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Ref. No. [UMCES] CBL 2016-013
ACT VS16-04
11
Reference Sample Analysis
The Winkler titration for quantifying dissolved oxygen was used as the standard for
comparison. The specific method is described in detail below and is based on the procedures
described in,
Measurement of primary production and community respiration in oligotrophic lakes
using the 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.