Author Version: Estuar. Coast. Shelf Sci., vol.153; 2015; 96–107 Effect of salinity induced pH/alkalinity changes on benthic foraminifera: A laboratory culture experiment Rajeev Saraswat*, Mamata Kouthanker, Sujata Kurtarkar, Rajiv Nigam, S.W.A. Naqvi, V.N. Linshy Micropaleontology Laboratory, National Institute of Oceanography, Goa, India * For Correspondence: Phone: 91-832-2450622; Fax: 91-832-2450603; Email: [email protected] Abstract The salinity of coastal waters in the vicinity of seasonally fresh water fed estuaries changes tremendously and reportedly affects the living calcite secreting organisms like foraminifera, as well as their dead remains. The precise mechanism of adverse effect of such seasonal salinity changes on calcite secreting organisms is, however not clear. The seasonal fresh water influx from the estuaries also affects the pH and alkalinity of the coastal seawater. Therefore, to understand the effect of salinity induced pH/alkalinity variations on benthic foraminifera, living specimens of Rosalina globularis were subjected to different salinity. Additionally, water samples were collected from an estuary during both monsoon and post monsoon season to understand the relationship between salinity, pH and total alkalinity (TA). The pH decreased with decreasing salinity during both the seasons. A similar decrease in TA with decreasing salinity was also observed but only till 20 psu salinity, below which the TA increased with decreasing salinity. Even though the maximum growth was reported in specimens kept at 35 psu salinity, growth of specimens maintained at >25 psu salinity, was same. Specimens kept at 10 psu and 15 psu salinity, however were much smaller and turned opaque within two days of lowering the salinity and later on their tests dissolved within 24 and 43 days, respectively. No specimen reproduced at 10 psu and 15 psu salinity, while only a few specimens (3%) reproduced at 20 psu salinity. As compared to 10-20 psu salinity, ~60 % reproduction was observed in specimens subjected to 25-40 salinity. The specimens maintained at 20 psu salinity took twice the time to reach maturity than those subjected to 25- 40 psu salinity. Since a big drop in pH was observed at 10-15 psu salinity (pH 7.2 and 7.5, respectively), while the alkalinity was still higher, we suggest that fresh water influx induced drop in pH adversely affects calcification and reproduction in benthic foraminifera. The response is, however not linear as beyond a certain limit, a further increase in pH does not affect benthic foraminifera; rather they respond to salinity as per their salinity tolerance range. It is further inferred that the time required to reach reproductive maturity increases at the extreme salinity tolerance limits. Dissolution of calcareous foraminifera below 20 psu salinity, suggests that salinity induced changes control the carbonate inventory in the coastal regions subjected to seasonal fresh water influx. 1 1. Introduction Benthic foraminifera, unicellular preferentially marine microorganisms are amongst the dominant group of organisms with calcareous exoskeleton (test), living in the shallow water regions of the world oceans. The foraminifera sequester huge amount of CO thereby removing it from the atmosphere, thus helping in 2 mitigating the effect of anthropogenic CO . All the foraminiferal tests however do not get buried in the 2 sediments. A large fraction of the tests remineralizes, before it gets buried and even after the burial in the sediments. Therefore, it is necessary to understand the factors that affect foraminifera. A change in seawater pH/alkalinity due to fresh water influx, re-suspension of bottom sediments, re-mineralization, aerobic/anaerobic oxidation of organic matter, have been suggested as some of the possible factors that control dissolution of benthic foraminiferal tests in shallow water coastal regions (Berkeley et al., 2007). Out of these several factors, the change in ambient pH affects the benthic foraminiferal tests to a very large extent, especially in the estuarine and coastal waters, where a large seasonal change in pH/alkalinity is reported. Additionally, anthropogenic derived ocean acidification (OA) will also affect calcification and subsequent burial of benthic foraminiferal tests. Benthic foraminifera are however capable of increasing the pH of vacuolized seawater at the site of calcification by one unit above seawater pH to secret calcite (Bentov et al., 2009; Nooijer et al., 2009), suggesting their capability to withstand a change in seawater pH. The capability of benthic foraminifera to modulate the pH of vacuolized seawater under different seawater pH conditions and the lowest pH that calcareous benthic foraminifera can tolerate is, however not clear (ter Kuile et al., 1989; Le Cadre et al., 2003; Kuroyanagi et al., 2009; Kurtarkar et al., 2011). Benthic foraminifera with hyaline and porcelaneous tests