8o

A METHOD FOR DETERMINING THE AMOUNT OFOXYGEN DISSOLVED IN i c.c. OF WATER

BY L. VAN DAM.(Zoological Laboratory of the University of Groningen, Holland.)

{Received istjtdy, I934-)

(With Three Text-figures.)

I. INTRODUCTION.IN the well-known Winkler method for determining the amount of oxygen dis-solved in water, an accuracy of about o-i c.c. O2 per litre may be reached in thecase of pure1 water, saturated with air. With very careful manipulation the accuracymay even be increased to o-oi c.c. O2 per litre. If, however, the water to be analysedhas a very low oxygen pressure, difficulties arise; if, during sampling, no particularprecautions are taken to avoid contact between water and air, the water will takeup oxygen and the figures found will be too high3. Various means are used toeliminate this error. Usually an excess of water is allowed to flow through thebottle; while, for limnological investigations, various special types of samplingapparatus have been designed (cf. Maucha, 1932). Bjerrum (1904) derived a cor-rection formula, and Powers (1918) used special sampling bottles which allow theaddition of reagents without exposing the water to air; Weinland (1918) collectedthe samples over mercury, Hogben and Zoond (1930) protected the water fromcontact with air by means of paraffin oil and Ellis (1934) described a techniquewhich allows both sampling and addition of reagents without exposing the sampleto air.

Some authors seem not to be aware of the fact that an inadequate samplingmethod may introduce serious errors. In the work of Helff (1928) and Helff andStubblefield (1931), for example, it is nowhere indicated that precautions weretaken to warrant accurate sampling. Hence the conclusion reached by theseauthors, that in the case of water of low oxygen pressure oxygen was sometimesgiven off by the animals under investigation, is probably erroneous3; in my opinionthe irregularity of their results is a strong indication that their sampling techniquewas defective.

Several authors have tried to adapt the original Winkler method (sample of1 If interfering substances, such as nitrites, organic matter, iron salts, etc., are present (t.g. in

polluted water), the unmodified method gives erroneous results. Concerning the merits of thedifferent correction methods worked out, no unanimity exists (cf. Alsterberg, 1925 and 1926;Theriault, 1925 and 1931; Maucha, 1932; Allee and Oesting, 1934).

* For similar reasons the figures found in the case of water with a very high oxygen pressure will,of course, be too low.

' As will be shown below, in the case of nearly oxygen-free water a faulty sampling techniquemay introduce an error of the order of 020 c.c. O, per litre. In the investigations of Helff and Helffand Stubblefield this error may have been somewhat smaller, since the water they analysed was notentirely oxygen-free, but on the other hand it must have been greatly increased by their use ofsamples of 30 c.c. only.

Method for determining amount of oxygen dissolved in i c.c. of water 81

250-100 c.c.) to smaller quantities of water1. Winkler himself (1924) modified hismethod for samples of 25 c.c, Allee(i929) analysed samples of 13 c.c, Lund(i92i),Thompson and Miller (1928) and Nicloux (1930) worked out micromethods inwhich samples of only 5-10 c.c. are required. Concerning these micromethods thefollowing general statement may be made: their accuracy has always been deter-mined by analysis of water saturated or nearly saturated with air2. And here wemeet with a serious drawback to all these micromethods: when analysing water ofa very low oxygen pressure, the error introduced by sampling is much more seriousthan when the macromethod is used3. Of course, here too this error may beeliminated by allowing a large excess of water to flow through the sampling vessel4;but since samples much larger than 5 c.c. are then needed, the typical advantage ofa micromethod is thus more or less lost. In the present paper a simple micromethodis described which requires samples of only £-1 c.c, and which has the furtheradvantage of avoiding all contact between water and air both during sampling andduring the addition of reagents; this means that it gives reliable results even in thecase of water with a very low (or very high) oxygen pressure.

II. DESCRIPTION OF THE MICROMETHOD1.

The sample is drawn by means of a modified Krogh and Keys syringe pipette andthe ordinary Winkler reagents are added without exposing the sample to air. Thetitra-tion of the liberated iodine is performed by means of Rehberg's microburette.

The original syringe pipette as described by Krogh and Keys (1931) wasdesigned to deliver small quantities of fluid with a very high degree of accuracy.

It consists of a glass tube with a closely ground glass plunger; to the tube, whichis fitted in a metal frame, an injection needle is cemented. The deliverance volumeof the syringe can be adjusted to any desired fraction of the maximum capacityby means of a screw, which is released or locked in position by a little set-screw.

In order to adapt this syringe to micro-analysis of dissolved oxygen, I havemodified it in the following way (cf. Fig. 1):

(1) In order to avoid the use of cement, which would, sooner or later, beattacked by the reagents, the injection needle was replaced by a strong, heavy-walledglass capillary (1) about 5 cm. long, which has a very small bore (inner diameter0-15-0-20 mm.) and which was welded to the tube.

