1.

PURPOSE: The purposes of this experiment are; To show the measurement of dissolved oxygen in order to use it in BOD determination and the measurement of biochemical oxygen demand and the concept of limiting substances in a bioassay test.

2. PROCEDURE: 1. We clean enough number of BOD bottles and set aside. 2. We prepare the required amount of dilution water. 3. The sample will be a synthetic or domestic wastewater sample and the appropriate dilutions of the sample will be prepared. 4. We use some BOD bottles for sample and rest of them for blank solution. After adding the necessary amount of sample, stopper the bottles containing the samples and blank solution such that no air bubbles remain in the bottles and incubate them for proper period of time, performing DO measurement for each of them at the end of the incubation period. 5. At the end of the incubation period, we add 1 mL MnSO4 solution to the bottles followed by 1 mL alkali iodide-azide reagent, well below surface of the liquid. 6. We stopper with care to exclude air bubbles and mix well by inverting the bottle several times. 7. After at least 100 mL clear supernatant is formed, we carefully remove the stopper and immediately add 1 mL concentrated H2SO4 by allowing the acid to run down the neck of the bottle. 8. We restopper, and mix by gentle inversion until dissolution is complete. The iodine should be uniformly distributed throughout the bottle before decanting the amount needed for titration. This should correspond to 200 mL of the original sample after correction for the loss of sample by displacement with the reagent has been made. 9. Thus, for a total of 2 mL (1 mL of each) of MnSO4 and alkali iodide-azide reagent in a 300 mL bottle, the volume taken for titration should be: 200*(300/ (300-2)) =201 mL 10. We titrate with 0.025 N thiosulphate to a pale straw color. 11. We add 1-2 mL starch solution and continue the titration to the first disappearance of the blue color. 3. THEORY: The presence of a sufficient concentration of dissolved oxygen is critical to maintaining the aquatic life and aesthetic quality of streams and lakes. Determining how organic matter affects the concentration of dissolved oxygen (DO) in a stream or lake is integral to waterquality management. The decay of organic matter in water is measured as biochemical or chemical oxygen demand. Oxygen demand is a measure of the amount of oxidizable substances in water sample that can lower DO concentrations.

The test for biochemical oxygen demand (BOD) is a bioassay procedure that measures the oxygen consumed by bacteria from the decomposition of organic matter (Sawyer et.al.1994). BOD of water or polluted water is the amount of oxygen required for the biological decomposition of dissolved organic matter to occur under standard condition at a standardized time and temperature. Usually, the time is taken as five days and the temperature is 20oC. The test measures the molecular oxygen utilized during a specified incubation period for the biochemical degradation of organic material (carbonaceous demand) and the oxygen used to oxidize inorganic materials such as sulfides and ferrous ion. It also may measure the amount of oxygen used to oxidize reduced forms of nitrogen (nitrogenous demand). (URL 3) The methods that are most commonly used to measure dissolved oxygen (DO) can be sorted into three major groups: colorimetric, titrimetric, and polarographic. Because oxygen can easily diffuse into water sample during handling (and alter the concentration of dissolved oxygen), samples have to be collected and processed without contact with air. Titrimetric: A water sample is trapped in a dissolved oxygen bottle, which is a bottle with specially-designed cap (or specially-designed mouth and glass stopper, often sold as BOD bottle), that allow for enclosure of liquids without contact with air. Chemical reagents added in excess interact with oxygen to form a product, and another chemical (the titrant) is used quantitatively to neutralize that product. The amount of titrant needed is proportional to oxygen concentration and is translated to mg/l. The best known titrimetric method for dissolved oxygen is the Winkler method; this method and its modifications are widely applied, both in the field (using a syringe to dispense the titrant) and in the lab (using high-precision burettes). Field kits are useful for routine monitoring of D.O. in creeks. Samples can be collected and fixed in the field, and titrated later at home or in the lab. Laboratory applications include D.O. electrode calibration. BOD measurements can be easily performed using an extra dissolved oxygen bottle from the Winkler method kit, or with electrodes specially equipped for sealing the speciallydesigned mouths of BOD bottles. (URL 4) Principle of Winkler Method: The chemical determination of oxygen concentrations in seawater is based on the method first proposed by Winkler (1888) and modified by Strickland and Parsons (1968). Oxygen in the water sample oxidizes iodide ion (I-) to iodine (I2) quantitatively. The amount of iodine generated is then determined by titration with a standard thiosulfate (S2O32-) solution. The endpoint is determined by using starch as a visual indicator. The amount of oxygen can then be computed from the titer: one mole of O2 reacts with four moles of thiosulfate. At the time of sampling, dissolved oxygen is fixed by the addition of Mn (II) under basic conditions, resulting in a brown precipitate, manganic hydroxide (MnO (OH) 2). Prior to analysis, the sample is acidified to pH 1.0-2.5. This causes the precipitated hydroxides to dissolve, liberating Mn (III) ions. Mn (III) ions oxidize previously added iodide ions to iodine. Iodine forms a complex (I3-) with surplus iodide ions. Iodine and the complex exist in equilibrium; thus, I3- serves as a reservoir of I2. The iodine is then titrated with