respond differently to change in seawater pH (Fujita et al., 2011; Hikami et al., 2011), suggesting that the response to OA varies from species to species as different benthic foraminiferal species are adapted to different habitats. While the drop in open ocean pH is the result of enormous input of CO in the 2 atmosphere by various anthropogenic activities (Caldeira and Wickett, 2003), local factors like fresh water runoff, coastal erosion, fertilizer input have also lead to the development of shallow water regions of increased ocean acidification (Doney et al., 2007; Naqvi et al., 2009; Cheung et al., 2009; Cooley and Doney, 2009; Doney et al., 2009; Zhang et al., 2010; Elliott and Kennish, 2011; Kelly et al., 2011). Dissolution of biogenic carbonate in such regional OA zones (Green et al., 1993; Alve and Murray, 1994; Barbieri 1996; 2001; Berkeley et al., 2007; 2009), unrelated to anthropogenic-derived OA makes it difficult to understand the possible response of calcite secreting organisms to anthropogenic-derived OA in such zones. Therefore, it is necessary to understand the effect of seasonal fresh-water influx induced physico-chemical changes on benthic foraminifera. It will help in assessing the relative contribution of OA and fresh-water influx induced dissolution of benthic foraminifera in shallow water coastal regions. Controlled laboratory culture studies can help to understand the response of benthic foraminifera to fresh water influx induced physico-chemical changes. Since riverine influx is one of the major factors for the development of localized zones of low seawater pH (Padmavati and Goswami, 1996; Kurtarkar et al., 2011), 2 here we have studied the response of benthic foraminifera to salinity induced seawater pH/alkalinity changes, under controlled laboratory conditions. 2. Materials and Method The experiment was conducted by following the guidelines provided by Riebesell et al (2010). To understand the relationship between seawater salinity, total alkalinity and pH, water samples were collected from the Mandovi-Zuari estuaries, which bring freshwater to the coastal regions off Goa during the southwest monsoon season. The water was collected from the mouth of the estuaries till the inner reaches (to cover a wide range of salinity) by using Niskin water sampler, during both monsoon (July) and post monsoon (October) season (Figure 1). The water was transferred to narrow mouth 1 l bottles and kept in dark and its salinity and pH was measured on the same day by using auto-cell and pH meter. Additionally, water samples were also collected for total alkalinity. The salinity of the media was measured by ATAGO hand-held salinity refractometer onboard, followed by auto-cell in the laboratory. The pH was measured by Labindia PHAN microprocessor controlled pH analyzer with a precision of ±0.02 pH units and ThermoScientific Orion Star A329 multi parameter meter which has a precision of ±0.01 pH units. For pH measurements, the electrode was standardized by using NIST buffers of pH 4, 7 and 10 at 25°C. The total alkalinity was measured by using VINDTA (Versatile INstrument for the Determination of Total Alkalinity) system. To get living benthic foraminifera, material including the top 1 cm of the sediments as well as sea grass attached to the perennially submerged rocky cliffs was collected from the waters off Dias Beach, Goa coast (Figure 1). The sea water was also collected for further use in laboratory as media. The sea grass was transferred to a plastic tub containing sea water and stirred vigorously to detach foraminifera attached to it. The entire material was sieved through 1000 µm and 63 µm sieves to remove extraneous material. After thoroughly cleaning the sample, 63-1000 µm fraction was transferred to glass beakers with sea water. In the laboratory, the material was observed under stereo-zoom microscope (Olympus SZX16) to separate living benthic foraminifera. Probable living specimens were picked using a micropipette or a very fine tipped brush and transferred to multi-well (6-wells) culture petri-dishes (Axygen). The living specimens separated under binocular microscope were subsequently observed under inverted microscope (Nikon ECLIPSE TE2000-U) for pseudopodial activity, movement, food collection, etc, to confirm their living status. Once confirmed to be alive, the specimens were divided into several batches and kept at different ecological conditions (salinity varying from 20 psu to 40 psu and temperature varying from 15°C to 30°C) to facilitate reproduction. The offsprings of the reproduced specimens, that is the juveniles with two-three chambers, were subjected to different salinities. All the juveniles were taken from specimens kept at 35 psu salinity and 27°C temperature as the maximum reproduction was observed at this salinity-temperature. The advantage of conducting experiments on juveniles is that there is ample time for the specimens to respond to physico- chemical conditions, before they mature and reproduce. 