2.1.

Liquid phase
In this section, the methods used for the determination of the parameters of
liquid phase are described. For soluble fraction analysis, the samples were
previously filtered using cellulose acetate membrane filters (MF-Millipore,
Millipore) with a pore size of 0.45 m in order to remove suspended solids.

2.1.1.Orthophosphate
Orthophosphate concentration (PO43--P) was analyzed following the method
4500-P E described in Standard Methods for the Examination of Water and
Wastewater (APHAAWWA-WPCF, 2012). Ammonium molybdate and antimony
potassium tartrate react with orthophosphate in an acid medium to form an
antimony-phospho-molybdate complex. This complex is reduced by ascorbic
acid into blue-colored complex. The intensity of the blue color is proportional to
the orthophosphate concentration, which can be determined using a
spectrophotometer at 880 nm.
Reagents preparation
- Solution A: Sulphuric acid 5N.
- Solution B: Antimony potassium tartrate. 1.3715 g of K(SbO)C 4H4O60.5H2O was
added to 500 mL of distilled water.
- Solution C: Ammonium molybdate. 20 g of (NH 4)6Mo7O244H2O was added to
500 mL of distilled water.
- Solution D: Ascorbic acid. 0.01 M. 1.76 g of ascorbic acid was added to 100 mL
of distilled water. This solution was stable for one week.
- Combined reagent: The above solutions were mixed in the following
proportions for 100 mL of the combined reagent: 50 mL of solution A, 5 mL of
solution B, 15 mL of solution C and 30 mL of solution D. The mixture was
mixed after the addition of each solution, following the mentioned order. This
reagent was stable during 4 hours.
Determination procedure
The volume of the sample was 5 mL (diluted if necessary, to get a maximum
concentration of 1 mg PO43--PL-1). One drop of phenolphthalein indicator
solution, 0.5-1.0 g phenolphthalein in 1 L of ethanol at 80% concentration, was
added to the sample. If a red color was developed, some drops of solution A
(H2SO4 5N) were added until the color disappeared. Then, 0.8 mL of the
combined reagent was added and the mixture was stirred with a vortex stirrer.
After at least 10 minutes but no more than 30 minutes, the intensity of blue
color was measured in a spectrophotometer (Shimadzu UV-1800, UVVisible) at

880 nm. A series of reference solutions, in the range from 0 to 1 mg PO 43--PL-1,

were prepared (Figure 2.1) using a commercial standard solution of PO 4-3 (1000
mg PO43--PL-1; CertiPUR, Merck). For these reference solutions, the combined
reagent was added following the procedure previously indicated. A calibration
curve that correlated absorbance versus phosphorus concentration of the
reference solutions should yield a straight line. Figure 2.1 shows an example of
one of the calibration curves used during this study.
The absorbance of the samples and a blank sample was measured and
phosphorus concentration of the samples calculated using the corresponding
calibration curve.

Figure 2.1. Example of a calibration curve for orthophosphate concentration

determination.

Interferences
Concentrations of arsenates as low as 0.1 mgL -1 react with the molybdate
reagent to produce a blue color similar to that formed with phosphate.
Hexavalent chromium and NO2- interfere to give results about 3% low at
concentrations of 1 mgL-1 and 10-15% low at 10 mgL -1. Filtration of the
samples should be done immediately after collection.

Calibration curve:
water vol.
(mL)
5
4,5
4
3,5
3
2,5
2
1,5
1

patron vol.
(mL)
0
1
1,5
2
2,5
3
3,5
4
4,5

ppm P-PO4
0,0000
0,1088
0,2176
0,3264
0,4352
0,5440
0,6528
0,7630
0,8704

1,0880