biochemical oxygen demand biochemical oxygen demand biochemical oxygen demand (also known as bod or biological oxygen demand) is an analytical parameter representing the amount of dissolved oxygen (do) consumed by aerobic bacteria growing on the organic material present in a water sample at a specific temperature over a specific time period. the bod value is most commonly expressed in milligrams of oxygen consumed per liter of sample during 5 days of incubation at 20 °c and is often used as a surrogate of the degree of organic water pollution. bod test bottles at the laboratory of a wastewater treatment plant biochemical oxygen demand (bod) reduction is used as a gauge of the effectiveness of wastewater treatment plants. bod of wastewater effluents is used to indicate the short-term impact on the oxygen levels of the receiving water. bod analysis is similar in function to chemical oxygen demand (cod) analysis, in that both measure the amount of organic compounds in water. however, cod analysis is less specific, since it measures everything that can be chemically oxidized, rather than just levels of biologically oxidized organic matter. background most natural waters contain small quantities of organic compounds. aquatic microorganisms have evolved to use some of these compounds as food. microorganisms living in oxygenated waters use dissolved oxygen to oxidatively degrade the organic compounds, releasing energy which is used for growth and reproduction. populations of these microorganisms tend to increase in proportion to the amount of food available. this microbial metabolism creates an oxygen demand proportional to the amount of organic compounds useful as food. under some circumstances, microbial metabolism can consume dissolved oxygen faster than atmospheric oxygen can dissolve into the water or the autotrophic community (algae, cyanobacteria and macrophytes) can produce. fish and aquatic insects may die when oxygen is depleted by microbial metabolism. biochemical oxygen demand is the amount of oxygen required for microbial metabolism of organic compounds in water. this demand occurs over some variable period of time depending on temperature, nutrient concentrations, and the enzymes available to indigenous microbial populations. the amount of oxygen required to completely oxidize the organic compounds to carbon dioxide and water through generations of microbial growth, death, decay, and cannibalism is total biochemical oxygen demand (total bod). total bod is of more significance to food webs than to water quality. dissolved oxygen depletion is most likely to become evident during the initial aquatic microbial population explosion in response to a large amount of organic material. if the microbial population deoxygenates the water, however, that lack of oxygen imposes a limit on population growth of aerobic aquatic microbial organisms resulting in a longer term food surplus and oxygen deficit. a standard temperature at which bod testing should be carried out was first proposed by the royal commission on sewage disposal in its eighth report in 1912: (c) an effluent in order to comply with the general standard must not contain as discharged more than 3 parts per 100,000 of suspended matter, and with its suspended matters included must not take up at 65°f more than 2.0 parts per 100,000 of dissolved oxygen in 5 days. this general standard should be prescribed either by statute or by order of the central authority, and should be subject to modifications by that authority after an interval of not less than ten years. this was later standardised at 68 °f and then 20 °c. this temperature may be significantly different from the temperature of the natural environment of the water being tested. although the royal commission on sewage disposal proposed 5 days as an adequate test period for rivers of the united kingdom of great britain and ireland, longer periods were investigated for north american rivers. incubation periods of 1, 2, 5, 10 and 20 days were being used into the mid-20th century. keeping dissolved oxygen available at their chosen temperature, investigators found up to 99 percent of total bod was exerted within 20 days, 90 percent within 10 days, and approximately 68 percent within 5 days. variable microbial population shifts to nitrifying bacteria limit test reproducibility for periods greater than 5 days. the 5-day test protocol with acceptably reproducible results emphasizing carbonaceous bod has been endorsed by the united states environmental protection agency (epa). this 5-day bod test result may be described as the amount of oxygen required for aquatic microorganisms to stabilize decomposable organic matter under aerobic conditions. stabilization, in this context, may be perceived in general terms as the conversion of food to living aquatic fauna. although these fauna will continue to exert biochemical oxygen demand as they die, that tends to occur within a more stable evolved ecosystem including higher trophic levels. taking samples from the influent raw wastewater stream for bod measurements at a wastewater treatment plant in haran-al-awamied near damascus in syria history the royal commission on river pollution, established in 1865, and the formation of the royal commission on sewage disposal in 1898 led to the selection in 1908 of bod5 as the definitive test for organic pollution of rivers. five days was chosen as an appropriate test period because this is supposedly the longest time that river water takes to travel from source to estuary in the u.k. in its sixth report the royal commission recommended that the standard set should be 15 parts by weight per million of water. however, in the ninth report the commission had revised the recommended standard: an effluent taking up 2–0 parts dissolved oxygen per 100,000 would be found by a simple calculation to require dilution with at least 8 volumes of river water taking up 0.2 part if the resulting mixture was not to take up more than 0.4 part. our experience indicated that in a large majority of cases the volume of river water would exceed 8 times the volume of effluent, and that the figure of 2–0 parts dissolved oxygen per 100,000, which had been shown to be practicable, would be a safe figure to adopt for the purposes of a general standard, taken in conjunction with the condition