4.1 PRINCIPLE OF MRT
OECD 301F: Manometric Respirometry Test (OECD, 1992) was selected as the most appropriate method of the RBTs due to the solubility status of the test compounds under investigation.
MRT operates on the principle that a measured volume of inoculated mineral medium, containing a known concentration of the test substance as the only source of organic carbon, is stirred at a constant temperature for 28 days. The microbes from the inoculum consume oxygen in the flask, which leads to carbon dioxide being released. The released carbon dioxide is absorbed by NaOH pellets. The oxygen consumption leads to a change in pressure which is required to maintain constant gas volume in the respirometer. The change in pressure in the flask is converted to BOD using the equation (3):
𝐵𝑂𝐷 = 𝑀(𝑂2)
𝑅×𝑇𝑚 × (𝑉𝑡𝑜𝑡− 𝑉𝑖
𝑉𝑖 + 𝛼𝑇𝑚
𝑇0) × ∆𝜌(𝑂2) (3) Where
M(O2) = Molecular weight of oxygen (32000 mg/mol) R = Gas constant (83.144 L·mbar/mol·K)
T0 = reference temperature (0 OC = 273.15 K) Tm = measuring temperature (20 OC ±2 = 293.15 K) Vtot = bottle volume (theoretical volume) [mL]
Vi = volume of sample
α = Bunsen absorption coefficient (0.03103)
Δρ(O2) = the difference of the partial oxygen pressure [hPa]
4.2 CLOSED RESPIROMETER
Manometric respirometry tests were carried out using Oxitop ® Control System (WTW, Weilheim, Germany). This system is composed of an Oxitop ® OC110 Controller (Figures 4 and 5) which is the interface between the measuring heads and the computer system, Oxitop
®-C measuring heads which are fitted to the BOD flasks contain the respirometer and measures the values automatically and a stirrer IS 12 which holds the BOD flasks in the incubator and make sure they allow for continuous and automated stirring of the content of the bottles with the magnets inserted.
Figure 4. The Oxitop ® OC 110 controller and Oxitop ® - C measuring heads.
Figure 5. BOD bottle fitted with Oxitop® - C measuring heads (left) and BOD bottles fitted with Oxitop ® - C measuring heads placed on the stirrers IS 12 in the incubator (right).
4.3 PREPARATION OF MINERAL MEDIUM FROM STOCK SOLUTION
Based on the guideline of OECD 301F (OECD, 1992), mineral medium was prepared from four stock solutions (A, B, C and D) using analytical grade chemicals.
To a flask filled with 800 ml of deionized water, Solution A (KH2PO4, K2HPO4, Na2HPO4·2H2O, NH4Cl), Solution B (CaCl2), Solution C (MgSO4·7H2O) and Solution D (FeCl3·6H2O) were added (Table 2), and the volume was then adjusted to 1000 ml with deionized water.
Table 2: Preparation of Stock solution and Mineral medium (OECD, 1992)
Solution Chemical Stock Solution Mineral medium
mass (g) volume (l) volume (ml) total volume (ml)
A
KH2PO4 8.50
1 10
1000 K2HPO4 21.75
Na2HPO4·2H2O 33.40
NH4Cl 0.50
B CaCl2 27.50 1 1
C MgSO4·7H2O 22.50 1 1
D FeCl3·6H2O 0.25 1 1
4.4 PREPARATION OF BOD FLASKS
Each analysis consisted of four set-ups: the “blank” series containing only inoculum in the mineral medium, the “reference” series containing readily biodegradable sodium acetate as the only carbon source in mineral medium with inoculum, the “toxicity control” containing the test compound and sodium acetate in mineral medium with inoculum and the actual “test” series, which was the compound under investigation as the only source of carbon in mineral medium containing inoculum.
4.4.1 Test compounds
The test compounds, - N10O and Clodronate, were provided by the research group of Professor Jouko Vepsäläinen of University of Eastern Finland.
The test compounds were dispersed directly to the final volume of mineral medium (1000 ml) to give a test concentration of 50 mg/l. Three test vessels were used for this analysis in each experiment.
The solubility of the compounds was a critical factor in selecting the type of analysis. Both compounds are poorly soluble in water. N10O has a solubility of 58 mg/l while Clodronate has a solubility value of 20.8mg/l.
4.4.2 Reference compound
Sodium acetate (C2H3NaO2) which meets the criteria for ready biodegradability was used as a reference compound to ensure the functionality of the test system, inoculum viability and procedure. The same concentration as test substance (50 mg/l) was used for the analysis. Four test vessels were used as replicates.
4.4.3 Blank series
The blank parallel was made to create allowance to check for the endogenous activity of the inoculum. This series in the test was just the inoculum in mineral medium. Two replicates were made for this series.