3 The experimental specimens were subjected to seven different salinity (10, 15, 20, 25, 30, 35, 40 psu). This salinity range was selected based on continuous monitoring of physico-chemical parameters in the field from where living specimens were collected (Rodrigues, 1984; Nigam et al., 2008). Temperature (27°C) was kept constant throughout the experiment. Five juvenile specimens were kept in each well of the 6-well culture petri-dish and thus each culture dish contained 30 specimens. Each well has a diameter of 35 mm, height 20 mm and the working volume of each well was 10 ml. One culture petri-dish was designated for a single salinity making a total of 7 dishes for the experiment. The experiment was carried out in replicate. Initially, all the specimens were maintained at 35 psu salinity as the juveniles were taken from specimens subjected to this salinity. Subsequently, the salinity was changed gradually (in steps of 2/3 psu) to reach desired salinity. The salinity was changed gradually in order to avoid experimental shock which specimen could experience if transferred directly to the desired salinity (Fig. 2). On second day, salinity in one culture dish was maintained as 35 psu and in other one, it was increased to 37 psu. The specimens subjected to 35 psu were treated as the control set. On the same day, the salinity in rest of the trays was lowered to 33 psu. On 4th day, salinity of the media in the culture-dish kept at 37 psu was increased to 40 psu and that of the remaining 5 sets was lowered to 30 psu. On 6th day, salinity of the four out of five sets kept at 30 psu salinity was further lowered to 27 psu. Likewise, the salinity was gradually lowered every alternate day until all the sets had desired salinity of 25, 20, 15 and 10 psu. All the culture trays were covered with polythene cling-film to avoid evaporation. The media of different salinity was prepared by mixing water of salinity varying from 5 psu to 35 psu, which was collected from the Mandovi estuary. Seawater of salinity >35 psu was prepared by natural evaporation of 35 psu salinity seawater. The stock culture bottles were air-tight and the pH of the media was checked every time before we changed media in culture dish. Even the pH of water that was removed from the culture trays was also checked. The pH variation was insignificant throughout the experiment. The incubation temperature was kept constant (27°C) throughout the experiment by maintaining the culture trays in cooling incubators (Sanyo make, Model MIR-154, temperature fluctuation ±0.2°C, temperature uniformity ±0.5°C) with 12 hour light and dark cycles. Living diatom Navicula (100 μl solution, ~20 cells) was added as food, every time the medium was changed. This diatom species was chosen for food since foraminifera feed on diatoms and Navicula species has been reported from the area from where material for living specimens was collected. Media was changed every alternate day whereas pseudopodial activity, growth and the maximum diameter was measured every fourth day. Foraminifers were examined using Nikon ECLIPSE TE2000-U inverted microscope and the photographs of the living specimens were taken with an Olympus DP21 camera. The maximum diameter of all specimens was measured by using Cell Sens StandardTM prior to incubation and weekly once thereafter until either the specimens reproduced, died or stopped responding. The experiment was stopped after 75 days when either the specimens had reproduced or died, or stopped growing. During the course of the experiment, a few specimen developed abnormal tests. The growth of such abnormal specimens was not considered to calculate average growth. But, these abnormal specimens were 4 monitored throughout the experiment, till they reproduced or died or stopped responding. Additionally, the chambers of a few specimens also broke in between. The growth of such specimens was also not considered to calculate the average growth. Since it was difficult to identify and track individual specimens kept in a well of the culture tray, average of the growth of all the specimens kept in each well was considered. 3. Results 3.1. Salinity-pH-Alkalinity Relationship The pH varies from 6.77 to 8.04 during the monsoon season, and from 6.93 to 7.97 during post-monsoon season. The pH decreases with decreasing salinity during both monsoon and post monsoon seasons (Fig. 3). The total alkalinity was measured for the post monsoon samples and varies from 731 µmol/kg to 2256 µmol/kg. The total alkalinity also decreases with decreasing salinity but only till 20 psu salinity. Below 20 psu salinity the alkalinity increases with a further decrease in salinity, probably due to soil-water interaction at shallow water depths. The relationship between salinity and pH of the seawater samples collected from the field matches with those prepared in laboratory, with a small difference probably due to the preparation of high salinity (40 psu) media by controlled evaporation in the laboratory. 