thiosulfate; iodine is reduced to iodide and the thiosulfate is oxidized to tetrathionate. The stoichiometric equations for the reactions described above are: Mn2+ + 2OH- Mn (OH) 2 2Mn (OH) 2 + O2 +H2O 2MnO (OH) 2 oxidation of Mn (II) to Mn (III) oxidation of I- to I2

2Mn (OH) 3 + 2I- + 6H+ 2 Mn2+ + I2 + 6H2O I2 + I- I3I3- + 2S2O32- 3I- + S4O62-

oxidation of S2O32- to S4O62- ; reduction of I3- to I-

The thiosulfate solution is not stable and therefore must be standardized with a primary standard, typically potassium iodate (KIO3). Standardization is based on the coproportionation reaction of iodide with iodate, thereby forming iodine. As described above, the iodine binds with excess iodide, and the complex is titrated with thiosulfate. One mole of iodate produces three moles iodine, which are consumed by six moles of thiosulfate. IO3- + 8I- + 6H+ 3I3- + 3H2O I3- + 2S2O32- 3I- + S4O62(URL 5) Interferences: Since the BOD test is dependent on biological activity, the major interferences will be those substances which inhibit the growth of the microorganisms. These will include chlorine, caustic alkalinity or acidity, mineral acids, and heavy metals (such as copper, zinc, chromium, and lead). Excessive nitrites can interfere with the BOD determination. Growth of algae in the presence of light can cause problems by actually increasing the DO of the sample before testing, which must be removed by deaeration. A common problem encountered in BOD testing results from residues building up in the BOD and dilution water bottles. To prevent this, all glassware should be acid cleaned on a regular basis. (URL 6) The requirements of the environmental conditions for the BOD test can be summarized as follows: - Sufficient nutrients, e. g. N, P, S, K, Na, and certain trace elements. - Free from toxins. - Presence of a mixed culture of microorganisms (seed). - Dissolved oxygen must be available in the sample throughout the period of the test. - No interference due to re-aeration. - 20oC incubation. The Significance of BOD: Measurement of BOD has long been the basic means for determining the degree of water pollution. It is the most important measurement made in the operation of a sewage

treatment plant. By comparing the BOD of incoming sewage and the BOD of the effluent water leaving the plant, the efficiency and effectiveness of sewage treatment can be judged. For example, in a typical residential city raw sewage has a BOD value of around 300 mg/L. If the effluent from the sewage treatment plant has a BOD of about 30 mg/L, the plant has removed 90 percent of the BOD. If water of a high BOD value flows into a river, the bacteria in the river will oxidize the organic matter, consuming oxygen from the river faster than it dissolves back in from the air. If this happens, fish will die from lack of oxygen, a consequence known as a fish kill. Thus, sewage treatment plants must remove as much BOD as possible from the sewage water. To check sewage treatment effectiveness and to study and control organic- matter pollution, millions of BOD tests are performed. (URL 7 ) Kinetics of the BOD Reaction: The kinetics of the BOD reaction, for practical purposes, have been formulated as a firstorder reaction, expressed as - dC/dt = k C (1)

