11 / 53
Ref. No. [UMCES] CBL 2016-012
ACT VS16-03
11
(5) Potassium iodate standard, 0.1000N ±0.005N commercially available stock (Fisher SP232-1).
Sample Fixing Procedures
(1) Samples were fixed immediately after collection into the BOD bottles. Filling order was noted
on log sheets along with bottle and sample IDs. 1.0 ± 0.05 mL of MnCl
2
was dispensed just below
the water surface, followed by 1.0 ± 0.05 mL of alkaline iodide using positive displacement
pipettors. The pipettors were washed with distilled water every day to prevent valve and plunger
malfunction due to salt crystallization.
(2) The bottle was immediately closed and shaken vigorously. The precipitate was allowed to settle
for about two thirds of the bottle and shaken again to re-suspend the precipitate a second time. A
water seal was immediately added to the neck of the bottle to prevent air suction by the contained
water sample.
(3) Samples were stored in the dark and room temperature (ca. 20
o
C) and temperature variations
were minimized. Samples were titrated within 18 - 24 hours of being fixed.
(4) Samples were acidified just prior to titration. With the precipitate settled to the lower third of
the bottle, 1.0 ± 0.05 mL of 23N H
2
SO
4
was added. The H
2
SO
4
was allowed to flow gently along
the neck of the bottle. The bottle was closed and shaken vigorously, until precipitate was dissolved
(5) If titration was delayed beyond the 24 hour window, the fixed sample remained stored in
darkness and at a temperature equal to or slightly lower than the temperature of the samples, with a
water seal maintained at all times. The sample was acidified only immediately before titration.
Storage at temperatures above the sample temperature cause the loss of I
2
due to the thermal
expansion of the solution of 0.025 mL ·°C
–1
for a 125 ml sample (Carignan
et.al. 1998).
Sample Titration Procedures
Whole bottles were titrated using a Metrohm automated model 916 Ti-Touch titrator
equipped with a 10-mL burette and a Metrohm Pt ITrode. The Pt ring of the electrode was polished
weekly. The titrator was used in the dynamic equivalence point titration (DET) mode, with a
measuring point density of 4, a 1.0-µL minimum increment, and a 2 mV·min
-1
signal drift
condition. In this method, the solution’s potential (controlled by the
I
2
/I
–
and
# %#& ' (#&
– redox
couples) was monitored after successive additions of titrant, where optimal increment volumes are
calculated by the titrator’s software. During titration, the size and rotation speed of the magnetic
stirring bar was controlled in such a way that complete mixing of the I
2
generated during
standardization occurred within 3 - 4 s, without vortex formation. To reduce the titration time (3 -
4 min) and I
2
volatilization, an initial volume of titrant equivalent to 85–90% of the expected O
2
concentration was added at the beginning of the titration. Because the molar volume of water and
the normality of the titrant vary appreciably with temperature, care was taken to standardize the
titrant and conduct all titrations of a given batch of samples at constant temperature (± 1°C).
(1) The stopper of the BOD bottle was removed and, using a wash bottle fitted with a 200-µL
pipette tip, the I
2
present on the side and conical part of the stopper was rinsed into the BOD bottle
with 1 - 2 mL of distilled water.
(2) BOD bottles (Corning No. 5400-125) had been selected to accommodate the displacement of
the electrode without having to remove any volume of the fixed sample.
(3) The stirring bar was inserted into the bottle using plastic or stainless steel forceps.
(4) The delivery tip and the electrode were immersed, the stirrer turned on and the titration begun.
The electrode was not allowed to touch the neck of the bottle.
(5) Once the titration was complete, the equivalence point volume (
V
T) was noted