3.2. Growth A considerable growth occurred in all the sets during the initial 15-20 days (Fig. 4). It should however be noted that during this period, salinity of the various sets was gradually changed to bring it to the desired salinity levels. The desired salinity levels in all sets were achieved after 20 days from the beginning of the experiment. A few of the specimens kept at >20 psu salinity were alive till ~75 days. The specimens subjected to 10 psu salinity responded till only 45 days whereas those kept at 15 psu salinity responded till 63 days from the beginning of the experiment. The average growth was highest (167±10 µm) in specimens kept at 35 psu salinity (Fig. 4). The average growth of specimens subjected to 25 psu (166±16 µm), 30 psu (151±25 µm) and 40 psu salinity and (164±1 µm) was nearly same as that of the specimens subjected to 35 psu salinity (Fig. 4). It should however be noted that during the later stages of the experiment, many of the specimens kept at 30 psu salinity reproduced. Such specimens were than not considered to calculate the average growth. As compared to this, less number of specimens kept at 25 psu salinity reproduced and most of the specimens were still growing, leading to higher average growth. The minimum growth (129±10 µm) was observed in specimens subjected to 20 psu salinity. The specimens at 10 psu and 15 psu salinity grew only when the salinity was still lowered to bring it to the desired levels. The growth in these specimens stopped immediately after attaining the desired salinity of 10 psu and 15 psu. The maximum diameter of any specimen at different salinity was also noted since the average growth was calculated from the living specimens and not all the specimens lived till the end of the experiment. The maximum diameter of the specimen subjected to 40 psu salinity was the largest (359±40 µm), followed by 5 that of 35 salinity (324±24 µm) (Fig. 5). The maximum diameter of the specimen subjected to 10 psu and 15 psu salinity is irrelevant as these specimens grew only when the salinity was still lowered to reach desired salinity levels. Except 25 psu salinity, the maximum diameter varied linearly with the salinity. 3.3. Dissolution Specimens grown at 10 psu and 15 psu salinity became opaque and began to dissolve within 2 days of lowering the salinity. After a few days, the number of pores in the last chamber these specimens also increased due to dissolution which rendered chambers completely transparent. The increase in porosity in the final chamber however can also be due to another physiological stressor, viz. oxygen, as it has been illustrated that some benthic foraminifera change their pore density as a function of oxygen levels (Glock et al., 2011; Kuhnt et al., 2013). The chambers which became transparent, began to dissolve. Dissolution progressed from the last chamber to initial chamber (Plate 1). Initially 4-5 chambers were dissolving one after another but later the whole test started to dissolve at a time. Dissolution was more prominent in specimen subjected to 10 psu salinity and 60% specimen died within 24 days from lowering the salinity. As compared to this, out of the entire specimens kept at 15 psu salinity, the tests of ~23% specimens dissolved within 39 days of lowering the salinity. However, later on, the rate of dissolution increased and the tests of ~93% specimen dissolved within next 11 days. The tests of all the specimens maintained at 10 psu salinity dissolved by this time. 3.4. Abnormality Several specimens subjected to various salinities (Plate 2) developed abnormalities from the 6th to 11th day onward. The number of abnormal specimens was very low at 25 psu to 40 psu salinity, with only 2-3 specimens having abnormal chambers. The maximum number of abnormal specimens was noticed at 20 psu salinity, wherein a total of 10 specimens had abnormal chambers. After lowering the salinity, abnormal chambers were observed in 4-5 specimens at 10 psu and 15 psu salinity as well, before calcification stopped and tests started to dissolve. Specimens maintained at 15 psu salinity developed abnormalities after 17 days of lowering the salinity while those kept at 10 psu salinity had abnormal chambers within 2 days. The abnormalities included exceptionally large or small chamber and addition of chambers in a plane other than the normal plane of addition of chambers. 