Environmental Protection Bureau Of Tainan City Government

NIEA W510.54B
(November 15, Year 89 of the Republic of China(89) wreath office check word No.67626 announce(since February 15, Year 90
of the Republic of China implementation) NIEA W510.54 B
I、Method outline
Biochemical Oxygen Demand，BOD5）。
(Water samples in 20 ℃ incubator and cultured for 5 days in the dark to determine aerobic microorganisms in water samples
during the oxidation of dissolved oxygen in water consumption of substances (Dissolved Oxygen, referred to as DO), 5 days
can be obtained BOD (Biochemical Oxygen Demand, referred to as the BOD5)

II、Applicable scope
This method is applicable to surface water, groundwater and marking and detection of biochemical oxygen demand in water.

III、Disturbance
（A）Acidic or alkaline water can cause errors, of sodium hydroxide or sulphuric acid should be used to adjust.
（B）If containing residual chlorine in water samples will consume oxygen and cause errors, you can use the sodium sulfite
exclude interference.
（C）If cyanide ion in water samples, hexavalent chromium ions and heavy metals will cause interference must be handled
properly, otherwise unsuitable for determination of biochemical oxygen demand.
（D）Dissolved oxygen saturation in water can cause errors. You can adjust the temperature to 20 ° c, then pass into the air
or fully shake to remove interference.
（E）Inorganic substances in water samples such as sulfide and ferrous iron oxidation of errors caused by the consumption
of dissolved oxygen; in addition, reducing oxidation of nitrogen in water sample also consumes oxygen and cause
errors, but you can use the nitrification inhibitors to avoid oxidation.
（F）Visible to the naked eye if included in biological water samples should be removed.

IV、Device
（A）BOD bottles:The glass bottle of 60 mls or larger capacity(with the 300 mls have glass to fill and the BOD bottle of
speaker-like in shape is good).should be to clean wash before use, and then to distilled water leaching clean and dry.
in the seal period shall be cut off from the air in water,way to add distilled water to have stamped with glass stopper
BOD bottles of Horn-shaped estuary. Water seals should be based on paper, plastic cups or thin metal cover of BOD
bottles shaped to reduce water evaporation during.(Note: In order to reduce errors, use a calibrated volume and the
same encoding BOD bottles and caps. If the cap without coding, self-engraved.)
（B）Constant temperature incubator:The temperature may control in 20 ±1 ℃, and may evade the light to prevent in the
BOD bottle the algae line of photosynthesis to cause the water sample to dissolve the oxygen to increase.
（C） Dissolved oxygen measuring device (see detection of dissolved oxygen in water- azide modification method).

V、Reagents
（A）Phosphate buffer solution: dissolve 8.5 g potassium dihydrogen phosphate (KH2PO4), 21.75 g potassium hydrogen
phosphate (K2HPO4), 33.4 g disodium hydrogen phosphate (Na2HPO4. 7H2O) and 1.7 g of ammonium chloride in
about 500 mL of distilled water, to dilute distilled water to 1 l, pH value should be a 7.2.solution or solution described
below, if signs of biological breed shall be discarded
（B）Magnesium sulfate solution: dissolve 22.5 g of magnesium sulfate (MgSO4.7H2O) in distilled water and diluted to 1 L
（C）Calcium chloride solution: Dissolves 27.5 g calcium chloride in the distilled water, and dilutes to 1 L.
（D）Ferric chloride solution: Dissolves 0.25 g ferric chloride (FeCl3.6H2O) in the distilled water, and dilutes to 1 L.
（E）Sulphuric acid solution, 1 n: slowly added 28 mL concentrated sulphuric acid in the mix of distilled water and dilute to 1
l(attention:Prepare process in will produce a great deal of heat).
（F）odium hydroxide solution, 1 n: dissolve 40 g of sodium hydroxide in distilled water and dilute to 1 l.
（G）Sodium sulfite solution, about 0.025 N: dissolve 1.575 g sodium sulfite (Na2SO3) in 1 L distilled water. This solution
instability, must be prepared using on the day.
（H）Nitrification inhibitor: Add 3 mg of 2 - chloro -6-- (trichloromethyl) pyridine 【Soft 2-Chloro-6-(trichloromethyl)
pyridine, referred to as the TCMP】 in 300 mL BOD bottle, and then covered with caps, Or add appropriate amount
of TCMP in the dilution water to a final concentration of 10 mg / L.Pure TCMP dissolution rate may be slower, may
also be floating on the surface. TCMP sold in some of the more soluble in water, but their purity might not 100%,
need to adjust their dosage.
（I）Glucose - glutamic acid standard solution: dissolve 0.1500 g 1 hour after 103 ℃ drying of glucose and glutamate in
distilled water and diluted to 1 L. This solution should be prepared before use.
（J）Ammonium chloride solution: 1.15 g of ammonium chloride dissolved in about 500 mL of distilled water, adjust pH
value to 7.2 with sodium hydroxide solution and dilute distilled water to 1 l. This solution concentration is 0.3 mgN/mL.
（K）Lugol's solution: Dissolves 10 g potassium iodide in 100 mL distilled water.
（L）Dilution water: water sample dilution used. Mineral water, distilled water, can be used after to chlorine by water or
natural water preparation, see seven, preparation step (a).

