# Applications of Sodium Humate in Present-day Aquaculture

Volumetric Methods

**1.Principle of The Method**

Dissolve sodium humate in water. Then oxidize the carbon in the humic acid solution to carbon dioxide with potassium dichromate in sulfuric acid solution. Finally calculate the humic acid content according to the consumption of potassium dichromate and the carbon coefficient of humic acid.**2.Reagents**

2.1 Potassium dichromate (GB 624-77) standard solution, C（1/6K2Cr2O7）=0.1mol/L (equivalent to 0.1M): Dry potassium dichromate at 130 ° C for 3 hours, then cool to room temperature in a drier, weigh 4.9036g and put them in a beaker, dissolve them with water, then transfer the solution to a 1000 ml volumetric flask, dilute to the mark and shake up the dilution.

2.2 Potassium dichromate (GB 624-77) standard solution, C（1/6K2Cr2O7）=0.4mol/L (equivalent to 0.1M). Weigh 20g of potassium dichromate, dissolve it in 1000ml of water and store the solution in a flask for use;

2.3 Sulfuric acid (GB 625-77);

2.4 Phenanthroline (GB 1293-77) indicator; weigh 1.5g of phenanthroline and 1g of ammonium ferrous sulfate; dissolve them in 100ml of water and store the solution in a brown bottle;

2.5 Standard solution of ammonium ferrous sulfate (GB 661-77); C (Fe2+) = 0.1 mol / L (equivalent to 0.1 M). Weigh 40g of ammonium ferrous sulfate hexahydrate and dissolve it in an appropriate amount of water, add 20ml of concentrated sulfuric acid to the solution and then dilute the solution to 1000ml with water; Shake the dilution and put it into a brown bottle, put two clean aluminum sheets or cable aluminum wire in the bottle to keep the dilution concentration stable for a long time and avoid frequent calibration. The concentration of the solution is calibrated as follows:

Put 25.0ml of C（1/6K2Cr2O7）=0.1mol/L potassium dichromate standard solution in a 250ml Erlenmeyer flask, add 70-80ml water and 10ml concentrated sulfuric acid; add 3 drops of phenanthroline indicator after cooling. Titrate the solution with a solution of uncalibrated ammonium ferrous sulfate until the solution becomes brownish red.

The concentration of ammonium ferrous sulfate standard solution C (Fe2+), expressed in mol/L, is calculated by the following formula:

C（Fe2+）= 25/v×0.1

Remarks: The letter V in the formula above refers to the volume of ammonium ferrous sulfate consumed in the titration, ml.

2.6 Analysis steps

2.6.1 Dissolution

Weigh 0.2±0.01g (accurate to 0.0002g) of humic acid and put it in a 250 Erlenmeyer flask; add 70ml of water and insert a small glass funnel into the flask mouth; heat the flask in a boiling water bath for 30 minutes and stir frequently; take out the flask; Transfer the solution and residue to a 200ml volumetric flask after cooling; dilute to the mark with water and shake well; dry filter the dilution with a quantitative filter paper (medium speed) and discard the original part of the filtrate (about 10 ml).

2.6.2 Oxidation

Put 5.0 ml of the filtrate obtained from 2.6.1 in a 250ml Erlenmeyer flask, add 5.0 ml of C（1/6K2Cr2O7）=0.4mol/L potassium dichromate solution and slowly add 15 ml of concentrated sulfuric acid; heat and oxidize the solution for 30 minutes in a boiling water bath.

2.6.3 Titration

Remove the oxidized solution in 2.6.2 from the water bath; cool to room temperature and add about 70 ml of water and 3-5 drops of phenanthroline indicator; titrated with ammonium ferrous sulfate standard solution until the color of titrant change from orange to green and finally brownish red.

Carry out blank test according to the above steps.

2.7 Calculation of Analysis Results

Humic acid content (calculated on a dry basis) in sodium humate, expressed in mass percent (%):

Remarks: V0 — volume of ammonium ferrous sulfate solution consumed by titration in blank test, ml.