Where C is the concentration of biodegradable organic matter at time t, and k is the rate constant. If the biodegradable organic matter is expressed in terms of its equivalent oxygen consumption potential, then Lt can be used in place of C, where Lt represents the oxygen equivalent of the organic matter (BOD) remaining at time t. [BODL - the ultimate BOD] Equation (1) can be written as - dLt/dt = k Lt Upon integration of Eq. (2), we have [k = k/2.303] Lt / L0 = e-k t = 10 k t The amount of BOD remaining at time t equals Lt = L (e-k t) The amount of BOD exerted at any time equals yt = L Lt = L (1 e-k t) or the 5-day BOD equal y5 = L L5 = L (1 e-k 5) (URL 8) (6) yt = L Lt = L (1 10-k t) (5) (4) (3) (2)

4. DATA ANALYSIS AND CALCULATIONS Table 1. Our data until 3rd day mLtitrant/ day Blank Sample 1 Sample 2 0 8.7 8.1 8.3 1 6.5 4.5 4.5 2 4.6 2.9 3.0

We must provide the right conditions for an appropriate BOD measurement.If we do not provide right condition, we will have high oxygen consumption in BOD test.In order to provide right condition,it is required to appropriate dilution. It must be done, the amount of oxygen in organic substances is to be enough for sample.At the end of the experiment, organic substances in the sample should not consume all dissolved oxygen in the sample. Because of not providing the suitable dilution proportion, the amount of dissolved oxygen decrease sharply in our experiment.(URL 11) Adding excess seed, not providing dark condition, pH,osmotic condition and suitable temperature (20 C) can cause high oxygen consumption.(URL 12) Table 2. Data given by asistants mL titrant/ day 0 1 2 Blank 8.4 7.6 7.4 Sample 1 8.2 6.1 5.0 Sample 2 8.2 6.2 5.2 1. At the end of the 5 days; (201 mL sample= 0.201 L) (1 mL 0.025 N Na2S2O3= 0.2 mg DO) For blank: mL thiosulphate used=6.9 DOf=(6.9*0.2)/0.201=6.87 mg/L For sample 1: mL thiosulphate used=3.0 DOf=(3.0*0.2)/0.201=2.99 mg/L For sample 2: mL thiosulphate used=3.2 DOf=(3.2*0.2)/0.201=3.18 mg/L 2. BOD(mg/L)= (DOb DOi)/P Where; DOb= Dissolved oxygen value found in the blanks, mg/L DOi= Dissolved oxygen value found in the diluted samples, mg/L P= Dilution ratio ( P= 0.04 for all calculations) 3 7.3 4.1 3.9 4 7.0 3.3 3.5 5 6.9 3.0 3.2

FOR DAY 0: For sample 1: DOb=(8.4*0.2)/0.201=8.36 mg/L DOi=(8.2*0.2)/0.201=8.16 mg/L BOD=(8.36-8.16)/0.04=5 mg/L For sample 2: DOi=(8.2*0.2)/0.201=8.16 mg/L BOD=(8.36-8.16)/0.04=5 mg/L

FOR DAY 1: For sample 1: DOb=(7.6*0.2)/0.201=7.56 mg/L DOi=(6.1*0.2)/0.201=6.07 mg/L BOD=(7.56-6.07)/0.04=37.25 mg/L For sample 2: DOi=(6.2*0.2)/0.201=6.17 mg/L BOD=(7.56-6.17)/0.04=34.75 mg/L FOR DAY 2: For sample 1: DOb=(7.4*0.2)/0.201=7.36 mg/L DOi=(5*0.2)/0.201=4.98 mg/L BOD=(7.36-4.98)/0.04=59.5 mg/L For sample 2: DOi=(5.2*0.2)/0.201=5.17 mg/L BOD=(7.36-5.17)/0.04=54.75 mg/L FOR DAY 3: For sample 1: DOb=(7.3*0.2)/0.201=7.26 mg/L DOi=(4.1*0.2)/0.201=4.08 mg/L BOD=(7.26-4.08)/0.04=79.5 mg/L