4.4.4 Toxicity batch
This batch of parallel was required as there was no available information regarding the toxicity of the test compound. A solution at a mass concentration of 50 mg/l was prepared. The solution was the mineral medium in which there was the test compound (25 mg/l) and reference compound (25 mg/l) mixed together and then inoculated with activated sludge. Duplicates BOD bottles were used for this batch in each test.
4.5 INOCULUM
Activated sludge which was the inoculum used for the studies was obtained from the aeration tank of Lehtoniemi Wastewater treatment plant, Kuopio. Three treatments were tested for the most effective inoculum – untreated activated sludge, filtered activated sludge and settled activated sludge (Appendix I).
Untreated activated sludge was 200 ml of freshly collected activated sludge left at room temperature in a magnetic stirrer to aerate and mix before use without any alteration. 0.5 ml of the activated sludge was dispensed into each BOD flask as an inoculum.
Filtered activated sludge was treated by passing 200 ml of freshly collected activated sludge through a Whatman® Filter paper (Grade 2). 0.5 ml of the filtrate was then introduced into each BOD bottle as the inoculum from the magnetic stirrer where it was left to aerate.
Settled activated sludge was treated by leaving 200 ml of the freshly collected activated sludge to settle for 10 minutes at room temperature. The supernatant was poured out while the residue was left on a magnetic stirrer until ready for use as an inoculum. 0.5 ml of the inoculum was dispensed into each BOD flask. This method was adopted after testing the effectiveness of various treatment on activated sludge. Fresh activated sludge was collected and prepared for use for each analysis.
4. 6 PROCEDURE
Using the 432 ml overflow measuring flask supplied by WTW, Weilheim (Germany), the various solutions were dispensed to their respective prelabelled BOD flask. A magnet bar (4 cm), to ensure proper stirring during incubation, was dropped into each bottle. In order to get more direct results of the carbonaceous demand (CBOD), nitrification was inhibited. Nine drops of allylthiourea (ATU) (5 g/L) was added to each bottle to prevent nitrification. 0.5 ml of inoculum (settled activated sludge) was then dispensed into each bottle. The quiver was inserted to each bottle. NaOH (3 pellets) were dropped into the quiver. The Oxitop ® - C measuring heads was then screwed onto each of the bottles. The Oxitop ® OC 110 controller was then used to start the experiments using the BOD28 programme. The bottles were placed on the Oxitop ® stirrer IS 12 in the incubator for 28 days at a temperature of 20±2 OC during which progress of the experiment was checked periodically. pH was measured on day 0 and day 28 using WTW pH 3210 pH meter (Weilheim, Germany). At the expiration of the 28-days test period, the measured data (BOD) were obtained from the ACHAT OC – v3.20 software for Oxitop ® control system designed by WTW (Weilheim, Germany). The analysis for N10O and Clodronates were repeated three times.
4.7 CALCULATIONS AND EXPRESSION OF RESULTS OF BIOLOGICAL OXYGEN DEMAND, THEORETICAL OXYGEN DEMAND AND PERCENT DEGRADATION
Mean values of the replicates for each batch was calculated daily from the data collected using Microsoft Excel application from Office 365 program. Biochemical Oxygen Demand (BOD) was calculated based on the mean values obtained, by subtracting the Oxygen depleted (mg O2/l) of the inoculum blank from that of the compound under investigation. The corrected depletion was then divided by the concentration (mg/l) of the compound understudy to give the specific BOD as mg Oxygen per mg test substance as in the OECD (1992) guideline (formula 4).
BOD = mg O2⁄l uptake by test substance - O2⁄l uptake by blank
mg test substance l⁄ in vessel =mg O2⁄mg test substance (4)
The theoretical oxygen demand (ThOD) of the hypothetical compound Cc Hh Clcl Nana Oo Pp Ss, of molecular weight (MW), without nitrification, is computed based on formula 5, according to OECD (1992).
ThODNH3 = 16[2c + ½(h – cl – 3n)+ 3s + 5/2p + ½na – o]mg/mg
MW (5)
Based on this information, the ThOD of the reference compound – sodium acetate and test compounds – N10O and Clodronate were calculated (Table 3).
Table 3: ThOD of compounds
Compound Molecular formula Molecular weight (g/mol) ThOD
Sodium acetate C2H3NaO2 82.034 0.78
N10O C11H27NO7P2·H2O 365.24 1.40
Clodronate CH2Cl2Na2O6P2·4H2O 360.83 0.089
OECD (1992) guidelines was referred to in calculating for the biodegradation percentage, which is the amount of oxygen uptake by the microbes (from inoculum) corrected for by deducting the oxygen uptake in the blank series, expressed as percent of the Theoretical Oxygen Demand (ThOD) as shown in formula (6):
Biodegradation (%) = 100 × BOD
ThOD (6)
Where:
BOD is biochemical oxygen demand of the compound under study (mg/L)
ThOD is the theoretical oxygen demand required to completely oxidize a compound (mg/L)