VI、Sampling and save
Water sample after gathering the microorganism will possibly decompose the organic matter to reduce BOD the value.If the
water sample starts in the sampling latter 2 hours to analyze, cannot refrigerate, because if the sampling site is far away from the
checkout room to be unable in 2 hours to start to analyze, then the water sample should refrigerate in 4 ℃ hidden places, and
analyzes as far as possible in 6 hours, but in any event, the water sample should carry on the analysis in the sampling latter
48 hours. Before the analysis, should return the water sample heats to 20 ±3 ℃.

VII、Steps
（A）The making of dilution water
Takes the right amount volume the water (see five, reagents (12) described) in the suitable vessel, in every 1 L water,
joins the phosphate buffer solution, the magnesium sulfate solution, the calcium chloride solution and ferric chloride
solution each 1 mL.Before preparation of dilution of water use by 20 to ± 3 ° c, shaking or leads to filtered air and
does not contain organic substances, making it dissolved oxygen saturated; or preparation of dilution of water can
be placed in a cotton bottle, save time, make it dissolved oxygen saturation.When preparation dilution water, should
use the clean glassware, guarantees the dilution water quality.

（B）The save of dilution water
The water sample dilution uses the water (see also five, reagents (12) to state) may store to the use, so long as dilution
of water blank value its preparation conforms to seven, steps (eight) of quality control scope the stipulation, this storing
may improve quality of the certain water source, but because possible the microorganism to develop to certain water
sources causes the quality degeneration.The water source after joining the nutrient salt, the mineral substance and the
buffer solution should better not store to surpass for 24 hours, unless the dilution water blank value has been able to
conform to seven, steps (eight) of quality control scope the stipulation. If the dilution water blank value surpasses
the quality control scope, should purify the improvement or changes to water of the other origin

（C）Glucose - glutamic acid standard solution the check
BOD determination system is a biological test, therefore, when the presence or use of toxic substances in the plant
bacterium strains of bad, have a great impact on the results of determination of BOD, such as distilled water is often
contaminated by copper or some wastewater sources of bacteria activity weak, if using this water or bacteria, will
result in lower BOD determination of results.Must be determined by glucose - glutamic acid standard solution of the
BOD values??, to check dilution water quality, effectiveness and strain analysis. Determination of glucose - glutamic
acid standard solution of 2% dilution of culture at 20 ℃ for 5 days BOD values, check their compliance with the
detection precision and accuracy requirements (BOD value of 198 ± 30.5 mg / L).

（D）Plants the fungus
1. Mold mushroom spawn origin
Use plant strains of the bacterium must contain on the biological decomposition of organic matter in water with oxidation
ability of micro-organisms.The family dirty water, wastewater treatment plant, without disinfection with chlorine or other
ways of releasing water discharges of wastewater containing ideal micro-organisms on the surface, some of untreated
industrial waste water, disinfection of waste water, high temperatures or extreme pH values of wastewater microorganisms
are insufficient.The ideal germ grows source to handle hybrid liquid or its liquid waste in the system for waste water
living creature, if can not obtain, can adopt the family dirty water to grow source for germ, must calm down to place
to make it clarify under the indoor temperature first before using, calming down to place time should at more than an
hour, but most long not longer than 36 hours, should take the upper level liquid while taking to use.The use of biological
wastewater treatment system or effluent within the mixture, the nitrification inhibitor should be added after collection.