For sample 2: DOi=(3.9*0.2)/0.201=3.88 mg/L BOD=(7.26-3.88)/0.04=84.5 mg/L FOR DAY 4: For sample 1: DOb=(7*0.2)/0.201=6.97 mg/L

DOi=(3.3*0.2)/0.201=3.28 mg/L BOD=(6.97-3.28)/0.04=92.25 mg/L

For sample 2: DOi=(3.5*0.2)/0.201=3.48 mg/L BOD=(6.97-3.48)/0.04=87.25 mg/L

FOR DAY 5: For sample 1: DOb=(6.9*0.2)/0.201=6.87 mg/L DOi=(3.0*0.2)/0.201=2.99 mg/L BOD=(6.87-2.99)/0.04=97 mg/L

For sample 2: DOi=(3.2*0.2)/0.201=3.18 mg/L BOD=(6.87-3.18)/0.04=92.25 mg/L

Table 3. Average BOD values DAY 0 1 36 mg/L 2 57.1 mg/L 3 82 mg/L 4 89.8 mg/L 5 94.6 mg/L

AVERAGE OF BOD 5 mg/L VALUE 3.

Table 4. Data for Thomas graphical method t day 0 1 2 3 4 5 BOD(mg/L) 5,00 36 57,1 82 89,8 94,6 (t/y)1/3 0 0,3028 0,3272 0,3319 0,3545 0,3753

time vs (t/y)1/3
0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 2 time (days) y = 0.0582x + 0.1365

(t/y)1/3

(t/y)1/3 Linear ((t/y)1/3)

Figure 1. Thomas graphical method (t/y)1/3 vs time graph

We gain this equation from the graph; A=0.1365 B=0.0582

y=0.0582x+0.1365

k=6B/A L0=1/(k*A3) Therefore, k=(6*0.0582)/0.1365=2.56 L0=1/(2.56*(0.1365)3)=153.5 mg/L

5. DISCUSSION AND CONCLUSION 1. The methods that are most commonly used to measure dissolved oxygen (DO) can be sorted into three major groups: colorimetric, titrimetric, and polarographic (see below). Because oxygen can easily diffuse into a water sample during handling (and alter the concentration of dissolved oxygen), samples have to be collected and processed without contact with air. ( URL 1) 2. The dissolved oxygen concentrations in a wastewater before and after treatment are very important. While dissolved oxygen concentrations are necessary to carry out the BOD determination, as described above, dissolved oxygen levels are also quite important in determining how satisfactory a biological wastewater treatment plant is operating. For example, for satisfactory biological wastewater decomposition (i.e. treatment) some dissolved oxygen must be present. If it is not, the system will be inefficient and is said to be anaerobic. Septic conditions follow, accompanied by a variety of nuisance conditions such as odor and color changes.

