Main laboratory analysis
B.M. D’Antoni, F. Iracà, M. Romero
Panta Rei Water Solutions
Biochemical oxygen demand (B.O.D.) is an empiric laboratory assay which measures the amount of organic matter contained in a water sample. This assay is the most widely used method for the assessment of water quality.
The equation above, describes the biochemical process behind the BOD assay. BOD is, in fact, a measure of the amount of oxygen consumed by heterotrophic bacteria for the oxidation of organic matter. Hence, BOD is expressed in mg O2/L and higher the mount of organic matter in water the higher the BOD value. Although there exist various methods for BOD measurement, the principle is the same for all of them: a volume of water sample is put in a recipient where the changes in the oxygen content are measured before and after incubation at 10°C for a certain time.
BOD value increase over the time as organic matter is progressively biodegraded. However, after five days the majority of the organic matter contained in the sample has already been degraded. For this reason, BOD5, which is measured after 5 days of incubation is the most widely used method. On the other hand, if the aim is to measure the total content of biodegradable organic matter, BOD21 is measured after 21 days of incubation.
The oxidation of other compounds present in the water sample can also contribute to the consumption of oxygen. In particular, nitrification could interfere in the measurement of BOD, leading to an overestimation of its value. To prevent this, the use of an inhibitor is required (N-Allylthiourea 98%).
The measurement of BOD5 in the lab, consists of pacing the sample in a recipient sealed with a manometer and in constant agitation in dark conditions. The manometer measures the decline of pressure inside the recipient caused by oxygen consumption. With the measured decline of pressure, the mass of oxygen that has been consumed is calculated using the ideal gas law. Sodium hydroxide (NaOH) is added to absorb the carbon dioxide produced in the process, which might interfere in the pressure measurement.
How to proceed:
- 1) Introduce a magnet inside the bottles, so that when they are placed on the magnetized tray, they remain constantly agitated.
- 2) Determine the volume of the sample which will be introduced in the recipient. For that purpose, we previously need to make an estimation of the expected BOD range if the sample.
Use range 0-800 for inlet end range 0-200 for outlet. With that information, we go to the table above which establishes the specific volume of sample to be put in the recipient for each BOD range. For example, we expect that the BOD of our outlet sample is below 200 mg O2/L. In that case we can see that the volume to incorporate to the recipient is 250 mL.
- 3) To measure the exact volume, it is necessary to use a graduated cylinder as volume measuring tool.
- 4) Once the volume is measured we must introduce it into the recipient using a funnel.
- 5) At this point we will add nitrification inhibitor (few drops or 1 little spoon) into the recipient.
- 6) Now, it is time to put the NaOH into the plastic enclosure located within the manometric cap.
- 7) Next, we can proceed to firmly close the bottles to guarantee an airtight environment.
- 8) After that we make sure that the manometric caps is reset to start measuring from afresh.
- 9) Once that done, we introduce all recipients containing our samples in the magnetized tray, which is already located inside the incubator at 20°C in dark conditions.
- 10) After 5 days in the incubator, it is time to take the measurements of the manometric caps.
If the value is out of range, no values will be displayed.
- 11) Once the value of the manometric caps have been noted down, one more step is needed to get the final BOD5 value. To that end, we will use the following equation:
Where the term "factor" in the formula, corresponds the number reported in the Standard table for our specific sample (see point 2).
- 12) In this case we used a sample volume of 250 mL since we estimated our BOD value would not exceed 200 mg O2/L.
- 13) The factor to be used, for this example, is 5 (See point 2). The BOD value obtained, for this example, is 25 (See point 10).
- 14) The final value of BOD5, for this example, is BOD5= 25 * 5= 125 mg O2/L.
The chemical oxygen demand (C.O.D.) is an empiric laboratory assay which indirectly measures the amount of total organic matter (biodegradable and non-biodegradable) contained in a water sample. In particular, COD measures the amount of oxygen consumed for the oxidation of total organic matter. Thus, it is measured in mg O2equivalent/L. COD is strictly linked to BOD, however, whereas BOD is a measure of the amount of oxygen that bacteria will consume while decomposing organic matter under aerobic conditions, COD does not differentiate between biologically available and inert organic matter, and it is a measure of the total quantity of oxygen required to oxidize all organic material into carbon dioxide and water. In this way, the COD values of a water can be typically related to its BOD values in a more or less constant ratio. COD values are always greater than BOD values, but COD measurements can be made in a few hours while BOD measurements take at least 5 days. Since the COD test can be performed rapidly, it is often used as a rough approximation of the water’s BOD, even though the COD tests measures some additional organic matter which is normally not oxidized by biological action. The basis of the COD test is that nearly all organic compounds can be fully oxidized to carbon dioxide with a strong oxidizing agent under acidic conditions at high temperature. After a digestion step, the concentration of organic substances in the sample is calculated from a spectrophotometric determination of the remaining oxidant. The spectrophotometric method consists of adding a strong oxidant (K2Cr2O7) to the water sample under acidic conditions (H2SO4) and using silver (Ag) as a catalyst.
The sample is then digested for 2h at 148°C, and its determination is done by using a spectrophotometer.
During the digestion, the sample’s organic carbon material is oxidized with the (Cr2O72-) found in potassium dichromate. The dichromate readily gives up O2 to bond with carbon atoms to create CO2. The oxygen transaction from Cr2O72- to CO2 reduces the Cr2O72- ion to Cr3+. The amount of Cr3+ is determined after oxidation is complete. In essence a COD test determines the amount of carbon based materials by measuring the amount of oxygen the sample will react with. COD procedure using PF-12 spectrophotometer (see the manual) and NANOCOLER COD Test (see program code to insert in PF-12 on COD kit).
For inlet water use NANOCOLOR COD 1500, while for effluent water use NANOCOLOR COD 160.
3. Total Solids
The most important physical characteristic of wastewater is its total solids content, which is composed of floating matter, settleable matter, colloidal matter, and matter in solution. Total solids (TS) are obtained be evaporating a sample of wastewater to dryness and measuring the mass of the residue, while a filtration step is used to separate the total suspended solids (TSS) from the total dissolved solids (TDS).
For our purpose we will perform only TSS and VSS in inlet and outlet of the plant. When we take the sample from the oxidation thank we will analyze MLSS and MLVSS (the procedure is the same).
What we need for determination of TSS:
- 1) Put the filter in the oven (105°C) for 10 minutes in order to remove humidity;
- 2) After 10 minutes, take out the filter and let it cool for 15 minutes at room temperature;
- 3) Weight the filter and note done the value in grams (M0);
- 4) Place the funnel on the top of the glass;
- 5) Place the filter, after folding it, inside the glass;
- 6) Use the pipette and take 5 mL of sample (V0) - (note done the volume used);
- 7) Pour the sample through the filter and wait the filtration time;
- 8) Once the sample is filtered, take the filter and put it in the oven for 1 day at 105°C;
- 9) After 1 day, take out the filter and let it cool for 30 minutes at room temperature;
- 10) Weight the filter and note done the value in grams (M1);
- 11) Use the following formula to calculate the TSS:
What we need for determination of VSS:
- 1) Put the dried filter (M1) into the crucible;
- 2) Weight the crucible + dried filter and note done the value in grams (M2);
- 3) Put crucible + dried filter into the Muffle at 550°C for 2h;
- 4) After 2h, take out the crucible and let it cool for 30 minutes at room temperature;
- 5) Weight the crucible + ash and note done the value in grams (M3);
- 6) Use the following formula to calculate the TSS:
Before starting is better prepare a simple table as follow