Certain water samples possibly include are unable mold mushroom spawn of by the family sewage origin by the normal
speed decomposition the organic matter, this time should use waste water biological treatment system the intermixture or
it has not put the running water after the disinfection to take the mold mushroom spawn origin. If no inanimate object
handling equipment ,then take water for 3 to 8 km below the Discharge point.If this mold mushroom spawn originates
is also unable to obtain time, you can on laboratory culture.Tests when voluntarily indoor raises the mold mushroom
spawn, by the soil suspension, the active sludge or the market mold mushroom spawn takes the initial mold mushroom
spawn, in passing through precipitates the family sewage continual aeration raise, and increases the few sewage added
every day.Determination glucose - glutamic acid standard solution of the BOD values ??increase with time until the
measured values ??to reach a stable value and in the 198 ± 30.5 mg / L range, namely, that bacteria develop successful.

2. Plants the fungus control
So-called plant fungus strains for control that is fluid as water determination of BOD value, from the plant bacteria
control values of diluted concentration strain and strain measurement of dissolved oxygen intake, ideally, different
strains diluted concentration and the maximum dilution of concentration to 50% of oxygen consumption.Train 5
days later, dissolved oxygen consumption is greater than 2 mg/L and residual control of dissolved oxygen in the
planting of more than 1 mg/L bacteria, dissolved oxygen consumption (mg/L) corresponding strain volume (mL)
drawing, can render linear relations, its slope represents the oxygen consumption of 1 mL bacteria, and intercept
of the dilution water dissolved oxygen consumption, it must be less than 0.1 mg/L.Or, may also train 5 days later,
dissolved oxygen consumption is greater than 2 mg/L and residual control of dissolved oxygen in the planting of
more than 1 mg/L bacteria, dissolved oxygen consumption (mg/L) divided by the spawn of volume (mL) and seek
mean value .Joins in each BOD bottle the mold mushroom spawn to cause it to dissolve the oxygen consumption to
be situated between 0.6 to 1.0 mg/in the L scope, but joined the strain should be adjusted to make the effects of
glucose-bran acid standard solution of BOD value falls in the range ± 30.5 198 mg/L.The water sample always
dissolves the oxygen consumption deduction mold mushroom spawn to dissolve the oxygen consumption only
then to dissolve the oxygen consumption reality of for the water sample

（E）Water handled before the kind
1. Corrosive alkaline (pH> 8.5) or acid (pH <6.0) of water samples: a 1 N sulfuric acid or sodium hydroxide solution, the
pH value of water sample adjusted to 6.5 to 7.5, adding the volume of the sample volume should not exceed 0.5%
2. The water samples containing chlorine: water samples should be collected before chlorination possible to avoid water
samples containing chlorine.If water containing chlorine, to add sodium sulfite (Na2SO3) removal .Use of volume the
sodium sulfite solution may decide by the following test result: Joins 10 mL 1 + 1 acetic acid solution in the every 1000
mL neutral water sample (or 1 + 50 sulfuric acid solution) and 10 mL lugol's solution, after the mix even, by 0.025 N
sodium sulfite solution titrate, when the iodine and the starch indicator form blue color compound vanishing, namely
for end point of titration.Titration of sodium sulfite solution, the volume of water sample dechlorination is the required
amount of sodium sulfite solution. Dechlorination with sodium sulfite solution after 10 to 20 minutes, must inspect the
water sample whether still to include -odd chlorine. (Note: excessive sodium sulphite solution forms of oxygen demand,
and will slowly and chlorinated water may be present in reacting organic chlorine compounds).

（F）Water sample dilution technique
In principle, after the dilution, the water sample, raises 5 days later, the vestiges dissolve the oxygen above 1 mg/L, when
dissolves the oxygen consumption is bigger than 2 mg/L the reliability to be biggest. Become water kind dilution with dilution
water according to experience several different concentrations, make remnants remaining to dissolve oxygen and dissolve
oxygen consumption to match at the above-mentioned scope. Usually measured by water dilution and COD values to
calculate the BOD values of concentration. Usually of diluted concentration each kind of water sample is: Of industrial waste
serious pollution 0.0 to 1.0%; Not and after precipitates Haven't yet  the waste water 1 to 5%; The living creature once
handles of liquid waste 5-25%; River of water pollution 25 to 100%. Water sample dilution methods are of two kinds, dosage
tube can be diluted, and then mount the BOD bottles, or diluted directly in the BOD bottles.

