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**BIOCHEMICAL OXYGEN DEMAND**

EPA METHOD 405.1 (5 DAYS, 20 C.)

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**SCOPE AND APPLICATIONS**

A. The biochemical oxygen demand (BOD) test is used for determining the relative oxygen requirements of municipal and industrial wastewaters.

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**SCOPE AND APPLICATIONS**

Application of the test to organic waste discharges allows calculation of the effect of the discharges on the oxygen resources of the receiving water.

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**SCOPE AND APPLICATIONS**

Data from BOD tests are used for the development of engineering criteria for the design of wastewater treatment plants.

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**DETECTION AND REPORTING LIMITS**

THE REPORTING LIMITS FOR THE BOD TEST IS <2.

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PRECISION AND BIAS Eighty-six analysts in fifty-eight laboratories analyzed natural water samples plus an exact increment of biodegradable organic compounds. At a mean value of 2.1 and 175 mg/l BOD, the standard deviation was + or and + or - 26 mg/l, respectively.

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WORKING RANGE A. The working range is equal to the difference between the maximum initial DO and minimum DO residual of 1 mg/l.

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SUMMARY OF METHOD A. The sample of waste, or an appropriate dilution, is incubated for 5 days at 20 deg. C in the dark. The reduction in dissolved oxygen concentration during the incubation period yields a measure of the biochemical oxygen demand.

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**SAMPLE COLLECTION,PRESERVATION AND HOLDING TIMES**

A. Samples for BOD analysis may degrade significantly during storage between collection and analysis, resulting in low BOD values. Minimize reduction of BOD by analyzing sample promptly or by cooling it to near freezing temperature during storage.

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**SAMPLE COLLECTION,PRESERVATION AND HOLDING TIMES**

B. Sample collection for BOD can be done in a glass or plastic container. The maximum holding time for BOD is 48 hrs.

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HELPFUL COMMENTS B. 24 hours before BOD is to be set up, measure out the required number of liters of water for the test into a glass carboy and aerate for 2 hours.

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HELPFUL COMMENTS D. Always dechlorinate samples which contain chlorine.

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HELPFUL COMMENTS C. Place the carboy into the BOD incubator for 24 hours to bring to 20 C.

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HELPFUL COMMENTS E. To measure less than 25 ml, use a wide-tipped pipette; for volumes greater than 25 ml, a graduated cylinder may be used.

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HELPFUL COMMENTS F. Samples containing large solids should be homogenized or blend the samples to increase precision and allow the use of a more consistent solution.

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**HELPFUL COMMENTS G. Liters to use = Number of anticipated samples + 5**

(example: if 5 samples are anticipated for the BOD test measure out 10 liters.)

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SAFETY ISSUES A. Use caution when lifting heavy BOD carboy.

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SAFETY ISSUES B. Use latex gloves when handling sewage samples and when filling BOD bottles. This test represents a biohazard.

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**INSTRUMENT/EQUIPMENT**

A. Operating instructions and service manuals for DO meters are kept in the right hand drawer of the BOD station.

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**INSTRUMENT/EQUIPMENT**

B. Change the membrane on the DO probes on a monthly basis.

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**INSTRUMENT/EQUIPMENT**

C. Check lab checklist for maintenance to meters or BOD probe.

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**REAGENTS, STANDARDS, AND SUPPLIES**

A. Hach BOD Nutrient pillows (0.5 mL) B. Hach BOD Nutrient pillows (3mL) C. Hach BOD Nutrient pillows (6mL)

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**REAGENTS, STANDARDS, AND SUPPLIES**

G. BOD Seed: order through NCL H. GGA: Order through NCL

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QA/QC REQUIREMENTS A. Run a GGA STANDARD with every BOD run. Plot the points on the QA/QC graph, daily.

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QA/QC REQUIREMENTS B. Run a duplicate on 5% of all samples run through the lab, or one sample per week.

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QA/QC REQUIREMENTS C. Unknown samples are ordered through ERA once a year.

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QA/QC REQUIREMENTS D. At least 24 hours before the test, bubble or aerate BOD water for 2 hours.

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TEST PROCEDURE A. On the day of the test remove carboy from the BOD incubator and add 1 ml per each liter (10 ml for 10 liters) of Hach Nutrient BOD pillows.

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TEST PROCEDURE B. Set up BOD Bottles in numerical order.