(2) A spiral spring (2) was introduced, which pulls the head of the plunger (3)up against the screw (4).

1 Manometric micromcthods for estimating dissolved oxygen, being in general less convenientthan the Winkler procedure, are here left out of consideration (cf. Oesting 1934).

1 When reading the proofs I noticed a paper by Kawaguti (1933) in which a description is givenof an apparatus for analysing samples as small as 0-2 c.c. In my opinion this method 18 far toocomplicated for general use.

* In small samples the surface exposed to the air will in general be relatively greater (cf. p. 84);in these micromethods the errors introduced, when the stopper is taken out and the reagents areadded, also become of importance. It is only in the micromethods of Thompson and Miller and ofKawaguti that the reagents are added without exposing the water to air.

4 In the method of Thompson and Miller an excess of water is always allowed to flow through theapparatus; not enough, however, to eliminate this error.

* A brief description was given by van Dam (1933).

JHB-xili 6

82 L. VAN DAM

(3) The little set-screw was functionally replaced by a metal collar (Fig. 1 (5)and Fig. 2 (2)). This collar can be taken off (Fig. 2), so that theplunger can be pressed in wholly by means of the screw; the samplemay then be drawn by means of the same screw. A small excessof water is drawn in, the collar is replaced in its original position,and the excess water is driven out. This method must be used ifthe sampling has to be carried out very carefully, e.g. if a samplemust be drawn from the siphons of a Lamellibranch (cf. van Dam(1935)). If special care is not required, the sample may be drawnmore rapidly in the following way: the collar is left in position andthe plunger is pressed down by hand; the spiral spring is thenallowed to pull up the plunger with a moderate velocity. If thisvelocity is too great, gas bubbles may appear in the water, owingto the diminished pressure (high resistance in the very narrowcapillary). When the collar meets the metal tube (Fig. 1 (6)), thesyringe has a very definite deliverance volume of about J-i c.c,to be measured by weighing the quantity of water delivered bypressing down the plunger of the filled syringe (cf. Krogh andKeys, 1931, p. 2438).

The dead space (Fig. 1 (7)) and also, if the syringe has beenkept dry, the space between plunger and tube is filled with ordinarywater. The dead space is rinsed three or four times with a smallquantity of the water to be analysed, and is then finally filled.

Since the dead space is very small (at most o-oi c.c), only asmall quantity of water (at most 0-2 c.c.) is required for a thoroughrinsing. The required quantity of manganese chloride solution isthen added by turning back the screw through a certain angle.Example: From the MnCl, solution about 0-5 per cent, of thesample volume must be added; the thread of the screw being about0-8 mm., the length of the filled part of the syringe tube about40 mm., the screw has to be turned over approx. 900. Sinceconsiderable latitude is allowed in the amount of reagents addedit is not necessary to measure them very accurately. The MnCla

solution which remained in the dead space is screwed into thewater of the syringe, care being taken that no air enters. The waterand MnCl, solution are then mixed by gently rotating the syringebackwards and forwards on an axis rectangular to its length. Thecapillary is again entirely filled by turning the screw, the potassiumhydroxide-potassium iodide solution is added (in the same way asdescribed for the MnCl, solution), and the rotating movements arerepeated in order to disperse the precipitated manganous hydroxidethroughout the sample.

Fig. 1. Modified syringe pipette (longitudinal section, f natural size).Metal parts black, ebonite stippled, glass parts white.

Method for determining amount of oxygen dissolved in i ex. of water 83

The mixing movements must not be performed with too great vigour, and attentionmust also be paid to contraction of the fluid, in order to prevent any air entering thesyringe; by means of the screw the meniscus can easily be kept in the capillary.

After a few minutes thorough mixing1 the meniscus is adjusted to the end ofthe capillary and sulphuric acid is added by turning the screw through an angle,about two and a half times greater than in the case of the first reagents. Again thefluids are mixed until the precipitate has dissolved completely, i.e. until all theiodine is set free.

The iodine is then carefully delivered into a small titration vessel (Fig. 3) bypressing down the plunger by hand. Since the reagents contain only traces ofoxygen and are added in relatively very small amounts, the quantity of the deliverediodine solution practically equals the deliverance volume of the syringe (cf. p. 82).

IFig. 2. Axis of the screw (i) and metal

collar (2) (somewhat enlarged).

Fig. 3. Titration vessel(somewhat enlarged).

This vessel is then placed in the slot of the arm of the microburette describedby Rehberg (1925) and the iodine titrated with sodium thiosulphate o-ozN.During titration the fluid is stirred by means of a moderate stream of air bubbles(cf. Rehberg, 1925); no more air than is necessary should be used in order to preventloss of iodine by volatilisation. For the same purpose a large part of the requiredquantity of sodium thiosulphate may first be put into the titration vessel; theamount required may be determined by a preliminary test, or, after some practice,roughly estimated by observing the colour of the iodine solution.