When dilutes the water sample by the graduated cylinder, and must implant the mold mushroom spawn, may join directly the
mold mushroom spawn dilutes before to diluting the water sample to join the mold mushroom spawn in the graduated cylinder
, implant  the mold mushroom spawn may avoid in the graduated cylinder because of increasing of diluted concentration the
water sample to reduce ratio of the mold mushroom spawn/water sample; When direct in the BOD bottle dilution water kind
and have to Implant bacteria , can directly join the germ kind into dilution water or directly join a BOD bottle, After the dilution,
in BOD bottle water sample volume, if surpasses 67%, after the dilution, in the water sample the nutrient salt possible
insufficient to affect the mold mushroom spawn activeness, At this time, directly (0.33 mL/300 - mL the BOD bottle) the
proportion joins the nutrient salt, the mineral substance and the buffer solution in the BOD bottle by 1 mL/L.

1.The graduated cylinder dilute water samples: If the determination of azide modified when dissolved oxygen in water
samples should be drawn carefully siphon dilution water (if necessary, dilution water shall plant bacteria) in the capacity
of 1 至 2 L of the measuring cylinder, filled to half Full, and avoid air bubbles to enter. After joining the appropriate volume
of mixed water sample, and then add dilution water to scale, mix carefully, and avoid bubbles to enter. By the siphon
absorption intermixture, puts in separately two BOD bottles. The initial determination of which the first bottle of oxygen,
the other bottle is placed in the water in the 20 ℃and cultured for 5 days, then measured the dissolved oxygen.
2.Directly in the BOD bottle dilution water: Takes the suitable volume by the pipette to mix the even water sample, puts in
separately two BOD bottles, Increases the right amount mold mushroom spawn in the individual BOD bottle or the dilution
water, dilutes the water again (when necessity dilutes Implant bacteria) to fill up the BOD bottle, So that, when you plug
into the CAP, all air can be discharged without residual bubbles within the BOD bottles. When the water is greater than
1:100 dilution ratio, water samples should be diluted to an initial volumetric flask and then diluted to the final BOD bottle.
If azide dissolves the oxygen by the  correction method determination water sample, then carries on time the water
sample dilution, must install two BOD bottles, takes bottle of determinations initial dissolve the oxygen, another bottle
postpositioned raises in the hydraulic packing in 20 ℃ constant temperature incubators for 5 days, measured again it
dissolves the oxygen

（G）Initial dissolves determination of the oxygen
If the water sample including with will dissolve material of rapidly the oxygen response, will fill up the BOD bottle after the
dilution water, should immediately determine initial dissolves the oxygen. If the initial determination of dissolved oxygen
does not obviously declined rapidly, the water sample dilution and determination of initial dissolved oxygen during the length
of that non-important factors, but should not exceed 30 minutes.

（H)Dilution water blank
Take dilutes the water as the blank test specimen, inspects after has not planted dilution of water quality and clean BOD
bottle the fungus. When examines each batch of water samples should simultaneously culture a bottle after  Implant bacteria .
After raise and raise (20 ℃, 5 days) determine dissolves the oxygen, it dissolves the oxygen consumption not to surpass
0.2 mg/L, should better below 0.1 mg/L.

（I）Training
After the dilution water kind, repeat analysis water kind and Implant bacteria control, dilution water samples waters, such
as blank and GGA standard solution...etc. to place at the box of the constant temperature development of 20 ± 1 ℃ after
sealing inside develop for 5 days.

（J）Dissolves determination of finally the oxygen
Diluted water samples, repeated analyses, Implant bacteria control, water sample dilution water such as blank and the
effects of GGA standard solution samples after the water seal,  at a constant temperature of 20 ± 1 ° c incubator
training within 5 days.

VIII、Results processing
After 5 days of training, oxygen consumption is greater than 2 mg/L and residual  of dissolved oxygen in 1 mg/L above,
depending on whether planting bacteria, select the formula and calculates the biochemical oxygen demand.