3.5. Reproduction Many of the specimens reproduced during the course of the experiment. Even though the paired specimens (prerequisite for sexual reproduction) were also observed in a few wells, none of such pairs reproduced, probably because not all the requirements for sexual reproduction were met. Thus all the specimens reproduced asexually. The specimens formed a cyst (of food material) prior to reproduction. The juveniles with three-four chambers came out by breaking the parent test (Plate 3). The percentage of reproduction was same at 30 psu, 35 psu and 40 psu salinity (60±9%, 60±9% and 63±5%, respectively) (Fig. 6). Although the percentage of specimen reproduced at 40 psu salinity, was same as that at 30 psu and 35 psu, a few of the 6 specimens reproduced abnormally. The number of juvenile in such abnormally reproducing specimens was less (only 5-8) as compared to other specimens (59±9). As compared to this, only 40±19% of the specimens subjected to 25 psu salinity reproduced. The least reproduction was noted at 20 psu salinity (~3%). None of the specimen reproduced at 10 psu and 15 psu salinity. Out of all the juveniles, those at 35 psu salinity have shown good growth. The commencement of reproduction varied with salinity (17 to 66 days). The earliest reproduction (17 days) occurred at 35 psu salinity, while the specimens kept at 20 psu salinity were the last to reproduce (66 days). Average time taken for reproduction was comparable for both 30 psu and 35 psu salinity (24 and 25 days respectively), whereas that for 40 psu salinity, it was more (29 days). The specimens kept at 25 psu salinity took still more time to reproduce (35 days). The majority of the specimens have reproduced by ~38 days. Time taken for reproduction at 20 psu salinity was twice that at higher salinities; by this time second generation was observed at higher salinities. All the specimens have reproduced asexually. There was no apparent relationship between average size of the specimen at the time of reproduction and salinity. The average number of juveniles produced per specimen was the highest (62±8) at 35 psu salinity, followed by those at 40 psu salinity (59±9) (Fig. 7). The average number of juveniles produced by the specimens maintained at 25 psu and 30 psu salinity was less (35±9 and 49±7, respectively) than those at 35 psu and 40 psu salinity. The least number of juveniles per specimen were observed at 20 psu salinity. 3.6. Mortality Out of all the specimens, 1 specimen each at 30 psu and 35 psu didn’t respond well. The one at 30 psu salinity grew by 17 µm before it died after 31 days while the other at 35 psu died within 24 days after a growth of 3 µm only. Even though initially all specimen showed good growth, the growth rate declined at all salinities after attaining a certain growth (Fig. 4). Further progress of experiment resulted in the death of 20% specimen at 30 psu and 35 psu salinity while ~13% specimens died at 40 psu and 25 psu salinity (Fig. 8). It was observed that, after 40 days of attaining the desired salinity of 20 psu, the specimens turned opaque and subsequently ~7% specimens died at 20 psu salinity. All the specimens (100%) kept at 10 psu salinity died within 45 days while all those at 15 psu died within 63 days. The death of the specimens kept at 10 psu and 15 psu salinity was preceded by dissolution of the entire test. 4. Discussion The linear relationship between salinity and pH during both the monsoon and post monsoon season suggests that freshwater influx from the land during monsoon season will decrease both the seawater salinity and pH in the shallow water coastal regions. The salinity, pH and alkalinity relationship however is region specific and in a few estuarine systems, deviations have been previously reported (Wong, 1979; Sarma et al., 2001; Kurtarkar et al., 2011). The salinity-pH-total alkalinity relationship depends on several factors including mixing, composition and re-suspension of bottom sediments, vegetation and anthropogenic activities in the catchment area, aerobic or anaerobic dissolution or organic matter (Sankaranarayanan and Qasim, 1969; 7 Wong, 1979; Pinol and Avila, 1992; Sarma et al., 2001; Berkeley et al., 2007). The increase in TA at stations with salinity lower than 20 psu is attributed to soil-water interaction as the water depth is only 5-6 m at these stations (Wong, 1979; Sarma et al., 2001). 4.1. Cause for dissolution at low salinity The TA at salinity lower than 20 psu is comparable with that at salinity >30 psu, thus suggesting that alkalinity may not be the factor which results in distinctly different response (smaller size, dissolution, mortality) of benthic foraminifera at 10 psu and 15 psu salinity. The linear decrease in seawater pH with decrease in salinity, suggests that salinity induced change in seawater pH affects the benthic foraminifera. This salinity induced pH change has been previously suggested as one of the most important ecological factors that affect foraminiferal population especially in the coastal areas (SenGupta, 1999). The fact that R. globularis was alive at salinity ranging from 20 psu to 40 psu (pH 7.7 to 8.2) shows that it can tolerate wide range of salinity (pH). A similar response was also observed in corals which survived well when subjected to a range of aragonite