DETERMINATION

OF PHOSPHATE IN WATER IN POLYETHYLENE1 J. Heron

Biological Association,
ABSTRACT

AFTER

STORAGE

Freshwater

Ambleside,

England

The changes in phosphate concentration which take place when lake water is stored in polyethyleno bottles have been studied. The reduction in phosphate concentration which occurs is attributed to bacterial action and not to absorption of phosphate by the polyethylene. Appreciable changes in phosphate concentration can be avoided by collecting the sample in a polyethylene bottle which has been treated with a solution of iodine in potassium iodide solution.
INTRODUCTION

Regular determinations of the phosphate content of natural waters provide information concerning the biological changes taking place in the water. In waters of the English Lake District the concentration of phosphate is often very low (less than 1 pg P/L) so that it is essential to adopt a sensitive and precise method, Of equal importance is the method of sampling and the avoidance of errors due to changes in the phosphate concentration in the water after the sample has been collected. The storage of sea water before the determination o,f phosphate has been discussed by Murphy and Riley ( 1956) and Harvey ( 1948).
SAMPLING AND MElTHOD OF ANALYSIS

the number of bacteria was reduced from 14,80O/ml to 6,OOO/ml. The differences in the figures for the phosphate concentration of centrifuged lake water and the same water which had been allowed to stand overnight are shown in Table 1.
RESULTS

The method of sampling has been fully described in a previous paper, Heron ( 1961). Phosphate was determined by the method of Procter and Hood ( 1954) modified by Heron ( 1961) . All measurments of optical density were made with a Unicam S.P. 500 spectrophotometer. New P.V.C. tubing was tested for the release of phosphate on contact with water. Experimental methods

The object of the following experiments was to eliminate any change in phosphate concentration due to bacterial action by adding preservatives to the water. Toluene chloroform and 1 : 2 dichloroethane were tried and the results are shown in Figure 1. Two experiments were made with chloroform, the initial phosphate concentrations were 4.6 pg P/L and 4.9 pg P/L. After the preliminary determination of phosphate, 7 ml of chloroform per liter were added and the phosphate determination was again carried out at the times indicated in the figure. The experiments using 1 : 2 dichlorocthane were made with original phosphate concentrations of 4.9 and 2.6 pg P/L. The results show that 1 : 2 dichloroTABLE

z--L--.

Comparison of phowhate content of 1. centrifuged and settled lake water __ _~.___~ -~ - ~_ -Centrifuged as soon as ossible after co r lection, analyzed immediately (Pug/L) Not centrifuged, allowed to stand overnight in

A sample of 510 ml lake water was centrifuged for 30 min at 3,000 rev/min; 200 ml were then siphoned off for the phosphate determination. In one particular case __-1 It is a pleasure to acknowledge the valuable criticisms of the paper made by Mr H. C. Gilson and Mr F. J, H. Mackcreth. I am also indebted to Miss V. G. Collins for the bacterial counts.

Winder-mere South basin North basin Esthwaite Water Blelham Tarn

5.2 2.7 2.3 4.7

0.9 0.6 1.0 --~-.0.7

316

PIIOSPI-IATE

IN

WATER

STORED

IN

POLYETHYLENE

317

x TOLUENE ---

- -*CHLOROFORM o----------oDICHLOROETHANE

.-

FIG.

toluenc,

1. Changes chloroform,

in phosphate concentration of lake water and dichloroethane as preservatives.

stored in 1-L polyethylene

bottles

using

ethane prevents significant decreases in phosphate concentration and that toluene and chloroform were not effective. It was also observed that increases in phosphate concentration occurred and the cause of this release of phosphate has not yet been established. As the increase was more pronounced when chloroform was used it was thought that the chloroform might contain phosphate or that the presence of chloroform enhanced in some way the sensitivity of the calorimetric reaction. Both of these possible explanations were investigated. In the first case the phosphate was determined in distilled water which had been in contact with chloroform for 24 hr. The amount of phosphate found was very small being equivalent to 0.11 lug P/L. Weighed quantities of phosphate were then taken and the phosphate determination carried out in the presence of 7 ml/L chloroform. The calculated amount of phosphate was found in each case, Because of the erratic changes in phosphate concentration which occurred

