ក្រុមហ្ុន មិសតា

ុ ហ្វូដ អីនផត អិចផត ឯ.រ

MISOTA FOOD IMPORT EXPORT CO., LTD.

Laboratory Experiment

Guideline by: Mr. OL TOLA

Prepared by: Position: Internship

Ms. CHUN ROTANA

Ms. CHROENG SREYNY

2023-2024
 Biochemical Oxygen Damaged (BOD)

1. Definition

The biological oxygen demand expresses the quantity of oxygen necessary for the
destruction or degradation of organic matter in a watercourse, under the action of developing
micro-organisms. The BOD is defined according to standardized conditions at a constant
temperature of 20°C, for 5 days.

• Principle
The method consists of filling with sample, to overflowing, an airtight bottle of the
specified size and incubating it at the specified temperature for 5 days. Dissolved oxygen is
measured initially and after incubation, and the BOD is computed from the difference between
the initial and final DO.

2. Materials and Methodology

2.1. Material and Reagents

• OxiTop measuring system. (No Material)

• Incubator at 20°C
• Sample Bottles
• Dilution water (dechlorinated water)
• Dissolved oxygen (DO) meter or titration equipment

2.2. Reagents

- Phosphate buffer solution

- Magnesium sulfate solution
- Calcium chloride solution
- Ferric chloride solution
- Acid and alkali solution,1N
- Sodium sulfite solution
- Nitrification inhibitor, 2-chloro-6-(trichloromethyl) pyridine
- Glucose- glutamic acid solution

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 - Ammonium chloride solution
- Dilution water

2.3. Methodology

- Sample Collection: Collect a representative sample of the wastewater to be tested in clean sample
collection bottles. Preserve the sample to minimize changes in BOD before analysis (usually by
chilling or adding chemicals to inhibit microbial activity). And take care to avoid contamination
during sampling and handling.

- Initial DO Measurement: Measure the dissolved oxygen (DO) concentration in the sample
immediately after collection using a DO meter. This reading serves as the initial DO concentration
(DOi).

- BOD Bottle Preparation: Rinse BOD bottles with the sample to be tested. Fill each BOD bottle
with appropriate volumes of sample and dilution water to achieve a specific dilution factor (usually
1:10 or 1:100). Record the volume of the sample and the dilution water added to each bottle.

- Incubation: Cap the BOD bottles tightly to prevent the exchange of oxygen with the atmosphere.
Place the bottles in an incubator set to 20°C ± 1°C for 5 days.

- Final DO Measurement: After 5 days of incubation, remove the BOD bottles from the incubator.
Measure the DO concentration in each bottle using the DO meter. This reading represents the final
DO concentration (DOf). Record the volume of the sample and the dilution water added to each
bottle.

• Sampling and storage

Samples for BOD analysis may degrade significantly during storage between collection
and analysis, resulting in low BOD values.

Grab samples If analysis is begun within 2 h of collection, cold storage is unnecessary. If

analysis is not started within 2 h of sample collection, keep the sample at or below 4°C from the
time of collection. Begin analysis within 6 h of the collection; when this is not possible because
the sampling site is distant from the laboratory, store at or below 4°C and report the length and
temperature of storage with the results. In no case start analysis more than 24 h after grab sample

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collection. When samples are to be used for regulatory purposes make every effort to deliver
samples for analysis within 6 h of collection.

• Sample preparation and pretreatment:

All samples Check pH; if it is not between 6.0 and 8.0, adjust sample temperature to 20
3°C, then adjust pH to 7.0 to 7.2 using a solution of sulfuric acid (H2SO4) or sodium hydroxide
(NaOH) of such strength that the quantity of reagent does not dilute the sample by more than 0.5%.
Exceptions may be justified with natural waters when the BOD is measured at in-situ pH values.
The pH of dilution water should not be affected by the lowest sample dilution. Always seed
samples that have been pH adjusted.