Normally, oxygen is not a very soluble gas and dissolved oxygen concentrations in wastewaters are very low. For example, dissolved oxygen concentrations of a few milligrams per liter (or parts per million) are commonplace in water. The solubility of oxygen is such that dissolved oxygen levels in clean water are affected by temperature and salt concentrations expressed as chlorides. Biochemical Oxygen Demand, BOD, as it is commonly abbreviated, is one of the most important and useful parameters (measured characteristics) indicating the organic strength of a wastewater. BOD measurement permits an estimate of the waste strength in terms of the amount of dissolved oxygen required to break down the wastewater. The BOD test is one of the most basic tests used in the wastewater field. It is essentially a measure of the biological and the chemical component of the waste in terms of the dissolved oxygen needed by the natural aerobic biological systems in the wastewater to break down the waste under defined conditions. (URL 2) 3. The standard oxidation (or incubation) test period for BOD is 5 days at 20 degrees Celsius (C) (BOD5). The BOD5 value has been used and reported for many applications, most commonly to indicate the effects of sewage and other organic wastes on dissolved oxygen in surface waters. The 5-day value, however, represents only a portion of the total biochemical oxygen demand. Twenty days is considered, by convention, adequate time for a complete biochemical oxidation of organic matter in a water sample, but a 20-day test often is impractical when data are needed to address an immediate concern. A 5-day duration for BOD determination has no theoretical grounding but is based on historical convention.Tchobanoglous and Schroeder (1985) provide the following background: "In a report prepared by the Royal Commission on Sewage Disposal in the United Kingdom at the beginning of the century, it was recommended that a 5-day, 18.3C, BOD value be used as a reference in Great Britain. These values were selected because British rivers do not have a flow time to the open sea greater than 5 days and average long-term summer temperatures do not exceed 18.3C. The temperature has been rounded upward to 20C, but the 5-day time period has become the universal scientific and legal reference.(URL 9) 4. 5. TABLO 21: EVSEL NTELKL ATIK SULARIN ALICI ORTAMA DEARJ STANDARTLARI Tablo 21.1: Sektr: Evsel Nitelikli Atksular (Snf 1: Kirlilik Yk Ham BO Olarak 5-60 Kg/Gn Arasnda, Nfus =84-1000) KOMPOZT KOMPOZT PARAMETRE BRM NUMUNE NUMUNE 2 SAATLK 24 SAATLK BYOKMYASAL OKSJEN (mg/L) 50 45 HTYACI (BO5) KMYASAL OKSJEN HTYACI (mg/L) 180 120 (KO) ASKIDA KATI MADDE (AKM) (mg/L) 70 45 pH 6-9 6-9

Tablo 21.2: Sektr: Evsel Nitelikli Atksular (Snf 2: Kirlilik Yk Ham BO Olarak 60-600 Kg/Gn, Nfus = 1000-10000) KOMPOZT KOMPOZT PARAMETRE BRM NUMUNE NUMUNE 2 SAATLK 24 SAATLK BYOKMYASAL OKSJEN (mg/L) 50 45 HTYACI (BO5) KMYASAL OKSJEN HTYACI (mg/L) 160 110 (KO) ASKIDA KATI MADDE (AKM) (mg/L) 60 30 pH 6-9 6-9 Tablo 21.3: Sektr: Evsel Nitelikli Atksular (Snf 3: Kirlilik Yk Ham BO Olarak 600-6000 Kg/Gnden Byk, Nfus=10000-100000) KOMPOZT KOMPOZT PARAMETRE BRM NUMUNE NUMUNE 2 SAATLK 24 SAATLK BYOKMYASAL OKSJEN (mg/L) 50 45 HTYACI (BO5) KMYASAL OKSJEN HTYACI (mg/L) 140 100 (KO) ASKIDA KATI MADDE (AKM) (mg/L) 45 30 pH 6-9 6-9 Tablo 21.4: Sektr: Evsel Nitelikli Atksular(Snf 4: Kirlilik Yk Ham BO Olarak 6000 Kg/Gnden Byk, Nfus > 100000) PARAMETRE KOMPOZT KOMPOZT BRM NUMUNE NUMUNE 2 SAATLK 24 SAATLK OKSJEN (mg/L) 40 35 90 25 6-9

BYOKMYASAL HTYACI (BO5) KMYASAL OKSJEN HTYACI (mg/L) 120 (KO) ASKIDA KATI MADDE (AKM) (mg/L) 40 pH 6-9

Tablo 21.5: Sektr: Evsel Nitelikli Atksular (Edeer Nfusun Ne Olduuna Baklmakszn Doal Artma (Yapay Sulak Alan) ve Stabilizasyon Havuzlar Sistemiyle Biyolojik Artma Yapan Kentsel Atksu Artma Tesisleri in) KOMPOZT KOMPOZT PARAMETRE BRM NUMUNE NUMUNE 2 SAATLK 24 SAATLK