（A）Not plants of biochemical oxygen demand the fungus water sample
BOD5（mg / L）=（D1-D2） / P.
（B）Implant bacteria water biochemical oxygen demand
BOD5（mg / L）=【（D1-D2）-（B1-B2）×f】 / P

D1：After the dilution, the water sample initial dissolves the oxygen（mg / L）
D2：After the dilution, the water sample raises 5 days after 20 ℃ constant temperature incubator dissolve the oxygen（mg / L）
P=【Water sample volume（mL）】 / 【Diluted water samples of the final volume（mL）】
B1：Implant bacteria controlled dissolved oxygen（mg / L）
B2：Controlled at 20 ° c constant temperature incubator Implant bacteria culture 5 days of dissolved oxygen（mg / L）
f：Increase after dilution mold mushroom spawn of the water sample with increases to control mold mushroom spawn ratio
f=【After the dilution, in water sample mold mushroom spawn percentage（﹪）】 / 【mplant  bacteria control the kinds of
bacteria in percentage（﹪）】
If the mold mushroom spawn direct increase or plants BOD of bottle in the fungus control in the sample
f=【After the dilution, in water sample mold mushroom spawn volume】 / 【Implant bacteria control the mold mushroom
spawn volume】

If water samples have more than one dilution of oxygen consumption in line with more than 2 mg / L and the residual
dissolved oxygen in the 1 mg / L or more, and higher dilution of the water is non-toxic and obvious signs of unusual
circumstances, within reasonable limits To issue a report on the average.

IX、Quality control
（A）Control Limit: In view of inter-laboratory comparison of the factors tested very much, BOD and out of the test results is
also large, should be compared to laboratory test results between the standard deviation as a single laboratory control limit.
But also by each individual laboratory to establish their own control limits, the method for each lab in a few weeks or months
at least of 25 glucose - glutamic acid standard solution, Calculate the mean and standard deviation, averaged ± 3 times the
standard deviation as the future of the laboratory determination of glucose - glutamic acid standard solution of the control limits.
Laboratory testing of these single control limit and inter-laboratory comparison test to compare the control limit, if the control
limits beyond 198 ± 30.5 mg / L outside the range, should the revaluation of the control limits, and to study the problem. If the
glucose - glutamic acid standard solution of the BOD values than the control limits should scope and diluted with water from
the strain of all the determination results.
（B）Blank samples, check samples, and sample to be determined simultaneously.
（C）Similar matrix and concentration of each batch of samples or at least every 10 samples to check the implementation of
a sample analysis.
（D）Matrix similar to the concentration of each batch of samples, or at least one out of every 10 samples 1 repeat analysis
to be performed.
（E）In the related quality control document should the record retention time and the preserved temperature.

X、Accuracy and accuracy
（A）Single domestic laboratory measurement examines the result of sample to is shown as form below:

Data source: the Environmental Protection Department of the Executive Yuan environment test routine inspection of the data.
*This statistical value please refer to ten, accuracy and the accuracy (three).

（B）The sole laboratory determination checks result of following table the sample to show:

Data source:Together the reference of this text.
*This statistical value please refer to ten, accuracy and the accuracy (three).
（C）The laboratory ratio measures:At a series of of compare to measure in, invite 2-112 laboratories(include many inspectors
and much germ to grow source) each time, it is Glucose and glutamic acid  1: 1 mix it to synthesize water kind BOD been
developing after 5 days value, synthesize the density scope of water kind as 3.3-231 mgs/ L.Income of average value and
standard deviation S of returning and returning the equation is as follows:

　　　﹦0.658 ×Add a density（mg / L）+ 0.280 mg / L
　　　S﹦0.100 ×Add a density（mg / L）+ 0.547 mg / L

以 300 mg / L GGA
standard solution After raises 5 days is the example, mean value of the substitution above equation its BODFor 198 mg / L，
standard deviation S For 30.5 mg / L。（Data source:Together the reference of this text.）

XI、Reference
（A）American Public Health Association， American Water Works Association & Water Pollution Control Federation.
Standard Methods for the Examination of Water and Wastewater，20th ED.，Method 5210B，pp.5-3～5-6.APHA，
Washington，D.C.，USA. 1998