V1 — volume of ammonium ferrous sulfate solution consumed by titration of the sample, ml.

c (Fe2+)— concentration of ammonium ferrous sulfate standard solution, mol / L.

0.003 — carbon mass equivalent to 1.00 ml of C(Fe2+) = 1.000 mol/L ammonium ferrous sulfate solution, g.

c—the pure humic acid carbon coefficient of different coal types (leonardite-base humic acid 0.64/ lignite-base humic acid, 0.58/ peat moss-base humic acid, 0.51).

a—the total volume of the sample solution, ml;

b——the volume of the sample solution taken during the test, ml;

m – the mass of the sample, g.

Mad – the amount of water in the sample, %.

2.8 Allowable Deviation

The absolute difference between the results of parallel tests and the results of different laboratory tests should not be greater than the values in the table below. The arithmetic mean of the parallel tests results is taken as the test result.

Allowable Deviation of Test Results

Humic Acid /% | Absolute Difference Between The Results of Parallel Tests /% | Absolute Difference Between The Results of Different Lab Tests /% |

＜25 | ≤1.0 | ≤1.5 |

25-50 | ≤2.0 | ≤3.0 |

＞50 | ≤3.0 | ≤4.0 |

**Appendix A**

1.Determination of Moisture Content

Weigh 1.0g of sample (accurate to 0.0002g) with a weighing bottle pre-dried to constant weight; Then open the lid, put the weighing bottle into an oven preheated to (105 ± 2) °C and dry for 2 hours; take out the weighing bottle and cover the lid. After cooling it in air for 2-3 minutes, put it into a dryer and cool it to room temperature, weigh it and then carry out inspection drying for 30 minutes each time until mass change of the tested sample is less than 0.001g.

Moisture content in the air-dried sample is calculated as is done in Formula A; moisture content in a dryer-dried sample is calculated as is done in formula B:

Remarks

Mad—the water content in the air-dried sample, %.

Md—the water content in the dryer-dried sample, %.

m———the value of the sample quality, g;

m3———The value of the dried sample quality, g.

The result of the calculation is round to the nearest tenth. The arithmetic mean of the parallel measurement results is taken as the measurement result.

Allowable deviation:

The absolute difference between the results of parallel tests shall be in accordance with Table 1.

Table 1 Allowable Deviation of The Results in Water Content Determination

Water Content /% | Absolute Difference Between The Results of Parallel Tests /% |

＜5 | ≤0.2 |

5-10 | ≤0.3 |

＞10 | ≤0.4 |

2.Determination of Ash

Weigh 1.0g (accurate to 0.0002g) of the sample, put it into a crucible pre-dried to constant weight, gently shake it and spread it to make its surface even. Then put the crucible into a box-type resistance furnace with a temperature of no more than 100 °C; slowly warm it up to 500 °C within 30 minutes and hold for 30 minutes; continue to raise the temperature to (815 ± 10) ° C, and then burn for 1 hour. After taking the crucible out, cool it in air for 5 minutes, then place it in a dryer, and cooled it to room temperature; weigh the ash; then conduct a check-up test for 0.5 hours until the mass change of the sample was less than 0.001 g.

Ash content Ad (dry basis), expressed as mass fraction (%), is calculated according to formula C:

Remarks:

m4——-the value of ash mass in grams (g);

m—the value of the sample mass in grams (g);

Mad—water content in the air-dried sample, %.

The result of the calculation is round to the nearest tenth. The arithmetic mean of the parallel measurement results is taken as the measurement result.

Allowable deviation:

The absolute difference between the results of the parallel tests and the results of different laboratory tests shall be in accordance with Table 2.

Table 2 Allowable Deviation of The Results in Ash Content Determination

Ash /% | Absolute Difference Between The Results of Parallel Tests /% | Absolute Difference Between The Results of Different Lab Tests /% |

＜15 | ≤0.2 | ≤0.3 |

15-30 | ≤0.3 | ≤0.5 |

＞30 | ≤0.5 | ≤0.7 |

**3.Determination of Water-insoluble Matter: Gravimetric Method**

3.1 Method Summary

Dissolved the tested sample in water and then centrifuge the solution to obtain the water-insoluble matter in it.