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BYOKMYASAL OKSJEN HTYACI (BO5) (ZNM) KMYASAL OKSJEN HTYACI (KO) ASKIDA KATI MADDE (AKM) pH (URL 13)

(mg/L) 75 (mg/L) 150 (mg/L) 200 6-9

50 100 150 6-9

I would not permit the discharge of this wastewater into a receiving stream because our BOD value is quietly higher than these parameters.

5. It is possible to correlate BOD and COD. BOD5/COD ratio is called Biodegradability Index and varies from 0.4 to 0.8 for domestic wastewaters. If BOD/COD is > 0.6 then the waste is fairly biodegradable and can be effectively treated biologically. If BOD/COD ratio is between 0.3 and 0.6, then seeding is required to treat it biologically. If BOD/COD is < 0.3 then it cannot be treated biologically.(URL 10) COD=328 mg/L and BOD=153.5 mg/L 153.5/328=0.47 so the sample is domestic wastewater and seeding is required to treat it biologically.

6. REFERENCES 1. URL 1.Dissolved Oxygen Measurement, retrieved 9 December 2012 from, http://www.swrcb.ca.gov/water_issues/programs/swamp/docs/cwt/guidance/311.pdf 2. URL 2. http://water.me.vccs.edu/courses/ENV149/bod.htm 3.URL 3. Experiment on Determination of BOD, retrieved 9 December 2012 from, http://nitttrc.ac.in/Four%20quadrant/eel/Quadrant%20-%201/exp13_pdf.pdf 4. URL 4.Dissolved Oxygen Measurement Principles and Methods, retrieved 9 December 2012 from,http://www.swrcb.ca.gov/water_issues/programs/swamp/docs/cwt/guidance/311.pdf 5.URL 5. Determination of Dissolved Oxygen by Winkler Titration, retrieved 9 December 2012 from, http://ocw.mit.edu/courses/earth-atmospheric-and-planetary-sciences/12-097chemical-investigations-of-boston-harbor-january-iap-2006/labs/dissolved_oxygen.pdf 6.URL 6. BOD, retrieved 9 December 2012 from, http://www.dep.state.pa.us/dep/deputate/waterops/redesign/NPDES%20Lab/Chapter%205. doc )

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7.URL 7. Introduction to BOD, retrieved 9 December 2012 from, http://www.advancelaboratorios.com/media/catalog/product/pdfs/IntroductionBiochemical OxygenDemand.pdf ) 8. URL 8.The Kinetics of the BOD Reaction, retrieved 9 December 2012 from, http://www.civil.ubc.ca/home/env_lab/The%20kinetics%20of%20the%20BOD%20reactio n.docx ) 9. URL 9. Five Day Biochemical Oxygen Demand, retrieved 9 December 2012 from, http://water.usgs.gov/owq/FieldManual/Chapter7/NFMChap7_2_BOD.pdf 10. URL 10. http://www.newagepublishers.com/samplechapter/001606.pdf 11.URL 11.evre Analiz Laboratuar,deney notlar,retrieved 9 December 2012 from http://194.27.49.11/cevre/%C3%87EVRE%20ANAL%C4%B0Z%20LABORATUVARI% 20II%20DENEYLER%C4%B0.pdf 12. URL 12.Clean Water. retrieved 9 December 2012 from www.cleanwater.com.tr 13.URL 13.Su kirlilii Kontrol Ynetmelii,retrieved 11 December 2012 from, www.ibb.gov.tr/tr-TR/kurumsal/Birimler/.../sukirliliiynetmelik.doc 14.Sawyer ,C.N.,Mc Carty,P.L.,Parkin,G.F.,Chemistry for Sanitary Engineers, 4th edition.,1994.

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