saturation levels, which depends on the seawater pH (Gattuso et al., 1998). Immediate dissolution of the test of specimens subjected to 10 psu and 15 psu salinity (pH 7.2 and 7.5, respectively) puts the lower limit of salinity tolerance for this species at 15 psu salinity (pH 7.5). The dissolution of the tests below 20 psu salinity was also observed in another species belonging to the same genus, i.e. Rosalina leei (Kurtarkar et al., 2011). Nigam et al (2006a) also reported dissolution and death of Pararotalia nipponica specimens below 15 psu salinity. A large increase in dissolution of benthic foraminiferal tests at lower pH was also reported by Haynert et al (2014). In a field study from the seas around the island of Ischia (Italy) where volcanic gas vents emit carbon dioxide from the sea floor lowering the pH, no calcareous forms were reported in the region with seawater pH below 7.6 (Dias et al., 2010). Even the large benthic foraminifera, Amphisorus hemprichii and Amphistegina lobifera which are usually reported from coral reefs, show a significant calcification only up to pH 7.6 (ter Kuile et al., 1989). However, this limit is well above than that reported by Le Cadre et al (2003) who observed decalcification of benthic foraminifer Ammonia beccarii at pH 7.0. Similarly, we conclude that benthic foraminifera can tolerate seawater with significantly low pH (7.5) than that reported by Uthicke et al (2013) (7.9). Additionally, we would like to mention that the lower pH tolerance limit of benthic foraminifera may be further lower as experiments suggest that the pH sensitivity of individual specimens isolated from the sediments, is much higher than that of the same species in its original habitat with sediments (Haynert et al, 2014). Therefore, we further conclude that it is highly unlikely that the benthic foraminifera will become extinct under the projected ocean acidification scenario, as suggested by Uthicke et al (2013). The lower pH tolerance limit of benthic foraminifera (7.5) is comparable with that of the corals as reef building also stops at pH below 7.7 (Fabricius et al., 2011). The response of benthic foraminifera is also similar to that of pteropods which also start dissolving under low pH conditions (Orr et al., 2005). The calcite production at increased CO concentrations (low pH) also declined in two dominant marine calcifying 2 phytoplankton species, the coccolithophorids Emiliania huxleyi and Gephyrocapsa oceanica (Riebesell et al., 2000). A similar preferential syndepositional dissolution of calcareous tests as compared to the agglutinated 8 forms has also been reported from shallow water regions of several places and was attributed to the fresh water influx, metabolization of organic matter and bacterial destruction (see Berkeley et al., 2007 for review). The tests of P. nipponica whose response to different salinity was studied by Nigam et al (2006a) are robust and thick walled as compared to that of both the R. globularis and R. leei. But all three of these are epifaunal species. It indicates that large freshwater influx can create localized zones of low seawater pH which may result in significant dissolution of all types of epifaunal benthic foraminiferal tests. Such large scale dissolution of benthic foraminiferal tests will however occur only if the low pH condition persists for longer time. Otherwise, the benthic foraminifera are capable of recovering and rebuilding the tests after short-term exposure to low saline (low pH) conditions (Kurtarkar et al, 2011). Such short-term low pH events will however, lead to abnormality in benthic foraminifera. The dissolution of the benthic foraminiferal tests, after prolonged exposure to 20 psu salinity indicates that hyposaline water (low pH) is detrimental to the survival of R. globularis. However the response time may vary from species to species. While the complete dissolution and death of all the specimens of both P. nipponica and R. globularis occurred within 25 days of lowering the salinity to 10 psu, the P. nipponica specimens survived longer (~100 days) than R. globularis (63 days) at 15 psu salinity (Nigam et al., 2006a). This difference in survival time at 15 psu salinity is probably due to the more robust tests of P. nipponica. It should however be kept in mind that the benthic foraminiferal response to change in seawater pH due to fresh water influx might be different than that because of higher CO levels. The response to change in 2 seawater pH due to elevated CO levels also depends on whether the species hosts symbionts, the type of 2 symbionts as well as the arrangement of calcite crystals in the tests. Both Baculogypsina sphaerulata and Calcarina gaudichaudii, which have a hyaline test and host diatom symbionts, secreted more calcite when subjected to pCO levels higher (580 and/or 770 μatm) than the present, whereas Amphisorus hemprichii, a 2 porcelaneous benthic foraminifera