Methods

Sample Collection

The samples of M. vertebralis were collected from Makuluva and Nukubuco reefs on the Suva Barrier Reef (Fig. 8.1). Sharma (2007) identified a total of 68 different species of benthic foraminifers in the Nukubuco Reef flat in Laucala Bay, including abundant M. vertebralis. Moreover, Sharma cultured living specimens to study growth, abundance and reproduction of marine benthic foraminifers.

The location of the collection site was determined using GPS data. Salinity and temperature of the sample collection area were assessed using an YSI-85 m. The map of Viti Levu is enlarged in Fig. 8.1c to show the research site. During low tide

a Map of Laucala Bay; b the mapping of the site of collection of Marginopora vertebralis; c algal flat habitat between Sandbank Island and Nukubuco Reef; and d M

Fig. 8.1 a Map of Laucala Bay; b the mapping of the site of collection of Marginopora vertebralis; c algal flat habitat between Sandbank Island and Nukubuco Reef; and d M. vertebralis attached to algae and coral (arrow). Scale bars a 5 km; b 0.06 km (Source: SOPAC 2012; Sharma 2007); d 1 cm

(0.3 m), M. vertebralis was easily collected as it is large enough to be identified in situ. The foraminifers and associated sediments were collected using hand scoops and immediately placed in an aerated aquarium with filtered seawater, where they were kept overnight. The next day, M. vertebralis specimens had crawled upward and attached themselves to the sides of the aquarium and therefore could be easily identified and removed, based on the methods of Sharma (2007).

Natural seawater (salinity 33.0), contained in 35 L reservoir tanks, was acidified by means of a bubbling system supplying CO2 gas (Fig. 8.2). The gas, saturated with water vapour (to limit evaporation) was injected through the water as very fine bubbles allowing the gas to rapidly dissolve. The seawater pH in the reservoirs was monitored using a flat-surface, combination pH electrode (YSI Environmental pH 100). Once the pH reached the required level, the supply of CO2 was halted via an automated feedback relay system (Accu-Max pH controller). As the acidified water was pumped from the reservoir to supply the experimental tanks, the overflow water was returned to the reservoir. Whenever the pH increased, CO2 bubbling was triggered to ensure that the desired pH level was maintained. Each reservoir contained a CO2 Reactor, an air pump and a water pump. The reservoirs were refilled with natural seawater (pH 8.1) every 10 days from a separate 300 m3 seawater tank, causing the pH in the reservoir tank to increase. This increase triggered the supply of CO2 to be restarted and CO2 continued to bubble through the water until the pH was reduced to the required level. Using this method it was possible to supply large quantities of CO2 acidified seawater with a consistent pH. Two seawater acidification reservoir tanks (pH 7.8, pH 7.5) and one control (ambient pH 8.1) were maintained. From each tank, water was supplied to the corresponding three small tanks (25 x 10 x 15 cm) where the M. vertebralis specimens were cultured.

The experimental setup diagram (Fig. 8.2) shows how the connections were made from the reservoirs to the connecting tanks and vice versa. The CO2 system is plugged with pH controllers (adjusted at pH 7.8 and 7.5). The CO2 is released with the help of the bubble counter, check valve and CO2 injector. The pH controller regulates and stabilizes the pH in the reservoirs and the peristaltic pump helps in the circulation of seawater, creating an ocean-like environment. Nine (4.5 L) tanks (3 treatments with 3 replicates each) were placed in a room (ambient temperature *27.5 °C) and connected to a pH controller system (Fig. 8.2). Each tank was individually supplied with seawater at a rate of approximately 3.3 mlmin-1 using a peristaltic pump. The M. vertebralis specimens were randomly assigned to one of the three pH treatment levels (8.1 [ambient seawater], 7.8, and 7.5).

The treatments were maintained under a 12-h light and 12-h dark cycle using a T5 Aquarium lamp with a light intensity of 42.5 wattsm-2. Acidification of the seawater did not begin until 24 h after the M. vertebralis specimens were placed in their treatment tanks. Seawater pH was reduced gradually over a period of two days and the experiment started when the final water chemistry for each treatment was reached. The experiment ran for 11 weeks, during which time, the pH of the water supplied to the tanks was monitored daily via a pH controller (Accu Max I). Samples for Total Alkalinity were taken at the beginning of the experiment and then once per week throughout the duration of the experiment. The values given by the

Experimental set-up model

Fig. 8.2 Experimental set-up model

pH electrodes in the reservoir tanks were cross-checked every week against values measured by a regularly calibrated pH meter (InLab 413SG, Mettler-Teledo).

The carbonate chemistry in the culture media was determined using the following procedures and calculations (Pawlowski 1994). Borosilicate bottles, used to collect the water samples for alkalinity measurements, were rinsed with concentrated HCl, followed by at least five rinses with deionised water. Seawater samples were collected from the experimental aquaria. Borosilicate bottles were fully filled and tightly capped. Analysis was carried out within 6 h of sample collection. To standardize acid used in titrations, 10 ml aliquot of 0.01 M sodium tetraborate was placed into a 250 ml conical flask and a few drops of mixed indicator were added. The aliquot was titrated with 0.01 M HCl until the end point was reached (color changed from green to purple). The titre was recorded for the calculation of HCl concentration:

To determine the total alkalinity (AT) of a sample, 50 ml of the water sample were pipetted into a 250 ml Erlenmeyer flask and few drops of phenolphthalein indicator were added. Once the solution color turned pink, it was titrated with 0.01 M HCl until the color disappeared and the volume of added acid was recorded. Then 2-3 drops of mixed bromocrescol green/methyl red indicator were added to the same solution and titrated with standardized 0.01 M HCl to a pink color. Then AT was calculated using this formula:

where A = Volume of standard HCl used (ml)

The MATLAB program CO2SYS (single-input mode directly adapted from Lewis et al. 1998) was used to calculate and present seawater parameters where the salinity was constant at 35 and temperature was constant at 27.5 °C. The input temperature and pressure were from measurements performed in the laboratory. There are four measurable parameters of the aquatic carbon dioxide system: pH, pCO2, total dissolved inorganic carbon (DIC) and total alkalinity (TA). The measured pH and alkalinity values were entered into the program. The equilibrium constants from Mehrbach et al. (1973) as refit by Lueker et al. (2000) on a total scale were used. The measurements made by Mehrbach et al. (1973) were made on real seawater.

Input variables (input conditions):

  • • Salinity (35), Temperature (27.5 °C) and Pressure (1 atm).
  • • Total Si (optional) and Total Phosphate (optional). If left empty, the total Si and total P concentrations are assumed to be zero in the calculations.
  • • Two (2) known CO2 parameters (TA, pH).

Output (for both “input” and “output” conditions):

  • • The other CO2 parameters (pCO2), total dissolved inorganic carbon [DIC].
  • • Contributions to the alkalinity.
  • • Carbonate speciation.
 
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