when preservatives such as toluene chloroform and 1 : 2 dichloroethane were used it was dicided to try the effect of freezing the water samples immediately after collection. As it would not be possible to centrifuge the samples in the field it was necessary to replace the centrifuging of the water by rapid filtration. In order to achieve this a filter was made by fusing a Pyrex glass tube to a Pyrex No. 2 sintered glass funnel. The dimensions of the complete filter were: 55 cm long, 7 cm bore, the capacity was approximately 1,540 ml. The phosphate concentration of the filtered samples was somewhat higher than the samples which had been centrifuged. The figures are given in Table 2. The rapid freezing was carried out in the field by first filtering the sample through the clean sintered glass funnel into a screwstoppered polyethylene bottle. When 200 ml had been collected, the bottles were tightly closed and carefully immersed in a mixture of solid CO2 and alcohol. This

318

J. HERON

TABLE 2. Comparison of phosphate content of of centrifuged and filtered lake water - .~ ___~____ ___C&i~~c~d
(/a/L) Filtered samples (/a/L) -- _~-

TABLE 4. Comparison of phosphate content in unfiltered samples of lake water before and after f reexing .~
Phosphate content immediately after collection ( /a/L 1 _ I.-Phosphate content after thawing (#a/L 1 Time in deep freeze cabinet (hr)

Windermere South basin North basin Esthwaite Water Blclham Tarn

3.0 1.9 0.9 3.5

3.8 2.3 0.9 4.5 -..-

---

mixture had been prepared beforehand in the laboratory and taken to the sampling point in a suitable Dewar flask. On return to the laboratory the polyethylene bottles were transferred to a deep freeze cabinet. Table 3 shows the results obtained with samples which had been kept frozen in a deep freeze cabinet for different periods of time. In this case the samples had been centrifuged and not filtered. It was thought that rapid freezing of the samples might result in the rupturing of cells which would give high results for phosphate in solution. If rupturing of cells did take place it would be reasonable to expect higher results from an unfiltered sample. The figures in Table 4 show that identical results were obtained from filtered and unfiltered aliquots of the same sample. The nature of the changes in phosphate concentration due to storage of lake water in polyethylene Two series of tests were carried out using filtered and unfiltered lake water. The phosphate concentration was determined
TABLE 3. Effect of freezing on the phosphate content of samples of lake water - -=---- --- -Phosphate content immedintely after centrifuging (fig/L) ---Phosphate content af tcr thawing (,ug/L) Time in deep freeze cabinet (days)

Windermcre South basin not filtered Windermere South --~ basin filtered --

4.4 3.6 ._ _

4.4 __ -- 3.6 ~ -.

20 20 -----

immediately and then at frequent intervals. The water was kept in a 5-L polyethylene bottle. Figure 2 shows the variation in the phosphate concentration, the changes are not as marked as in Table 1 and this can be explained by the different ratios of volume/surface area of the 5-L and I-L bottles. The two lines in Figure 2 suggest that the changes in phosphate concentration are not simply due to absorption by the polyethylene since one would expect the reduction in phosphate concentration to start in some degree as soon as the bottle was left undisturbed. Autoclaving of lake and distilled water containing added phosphate Four liters of lake water were autoclaved for 1 hr at 15 lb pressure in a Pyrex flask, after cooling, the water was transferred to a 5-L polyethylene bottle which had been washed out with alcohol and drained. The phosphate concentration was determined immediately and a quantity of the water was taken for a bacterial count which was subsequently shown to be nil. The initial phosphate concentration of 6.9 pg/L was high due to release of phosphate during After 17 hr the figure had autoclaving. fallen to 4.7 pg/L and then remained more or less constant until the end of the experiment. The results for autoclaved unfiltered lake water and also for autoclaved distilled water containing added phosphate are illustrated in Figure 3. The two figures clearly indicate that a viable bacterial population is essential to bring about changes in phosphate concentration. Figure 3 demonstrates that the phosphate concentration of

Windcrmerc South basin North basin Esthwaite Water Blelham Tarn Windermere South basin ---

2.6 1.0 1.2 3.6 _3.4._ -- -

2.7 1.4 1.5 3.1 3.0 -- -- -. --

3 3 8 8 5

PHOSPHATE

IN

WATER

STORED

IN

POLYETHYLENE

319

- - --

Filtered Not Filtered

FIG.