3.2 Instruments & Equipment

Common laboratory equipment (Analytical reagent, distilled water or purified water with equivalent purity are used, unless otherwise stated)

thermostat drying box (temperature control range: room temperature ~ 110 ° C, temperature control accuracy ± 2 ° C, with blast function); centrifuge (minimum rotational speed: 2000r / min, centrifuge cup volume >150mL) ; thermostatic water-bath (temperature control range: room temperature to 100 °C) ; box type resistance furnace (temperature control range: room temperature ~ 1000 ° C); quantitative filter paper (medium speed); weighing bottle with a ground-glass stopper (diameter: 50mm, height: 30mm).

3.3. Analysis Steps

3.3.1 dissolution

Weigh 1.0 g of the tested sample (accurate to 0.0002 g), place it in a 250ml Erlenmeyer flask, add 100 ml of water and heat in a boiling water bath for 30 min to dissolve the sample.

3.3.2 Filtration & Washing

Take the Erlenmeyer flask out, cool it to room temperature, then transfer the solution (and the insoluble matter in it) into the centrifuge cup and centrifuge at 2000r/min for 30 minutes; dry the quantitative filter paper and weighing bottle to constant weight in a dryer at 105-110 °C. Then decant the supernatant of the solution and put all the insoluble matter on a filter paper for filtration, Washed the insoluble repeatedly with water until the washing becomes colorless. Thus water-insoluble matter in the tested sample is obtained.

3.3.3 Drying

Put the insoluble matter together with the filter paper into the weighing bottle and dry it at 105-110 °C for 2 hours; take out the weighing bottle and cover it and cool it in air for 2-3 minutes; then put it into a dryer and cool it to room temperature (which takes about 20 minutes), weigh, dry repeatedly, cool and weigh until the difference between two consecutive weighings is ≤ 0.001 g, and calculate the mass of insoluble matter (m1) as is done in the formula below.

Remarks：

m1—the value of the mass of water-insoluble matter in grams (g);

m—the value of the sample mass in grams (g);

Mad—the amount of water content in the air-dried sample, %.

The result of the calculation is round to the nearest tenth. The arithmetic mean of the parallel measurement results is taken as the measurement result.

Allowable deviation:

The absolute difference between the results of the parallel tests and the results of different laboratory tests shall be in accordance with Table 3.

Table 3 Allowable Deviation of The Results in Wash-insoluble Matter Determination

Water-insoluble Matter/% | Absolute Difference Between The Results of Parallel Tests /% | Absolute Difference Between The Results of Different Lab Tests /% |

≤20 | ≤1.0 | ≤1.5 |

＞20 | ≤2.0 | ≤3 |

**4.PH Value Determination**

4.1 Method summary

Test the pH value of the tested sample by measuring 1% of its solution with a pH meter.

4.2 Instruments & Equipment

pH meter (sensitivity: 0.001 PH) ; electromagnetic stirrer; common laboratory equipment.

4.3 Reagents

4.3.1 phthalate standard buffer solution: c (C6H4CO2HCO2K) = 0.05mol / L;

4.3.2 phosphate standard buffer solution: c (KH2PO4) = 0.025 mol / L, c (NaHPO4) = 0.025 mol/L;

4.3.3 borate standard buffer solution: c (Na2B4O7) = 0.01 mol / L.

4.4 Analysis steps

Commission the pH meter according to its specification, then position and correct it by using the above-mentioned standard buffer solutions. Weigh about 1g of the tested sample (accurate to 0.001g) and put it in a 150ml beaker, add 100ml of distilled water or purified water and place the beaker on a electromagnetic stirrer; stir the solution for 10 minutes and then measure the pH value of the tested sample with a pre-commissioned and pre-calibrated pH meter.

The arithmetic mean of the parallel measurement results was taken as the measurement result.

4.5 Allowable Deviation

The absolute value of the parallel measurement results is not more than 0.2 PH.