• Preparation of dilution water

- Place the desired volume of water in a suitable bottle and add 1 mL each of phosphate
buffer, MgSO4, CaCI2, and FeCI3 Solution, and Seed dilution water, desired.
- Before use bring dilution water temperature to 20 ± 3°C.
- Saturate with DO by shaking in a partially filled bottle or by aerating with organic-free
filtered air. Alternatively, store in cotton-plugged bottles long enough for water to become
saturated with DO.
• Dilution water storage
- Source water may be stored before use as long as the prepared dilution water meets
quality control criteria in the dilution water blank.
- Discard stored source water if dilution water blank shows more than 0.2 mg/L DO
depletion in 5 d
• Glucose-glutamic acid check
- To check the dilution water quality.
- Periodically check dilution water quality, seed effectiveness, and analytical.
- Technique by making BOD measurements on a mixture of 150 mg glucose/L and 150 mg
glutamic acid/L as a "standard" check solution. Glucose has an exceptionally high and
variable oxidation rate but when it is used with glutamic acid, the oxidation rate is stabilized
and is similar to that obtained with many municipal wastes.
- Determine the 5-d 20°C BOD of a 2% dilution of the glucose-glutamic acid standard
check solution.
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 • Seeding
- Seed source: Some samples do not contain a sufficient microbial population (for
example, some untreated industrial wastes, disinfected wastes, high-temperature
wastes, or wastes with extreme pH values). For such wastes seed the dilution water or
sample by adding a population of microorganisms.
- Where such seed is not available, use supernatant from domestic wastewater after
settling at room temperature for at least 1 h but no longer than 36 h.
• Dilution technique
- Using a wide-tip volumetric pipet, add the desired sample volume to individual BOD
bottles.
- Add appropriate amounts of seed material either to the individual BOD bottles or to the
dilution water
- Fill bottles with enough dilution water, seeded if necessary
- Determine the initial DO on one bottle
- Stopper tightly, water-seal, and incubate for 5 d at 20°C.

3. Result and Discussion

3.1. Result

For each test bottle having 2.0 mg/L minimum DO depletion and at least 1.0 mg/L
residual DO, calculate BOD as follows:

(𝐷1 − 𝐷2) − (𝑆)𝑉𝑠

BOD5, mg/L = 𝑃

where:

D1 = DO of diluted sample immediately after preparation, mg/L,

D2 = DO of the diluted sample after 5 d incubation at 20°C, mg/L, S = oxygen uptake of

seed, DO/mL seed suspension added per bottle (6d) (S = 0 if samples are not seeded),

Vs = volume of seed in the respective test bottle, mL, and

P = decimal volumetric fraction of sample used; 1/P = dilution factor.

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 Total Dissolved Solid (TDS)

1.Definition

Total Dissolved Solids (TDS) is a measure of the total concentration of dissolved side
water, and mainly inorganic, but could be organic as well in the ionic form.

-TDS is represented usually as mg/l or ppm

- Dissolved solids include ions as: Na+, Cl-, Ca+2, SO4-2, HCO3-, NO3-, etc.
• Principle
Total dissolved solids (TDS) are measured as a volume of water with the unit
milligrams per liter (mg/L), otherwise known as parts per million (ppm). 500 ppm is the
recommended maximum amount of TDS for your drinking water. Any measurement higher
than 1000 ppm is an unsafe level of TDS. If the level exceeds 2000 ppm, then a filtration
system may be unable to properly filter TDS.

2. Materials and Methodology

2.1. Material and Reagents

-Weigh Balance
- Standard Digital Oven
- Desiccator
- Apparatus used-crucible
- Whatman Filter paper
- Beaker
2.2. Methodology
- Note down the initial dry weight of the crucible (W1)
- Filter the sample using the Whatman filter
- Take a 10ml filtered sample in a crucible which contains TDS
- Place the crucible in the side Oven land heat at 1030C-1050C
- After drying in the oven cool the sample to room Temperature in desiccator
- Note down the final dry weigh of the crucible(W2)

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3. Result and Discussion

Calculation for TDS

- Initial Weight of Container: W1(g)
- Final weight of the container W2(g)
(𝑊2−𝑊2)×100
- Amount of total Dissolve Solids present in the sample =𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑇𝑎𝑘𝑒𝑛 × 100

- The measurement method, units of reporting, interpretation of results, factors

influencing TDS levels, and strategies for monitoring and management.