2.

Variation

in phosphate

concentration

of lake

water

stored

in 5-L

polyethylene

bottles.

8e-0 AUTOCLAVED LAKE WATER NOT FILTERED

)(-++-X--XAUTOCLAVED DISTILLED WATER WITH ADDED PHOSPHATE ,x-----x-----x-X --a--d-C X ---3(

N/L

4 ::

4---j+--

-*--w. x---x ---e -x--

c-

x ---mm

x----x

2.

6 12 18 24
FIG.

4 , I ,

48

72

HOURS

120

144

168 distilled

Id0 water

with

3. Changes in phosphate concentration of autoclaved lake water added phosphate. Both stored in 5-L polyethylcnc bottles,

and autoclaved

J. HERON

123456
FIG.

D&S in phosphate concentration

9 stored

10

11

12

13

14 1-L poly-

4.

ethylene

Variation bottle.

of lake water

in an iodine

treated

a sterialized solution does not change appreciably on standing for 5 or 6 days. It seems possible therefore that the changes in the phosphate concentration of lake water when stored in bottles arc brought
8

about by the activity of a film of bacteria on the walls of the vessel. As expected the changes are more marked in a 1-L bottle than in a 5-L bottle. It has been shown that the bacterial ac-

FIG.

5.

ethylene

V,ariation bottle,

in phosphate

concentration

of lake water

stored

in an iodine-treated

1-L poly-

PHOSPHATE

IN

WATER

STORED

IN

POLYETHYLENE

321 are shown in Figures 4 that a release of phosunder these conditions effect is not noticeable

tivity in lake water can be suppressed by quickly freezing the samples as soon as possible after collection. For many investigations such a procedure would be inconvenient, and it was thought that a treated sample bottle reserved for phosphate determinations, would be far more practicable. The possibility that a sample bottle for phosphate determinations might be prepared by treating a polyethylene bottle with a solution of iodine in potassium iodide solution was suggested b,y Mr F. J. H. Mackereth. A clean polyethylcnc bottle was filled with a 5% solution of iodine in an 8% solution of potassium iodide and allowed to stand for one week. The bottle was well washed with distilled water and then used to store filtered lake water. It was found that the use of a polycthylene bottle treated in the way described above involved a slight modification to the procedure for the determination of phosphate. During the extraction of the phosphomolybdate complex by n hexanol it was found that any iodine in the water was also extracted so that after the 20-min shaking period the hexanol layer was distinctly colored. After the acid wash step the color of the hcxanol layer was discharged with the minimum number of drops of stannous chloride solution and the reduction completed in the usual manner by the addition of the required amount of stannous chloride solution. Several experiments were made using an iodine-treated polyethylene bottle to store lake water, phosphate determinations were carried out at intervals over a period of 10

weeks. The results and 5; they indicate phate takes place of storage but the for several weeks.

CONCLUSION

The sensitivity of the calorimetric method for the determination of phosphate in solution has been improved so that concentrations of less than I lug P/L can be determined. Water samples may bc collected and stored in polyethylene bottles which have been treated with a solution of iodine in a solution of potassium iodide. Under these conditions of storage, the phosphate concentration of the sample slowly increases possibly due to release of phosphate from organic phosphorus compounds in the water. When a number of other determinations are required the samples may be frozen by careful immcrsion in a mixture of solid CO2 and alcohol and then stored in a deep freeze cabinet.
REFERENCES

H. W. 1948. The estimation of phosphatc and of total phosphorus in sea waters. J. Mar. Biol. Assoc., 27: 337-359. HERON, J. 1961. The seasonal variation of phosphate, silicate, and nitrate in waters of the English Lake District. Limnol. Oceanogr., 6: 338-346. MURPE-IY, J., AND J. P. RILEY. 1956. The storage of sea water samples for the determination of dissolved inorganic phosphate. Anal. Chem. Acta, l$: 818-819. PROCTOR, C. M., AND D. W. HOOD. 1954. The determination of phosphate in sea water by an iso-butanol extraction procedure. J. Mar. Res., 13: 122-132.
HARVEY,