11 / 73
Ref. No. [UMCES] CBL 2016-010
ACT VS16-01
11
Winkler method
(Carignan et al. 1998). All Winkler titrations were done at the individual laboratory
and field sites by trained ACT staff using standardized techniques and equipment.
Initial Preparation
The volumes of each BOD bottles (≈ 125 mL) were determined with a precision better than
0.005%. The volume of each bottle was measured gravimetrically (± 0.01 mL) near 20
°
C, after filling
with degassed (boiled 10 min and cooled) distilled water. Since the procedure’s precision approaches 1
µg O
2
·
L
-1
, particular care was taken to avoid contamination of the glassware and working space from
any trace amounts of thiosulfate, iodate, I
2
, and manganese. Reagents recommended by Carritt and
Carpenter (1966) were used and whole bottles titrated to minimize the loss of volatile I
2
and the
oxidation of iodide to I
2
at low pH.
Reagents
(1) Manganous chloride solution (3M Mn
2+
): dissolve 300 g of MnCl
2
·4H
2
O in 300 mL of distilled
water. Bring to 500 mL.
(2) Alkaline iodide solution (8M OH
-
, 4M I
-
): separately dissolve 160 g of NaOH and 300 g of NaI in
ca 160 mL of distilled water. Mix with stirring and bring to 500 mL.
(3) 23N Sulfuric acid solution: slowly add 313 mL of concentrated H
2
SO
4
to 175 mL of distilled water.
Carefully mix and cool and bring to 500 mL.
(4) Thiosulfate titrant 0.03N: add 300 mL 0.1N Na
2
S
2
O
3
·5H
2
O (Fisher SS368-1) to 700 mL DI. The
thiosulfate is standardized daily with KIO
3
according to the procedure described below. Note: The
normality of thiosulfate will be adjusted to ensure that a complete sample can be titrated within one
burette volume (less than 10 mLs), but kept as low as possible to maximize precision.
(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).