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 Ammonia (NH3)

1. Definition

Ammonia in wastewater refers to nitrogen in the form of free ammonia and ionic
ammonium, mainly from the decomposition of nitrogen-containing organic matter in domestic
sewage, coking, ammonia synthesis and other industrial wastewater, as well as farmland drainage.
Some drinking water treatment processes add small amounts, generally less than 0.4 mg/l (ppm)
of ammonia to water to increase and extend the disinfecting ability of chlorine.

1.1. Objective

In this experiment fucose on determine ammonia in wastewater.

2. Materia and Method

2.1. Materia

- 0.2M HCl or H2SO4

- Wastewater Sample

- Methyl Orange

2.2. Method

- Take 10ml of sample into conical flask

- Add 2 or 3 drops of Methyl Orange indicator into the same conical flask

- Then take mixture to titrate with 0.2M HCl and record the volume to calculate.

3. Result and Discussion

- How to calculation ammonia in wastewater

𝑚
Molarity(ammonia) = 𝑉𝑜𝑙𝑢𝑚𝑒(𝐿), m=V×M

- V: volume of 0.2M HCL during titration

- M: mass mol of HCl

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 Chemical Oxygen Demand (COD)

1. Definition

COD is a measure of the oxygen equivalent of the organic matter in a water sample that is
susceptible to oxidation by a strong chemical oxidant. Higher COD levels mean a greater amount
of oxidizable organic material in the sample, which will reduce dissolved oxygen (DO) levels.

1.2. Objective

The chemical oxygen demand (COD) test is commonly used to indirectly measure the
number of organic compounds in liquid waste.

2. Materia and Method

2.1. Materia

- Sample

- Potassium dichromate

- Sulfuric acid

- Pharaon solution

- Ferrous ammonium sulfate solution

2.2. Method

- Firstly, Sample Collection as a wastewater sample at initial storage tank. Ensure proper labeling
and record relevant information such as date, time, and location of sampling.

- Second, Sample preparation that measure a known volume (typically 10-20 mL) of the
wastewater sample using a graduated cylinder or pipette.

- Transfer the sample into a flask. Next, by add a precise amount of digestion reagent (e.g.,
potassium dichromate) to the flask. The quantity depends on the expected COD levels in the
sample.

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- And then, add sulfuric acid (typically 5-10ml) into the same flask.

- Furthermore, add indicator 2 or 3 drop (typically, Pharaon) by mix as well.

- Transfer mixture solution titrates with Ferrous ammonium sulfate solution until change color
from green to reddish brown. And next, blank is re-do again as the same process without sample.

- Lastly, record volume to calculations, and any relevant information in a laboratory notebook or
data sheet.

3. Result and Discussion

- How to calculate COD

(𝐵𝑙𝑎𝑛𝑘−𝑉𝑜𝑙𝑢𝑚𝑒 𝑟𝑒𝑐𝑜𝑟𝑑)×𝑁×8000
COD = 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒

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 TSS (Total Suspended Solids)

1. Definition

Total suspended solids (TSS) are defined as solids in water that can be trapped by a filter.
To measure TSS, the water sample is filtered through a pre-weighed filter.

1.1. Objective

In this experiment to measure TSS for wastewater treatment operations and environmental
health by Turbidity Meter.

2. Materia and Method

2.1. Materia

- Sample

- Turbidity Meter

- Distilled Water

2.2. Method

- The measure of TSS by using Turbidity Meter, first press on “No” button to turn the device on.

- The turbidity meter is had to calibration by fill the vial with the distilled water (DW) and attach
the cap tightly then insert the vial with DW into the holder of the turbidity meter and then press
the button “READ”. The result of DW will show zero which indicator that the turbidity meter is
calibration and give the accurate result.