that the effluent should not contain more than 3–0 parts per 100,000 of suspended solids. this was the cornerstone 20:30 (bod:suspended solids) + full nitrification standard which was used as a yardstick in the u.k. up to the 1970s for sewage works effluent quality. the united states includes bod effluent limitations in its secondary treatment regulations. secondary sewage treatment is generally expected to remove 85 percent of the bod measured in sewage and produce effluent bod concentrations with a 30-day average of less than 30 mg/l and a 7-day average of less than 45 mg/l. the regulations also describe "treatment equivalent to secondary treatment" as removing 65 percent of the bod and producing effluent bod concentrations with a 30-day average less than 45 mg/l and a 7-day average less than 65 mg/l. typical values most pristine rivers will have a 5-day carbonaceous bod below 1 mg/l. moderately polluted rivers may have a bod value in the range of 2 to 8 mg/l. rivers may be considered severely polluted when bod values exceed 8 mg/l. municipal sewage that is efficiently treated by a three-stage process would have a value of about 20 mg/l or less. untreated sewage varies, but averages around 600 mg/l in europe and as low as 200 mg/l in the u.s., or where there is severe groundwater or surface water infiltration/inflow. the generally lower values in the u.s. derive from the much greater water use per capita than in other parts of the world. use in sewage treatment the bod is used in measuring waste loadings to treatment plants and in evaluating the bod-removal efficiency of such treatment systems. methods winkler published the methodology of a simple, accurate and direct dissolved oxygen analytical procedure in 1888,. since that time, the analysis of dissolved oxygen levels for water has been key to the determination of surface water. the winkler method is still one of only two analytical techniques used to calibrate oxygen electrode meters; the other procedure is based on oxygen solubility at saturation as per henry's law. there are two recognized methods for the measurement of dissolved oxygen for bod and a number of other methods not currently internationally recognised as standard methods dilution method disposable bod bottle glass bod bottle this standard method is recognized by epa, which is labeled method 5210b in the standard methods for the examination of water and wastewater. in order to obtain bod5, dissolved oxygen (do) concentrations in a sample must be measured before and after the incubation period, and appropriately adjusted by the sample corresponding dilution factor. this analysis is performed using 300 ml incubation bottles in which buffered dilution water is dosed with seed microorganisms and stored for 5 days in the dark room at 20 °c to prevent do production via photosynthesis. the bottles have traditionally been made of glass, which required cleaning and rinsing between samples. a sm 5210b approved, disposable, plastic bod bottle is available which eliminates this step. in addition to the various dilutions of bod samples, this procedure requires dilution water blanks, glucose glutamic acid (gga) controls, and seed controls. the dilution water blank is used to confirm the quality of the dilution water that is used to dilute the other samples. this is necessary because impurities in the dilution water may cause significant alterations in the results. the gga control is a standardized solution to determine the quality of the seed, where its recommended bod5 concentration is 198 mg/l ± 30.5 mg/l. for measurement of carbonaceous bod (cbod), a nitrification inhibitor is added after the dilution water has been added to the sample. the inhibitor hinders the oxidation of ammonia nitrogen, which supplies the nitrogenous bod (nbod). when performing the bod5 test, it is conventional practice to measure only cbod because nitrogenous demand does not reflect the oxygen demand from organic matter. this is because nbod is generated by the breakdown of proteins, whereas cbod is produced by the breakdown of organic molecules. bod5 is calculated by: unseeded : b o d 5 = ( d 0 − d 5 ) p _=-d_)} seeded: b o d 5 = ( d 0 − d 5 ) − ( b 0 − b 5 ) f p _=-d_)-(b_-b_)f} where: d 0 is the dissolved oxygen (do) of the diluted solution after preparation (mg/l) d 5 is the do of the diluted solution after 5 day incubation (mg/l) p is the decimal dilution factor b 0 is the do of diluted seed sample after preparation (mg/l) b 5 is the do of diluted seed sample after 5 day incubation (mg/l) f is the ratio of seed volume in dilution solution to seed volume in bod test on seed manometric method this method is limited to the measurement of the oxygen consumption due only to carbonaceous oxidation. ammonia oxidation is inhibited. the sample is kept in a sealed container fitted with a pressure sensor. a substance that absorbs carbon dioxide (typically lithium hydroxide) is added in the container above the sample level. the sample is stored in conditions identical to the dilution method. oxygen is consumed and, as ammonia oxidation is inhibited, carbon dioxide is released. the total amount of gas, and thus the pressure, decreases because carbon dioxide is absorbed. from the drop of pressure, the sensor electronics computes and displays the consumed quantity of oxygen. the main advantages of this method compared to the dilution method are: simplicity: no dilution of sample required, no seeding, no blank sample. direct reading of bod value. continuous display of bod value at the current incubation time. alternative methods biosensor an alternative to measure bod is the development of biosensors, which are devices for the detection of an analyte that combines a biological component with a physicochemical detector component. enzymes are the most widely used biological sensing elements in the fabrication of biosensors. their application in biosensor construction is limited by the tedious, time-consuming and costly enzyme purification methods. microorganisms provide an ideal alternative to these bottlenecks. many micro organisms useful for bod assessment are relatively easy to maintain in pure cultures, grow and harvest at low cost. moreover, the use of microbes in the field of biosensors has opened up new possibilities and advantages such as ease of handling, preparation