food web structure of the pertinent ecosystem by altering the grazing on phytoplankton communities and affecting the food supply of important predators. This is because M. mongolica cultured as a food organism for aquaculture is a primary and secondary consumer and an important food source for higher trophic levels, forming the “link” and being one of the major contributors to the transfer of energy from primary producers to higher-level consumers in the pelagic food web. From the viewpoint of a total food web, population decline of zooplankton invertebrates would negatively affect the energetic transfer from primary production of the smallest phytoplankton into secondary production and result in decrease of available food for many carnivores, which, in turn, affects fisheries and industries (Pedersen

et al., 2014; Nagelkerken and Connell, 2015). On the contrary, it is also important to remember that extinction does not require the instantaneous death of all individuals in a species. A slight density decrease of 1% per generation may reduce many animal populations to unsustainable numbers in little more than a century. Overall, from an ecological perspective, the results obtained in this study are relevant to the predicted decline in population size of M. mongolica, which is likely to reduce chances of survival and increase the vulnerability to extinction in a stressful environment with negative even dire consequences to coastal community expected in the immediate near future. Nevertheless, care should be taken when extrapolating laboratory results to natural populations, which had complex variations under natural conditions and could react differently. Particularly, OA is not a sudden change and it is still unclear whether M. mongolica will be able to adapt to the changing ocean environments. Clearly, relating the overall effects of marine environmental changes to the ecological significance of M. mongolica in the coastal ecosystems that are predicted to occur with increased seawater CO2 concentrations, more extensive evaluation might be required to achieve this goal, and adaptation potential of species with short generation times should not be neglected (Kurihara, 2008). At present, prolonged bouts of extreme hypercapnia associated with the acidification and measurable change in seawater carbonate chemistry and pH reduction down to 7.0 are typical for many coastal and near-shore areas, including several hatcheries and aquaculture farms in southeastern part of China (Cai et al., 2011), where M. mongolica is a widely cultured economically important crustacean. In some of these areas, low pH values (7.8e7.3) can persist for several months from late spring until early autumn (Li et al., 2014). Taken together, the findings of this study, which indicated that the reproduction and progeny of M. mongolica could be vulnerable to projected near-future OA, may have important realistic implications for environmental policy. Despite the aforementioned caveats, a precise understanding of the magnitude and sources of variation in the responses to OA will help managers and policy-makers make more accurate generalizations. In turn, this will substantially improve the accuracy of models and forecasts for the effects of OA in future acidified oceans. Acknowledgments This study was mainly supported by the Natural Science Foundation of Fujian Province (2012J01183) and Ningbo City (201301A6107013), and partly funded by the open project from the Key laboratory for Ecological Environment in Coastal Areas (Project No. 201210) and the State Key Laboratory of Tropical Oceanography in South China Sea Institute of Oceanology, Chinese Academy of Sciences (Project No. LTO1203). Financial support was also provided by Xiamen Engineering Center (Institute of Urban Environment) (Grant No. 3502Z20110005) to purchase the AT Control System. The authors would like to express their sincere gratitude to these financial assistances. Anonymous reviewers are thanked for their helpful comments. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.chemosphere.2016.04.086. References  nimo, F., 2012. Energy resource reallocation in Arzate-C ardenas, M.A., Martínez-Jero Daphnia schodleri (Anomopoda: Daphniidae) reproduction induced by exposure to hexavalent chromium. Chemosphere 87, 326e332.

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