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Test Procedure C. Obtain BOD WORKBOOK.

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TEST PROCEDURE D. Fill out each page completely. ( Facility , date, sample date, initials time on, time off, date off. )

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TEST PROCEDURE E. Use the following formula to determine the aliquot to be used for the center column for each sample. ml of sample added =1200/estimated BOD, mg/l

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TEST PROCEDURE F. The first column of each sample then should be about 1/2 this value and the third about 2 x as much.

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TEST PROCEDURE G. Use 6 ml per each GGA bottle.

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TEST PROCEDURE H. Prepare Seed according to manufacturers instructions.

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TEST PROCEDURE I. Calculate % concentration for each bottle and record. (Volume of sample/300)

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TEST PROCEDURE J. Line up BOD bottles corresponding to each page of workbook, keep numbers on bottles sequential in ascending order, and also try to keep numbers for each day sequential as well.

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TEST PROCEDURE This will facilitate reading out BOD. Carefully record in workbook the number of the bottle each sample will go in.

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TEST PROCEDURE K. For samples with very high BOD values, it may be difficult to accurately, measure small volumes or to get a truly representative sample. In such a case, initial dilution should first be made on the sample.

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TEST PROCEDURE K. For samples that are supersaturated, bring the DO down to a range of 7 to 9 mg/L. Never set up a set of bottles, above or below this range.

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TEST PROCEDURE L. Check the chlorine residual on samples, before checking pH. Samples containing large amounts of chlorine, will destroy the pH probe.

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TEST PROCEDURE L. Check Cl2 residual and determine the amount of Na2SO3 that will be needed. ( add one ml of Na2SO3 per liter of sample for each mg of Cl2 residual.

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TEST PROCEDURE M. Check the pH of the sample. It must be between 6.5 and Use 1N H2SO4 or 1N NaOH to lower or raise pH. Record initial pH and final pH in the workbook.

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TEST PROCEDURE N. Add seed to the BOD bottles. Fill BLANK bottle with water from the distilled water carboy, and nothing else, place in the BOD incubator.

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TEST PROCEDURE After seed is added you have 5 minutes to have bottles filled and read, do not allow sample aliquots and seed to sit more than 5 minutes.

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TEST PROCEDURE O. Measure aliquots required for each sample. Add well- mixed samples to clean BOD bottles. For the unseeded bottles use only dilution water.

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TEST PROCEDURE P. Place the dilution water carboy on the top counter above the DO meters. (Stir gently.) Connect the vacuum line to the compressed air and gently add air to start siphon.

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TEST PROCEDURE Discard the first 100 ml. Fill all BOD bottles to the half way point of the ground glass neck, do not place hose inside bottles.

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TEST PROCEDURE Q. Check the initial DO in each bottle by inserting the probe into the neck of the bottle and turning on the stirrer.

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TEST PROCEDURE Do not force the probe all the way in, as this displaces to much sample. Check for bubbles at the membranes. Let reading stabilize and then record reading.

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TEST PROCEDURE Bottles must be read within 3 to 5 min. after being filled, as oxygen depletion begins immediately.

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TEST PROCEDURE R. Stopper the bottle, making sure there are absolutely no bubbles inside. Water-seal the bottle by adding water above the stopper and use a plastic cap to inhibit evaporation.

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TEST PROCEDURE Place BOD bottles in a 20 deg. C. incubator. Be sure and place bottles on shelves that are labeled with the day of the week that they are to come out on.

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TEST PROCEDURE S. After bottles have been incubated for 5 days, determine final DO. Record the time off.

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Calculations A. Calculate for each bottle the BOD as below and then average the results for three bottles to obtain the final value.

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CALCULATIONS For any samples which deplete less than 2 mg/L throw out the results. And do not use them.

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CALCULATIONS For any samples which deplete to less than 1 mg/L DO throw out the value and do not use in the average.

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**CALCULATIONS Get the average for the seed bottles. Example:**

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CALCULATIONS Divide the seed bottles by the amount of seed used per bottle. example: 2.3 / 9=0.26*3=0.78 (0.8)

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CALCULATIONS Calculation when seeding BOD Bottles: (B-C)/(A) = BOD mg/L A= Percent sample concentration B= Initial- Final DO C= Seed depletion

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CALCULATIONS Percent sample concentration = sample size/ example: 6 mL/300 = 0.02

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