Starch in a suitable, invariable quantity (e.g. 0-025 c-c< of a 1 per cent, solution)is used as an indicator, a piece of filter paper serving as a background. For com-parison a similar vessel, filled with the fluid decolorised in a former titration, ishung beside the titration vessel. After each determination the syringe is rinsedthoroughly with water.

The accuracy of the method described was compared with that of the ordinaryWinkler method and of the micromethod of Nicloux (Tables I and II).

1 It is not necessary to mil continuously; it is sufficient if the precipitate, after having beenthoroughly dispersed through the fluid, be allowed to settle and is then dispersed again and thisprocess repeated three times.

6-2

84 L. VAN DAM

Table I. Comparison of results, obtained by macro- and micro-analysis of thesame water, saturated with air.

OrdinaryWinklermethod

Newmicro-method

BottleNo.

i

2

SyringeNo.

i2

3456

Capacity(cc.)

14921527

Capacity(c.c.)

1-2430-375042508260-8850-460

DissolvedO, (c.c.

per litre)

7 6 1

7-6:

DissolvedO, (c.c.

per litre)

7-677-667617667667-62

Mean(c.c. per

litre)

-

765

Maximumdeviation

from mean(c.c. per litre)

-

0 0 4

Table II. Comparison of results, obtained by macro- and micro-analysis of thesame water, made nearly oxygen-free by bubbling through pure nitrogen.

New micro-method

Macromethodof Winkler

Micromethodof Nicloux

Bottle filled b\

Bottle rapidly filledby means 01 a verywide siphon, reach-ing to the bottom

immersion

No water allowedto overflow

Bottle overflowedwith over twiceits volume

No water allowed to overflow

Dissolvedoxygen

(c.c. per litre)

0-03-004

056

025

0-06-0-10

O-53

Table I shows an accuracy amply sufficient for most purposes1.Table II shows that the macromethod and (especially) the micromethod of

Nicloux give too high figures in the case of water with a very low oxygen pressure,if no water is allowed to overflow the sample apparatus.

That the water of the filled syringe is practically shut off from the air by thewater in the long and very narrow capillary was proved by the following test: asyringe was filled with oxygen-free water and analysed 24 hours later, after thewater in the top of the capillary had been screwed out (some excess of water hadbeen sampled); it was found that no detectable amount of oxygen had dissolvedinto the water.

1 The divergence between individual results would probably be somewhat smaller still if thesame syringe had always been used.

Method for determining amount of oxygen dissolved in i c.c. of water 85

III. SUMMARY.

A description is given of a simple micromethod for determining the amount ofoxygen dissolved in water. The method is based on the Winkler procedure andrequires only 1 c.c. of water for each analysis. It has the further advantage ofavoiding all contact between water and air; consequently, even water of a very lowoxygen content may be analysed accurately.

REFERENCES.ALLEE, W. C. (1929). Ecology, 10, 14.ALLEE, W. C. and OESTINO, R. (1934). Phytiol. Zool. 7, 509.ALSTERBERC, G. (1925). Biochem. Z. 159, 36.

(1926). Biochem. Z. 170, 30.BJERRUM, N. (1904). Medd. Komm. Havunderstg. Kbh. 1, No. 5.DAM, L. VAN (1933). Hand. XXIV Ned. Nat. Geneetk. Congres, p. 150.ELLIS, W. G. (1934). J- Phytiol. 82, 5 P.HELFF, O. M. (1928). Phytiol. Zool. 1, 76.HELFF, O. M. and STUBBLKFIELD, K. I. (1931). Physiol. Zool. 4, 271.HOGBEN, E. and ZOOND, A. (1930). Trans. Toy. Soc. S. Afr. 18, 283.KAWAGUTI, S. (1933). j . Fac. Set. Imp. Univ. Tokyo, Sect, rv, Zool. 3, 183.KKOGH, A. and KEYS, A. B. (1931). J. chem. Soc. (Sept.), p. 2436.LUND, E. J. (1921). Proc. Soc. exp. Biol. N.Y. 19, 63.MAUCHA, R. (1932). Hydrochemitche Methoden in der Limnologie, mit besonderer Berilcksichtigung der

Verfahren von L. W. Winkler. Stuttgart. Die Binnengeujaster, 12.NICLOUX, M. (1930). Bull. Soc. Chim. biol. Paris, 12, No. 10.OESTING, R. B. (1934). Physiol. Zool. 7, 542.POWERS, E. B. (1918). Bull. III. Lab. nat. Hist. 11, 577.REHBERG, P. B. (1925). Biochem. J. 19, 2, p. 270.THERIAULT, E. J. (192s). Publ. Hlth Bull. Wash. No. 151.

(1931)- Supplement No. 90 to the Publ. Hlth Rep.THOMPSON, T. G. and MILLER, C. R. (1928). Industr. Engng Chem. 20, 774.WEINI-AND, E. (1918). Z. Biol. 69, 1.WINKLER, L. W. (1924). Z. Untersuch. NaJtr.- u. Genustm. 47, 257.