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Ref. No. [UMCES] CBL 2016-011
ACT VS16-02
12
(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
Thiosulfate Standardization
The Thiosulfate was standardized at room temperature as the first and last step in daily analysis.
Either triplicate assays of a fixed volume of iodate standard was run, or a range of volumes
(≥ 3) bracketing the normal sample titration range (eg. 0.500, 1.000, 1.500, 2.000 mL for well
oxygenated waters.) A clean BOD bottle and clean glassware were dedicated to this purpose.
(1)
Insert a stirring bar into a 200 mL beaker.
(2) With mixing add 1.0 mL of the H
2
SO
4
reagent followed by 1.0 mL of the alkaline iodide and
then 1.0 mL Mn
2+
reagent.