- Wash the vial with sample water several time and fill the sample with attach the cap tightly then
insert the sample into the holder of turbidity meter by press the button “READ” and result will
show on the screen in unit FTU or NTU.

3. Result and Discussion

- To compare the result to standard value of TSS.

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 pH (Potential Hydrogen)

1. Definition

pH measurement is an important parameter in nearly every water quality application. In

wastewater treatment, pH is regulated as part of discharge permitting and many treatment
processes are pH dependent. In environmental sampling and monitoring, high or low pH values
can be indicative of pollution.

1.1. Objective

This purpose of experiment is:

- To ensure optimal wastewater treatment efficiency, meet discharge standards, and preserve the
environment.

- To measure the pH of various solutions using pH indicators and meter.

- To determine the value of Ka for an unknown acid.

2. Materia and Method

2.1. Materia

- Sample

- pH meter

- Distilled Water

2.2. Method

- The pH meter is need to calibrate before measure the wastewater water simple. pH buffers have
two usually pH 4 and 7.

- Firstly, remove the protective cap from the pH probe by wash the probes with distilled water and
dry with tissue paper, show the pH meter in this case is pH 7.

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- Then take the probe into the water sample and make sure the probe is submersed but not touching
the bottom of the beaker the press “Cal” for adjust reading and a bit mix the sample in the probe
and press “Cal” for final time again, the pH meter adjust reading to match the buffer take
measurement the water sample, the pH value will show on screen after reading and lastly don’t
forget to wash with dry the probe as before.

3. Result and Discussion

- To discussion of pH in wastewater that can have a significant impact on the environment and the
effectiveness of wastewater treatment processes.

- Monitoring and controlling the pH of wastewater is essential to ensure compliance with

environmental regulations, protect aquatic ecosystems, and optimize the performance of treatment
systems.

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 Total Nitrogen (TN) by Kjedahl Method

1. Definition

Total Nitrogen (TN) in wastewater refers to the sum of all nitrogen compounds present in
the water, including organic nitrogen, ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen. TN
is an important parameter to measure in wastewater because nitrogen compounds can contribute
to water pollution and eutrophication if not properly treated. High levels of nitrogen in wastewater
can lead to excessive algae growth, oxygen depletion in water bodies, and other environmental
problems.

1.1. Objective

In this experiment to determine total nitrogen by Kjedahl method and determining the
nitrogen contents in organic and inorganic substances.

2. Materials and Method

2.1. Material and reagents

- E-flask

- Burette

- Weigh Balance

- Distillation

- Titrate

2.2. Reagents

- CuSO4

- K2SO4

- H2SO4

- Aqudest

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 - NaOH

- Green methyl red bromocresol indicator

2.3. Method

- 0.8g of CuSO4, 7g of K2SO4, 1g of sample were weighed

- Add CuSO4, K2SO4, and sample into Kjeldahl flask.

- Pure the CuSO4, K2SO4, and sample and add H2SO4 and destruction using H2SO4 for
60-120min at 4200C after that got ammonium sulfate change of solution dark
transparent and no smoke.

- Add 75ml of Aqudest

- Add 60ml of NaOH 40%

- Ammonium sulfate solution moves into the distilling flask.

- During distillation process chemical reaction between NaOH and ammonium sulfate
produced ammonium gas. And ammonium gas was captured by a 25ml of 1% boric
acid solution and green methyl red bromocresol indicator.

- The solution titrated with 0.1 HCl end point of the titration change color from green to
colorless until pink.

- The mole of HCl reacts is equals the mole of Nitrogen in the sample.

3. Result and Discussion

Formula

(𝑚𝐿𝐻𝐶𝑙(𝑠𝑎𝑚𝑝𝑙𝑒)−𝑚𝐿𝐻𝐶(𝑏𝑙𝑎𝑛𝑘))×𝐻𝐶𝑙×14.01×100
%N= 1000×𝑤𝑒𝑖𝑔ℎ 𝑠𝑎𝑚𝑝𝑙𝑒(𝑔)

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