that hosts dinoflagelate symbionts secreted less calcite at these pCO 2 levels. The net calcification, however also decreased in both B. sphaerulata and C. gaudichaudii when subjected to very high pCO (970 μatm) (Fujita et al., 2011). A contrasting response of two reef‐dwelling 2 foraminifera, Amphisorus kudakajimensis and Calcarina gaudichaudii, to different pCO2 levels (245-907 μatm) was also attributed to the different types of algal symbionts and species specific sensitivity (Hikami et al., 2011). We suggest that CO induced drop in seawater pH to a certain level may be favorable to a few 2 benthic foraminiferal groups, due to the ease in calcification as a result of associated symbionts which favour elevated pCO levels. The fresh water influx induced drop in pH will, however adversely affect all benthic 2 foraminifera. 4.2. Morphological changes in response to different salinity The maximum growth was observed in the specimens maintained at 35 psu salinity while the growth was low in specimens kept at both the higher and lower than 35 psu salinity. Non-linear response to salinity induced pH change indicates that within a certain range of seawater pH, other factors (here salinity), affect the growth 9 and reproduction (Nigam et al., 2008). A similar non-linear response of this species to another physical parameter viz. temperature was also observed (Saraswat et al., 2011). A few other benthic foraminiferal species also show a non-linear response to seawater pH (Kuroyanagi et al., 2009). Bradshaw (1961) also reported that the highest growth rate was observed in cultures of benthic foraminifera Ammonia beccarii tepida at normal salinity (34 psu) and that the growth rate decreased at lower salinity. Less growth at high salinity is in agreement with the results of Stouff et al (1999) who found that Ammonia tepida specimens showed less growth when subjected to salinity higher than normal. A similar decrease in test growth was also noted in modern planktic foraminifera collected in sediment traps deployed in the Southern Ocean as compared with the Holocene counterparts of the same species (Moy et al., 2009). Recently, is was shown that the planktic foraminiferal shell growth is directly proportional to [CO 2-] concentration, which varies with pH, 3 thus suggesting a strong effect of seawater pH on calcification efficiency of foraminifera (Marshall et al., 2013). The reduced planktic foraminiferal test growth during modern times was attributed to the high CO 2 concentration as compared to the preindustrial levels. Even during older times, the foraminiferal test weight varied with changing atmospheric CO concentration (Gonzalez-Mora et al., 2008). Laboratory culture 2 experiments on planktic foraminifera (Orbulina universa and Globigerinoides sacculifer) also show significant drop in calcification under high CO (low pH) condition (Lombard et al., 2010). 2 The maximum size attained by any specimen was the largest at the highest salinity, again suggesting a dominant effect of seawater salinity induced pH changes on benthic foraminifera. The highest saline water (40 psu) has the most alkaline pH (8.2). It is easier for benthic foraminifera to secrete tests under alkaline conditions. A similar response was also observed in another large benthic foraminifera Marginopora kudakajimensis, wherein maximum size was noted at highest pH (Kuroyanagi et al., 2009). The larger size, however, may also reflect growth of specimens without undergoing reproduction. Since reproduction occurs under narrow range of physico-chemical conditions, the specimens continue to grow without reproducing at conditions other than those favorable for reproduction, leading to a larger size. This explanation is supported by the larger size of the specimens subjected to 25 psu salinity as compared to those at 30 psu and 35 psu salinity. Here also, the specimens took longer time to reproduce as compared to the specimens subjected to 30 psu and 35 psu salinity. The number of abnormal specimens increased with decreasing pH. A similar response has also been observed in several benthic foraminiferal species collected from ecologically stressed environments (Boltovskoy et al., 1991; Dolven et al., 2013). Laboratory culture studies also confirmed the development of abnormal tests under ecologically stressed conditions (see Linshy et al., 2007, for review). Other calcareous organisms like coccoliths also showed a similar response wherein an increased proportion of malformed coccoliths and incomplete coccospheres developed under low pH condition (Riebesell et al., 2000). This work however does not help in delineating the abnormalities in benthic foraminifera as reported from polluted environments (Nigam et al., 2006b), and also confirmed in laboratory culture (Saraswat et al., 2004) with that from naturally ecologically stressed regions (Boltovskoy et al., 1991; Dolven et al., 2013). Additionally, 10