Washington State Department of Ecology

Епviшnшепtаl AssessmentJ Ршgгаm

Lab Accreditation Section

Supp!ementa! Guidance [от the Determination of

BIOCHEMICAL OXYGEN DEMAND (BODs)

and

CARВONACEOUS BOD (CBODs)

Water апд Wastewater

Plepared Ьу

Репу F., Вшkе, Chemist

Lab AccIeditation Section

March 1998

Publication No. 98-307

Р, inted оп Recyc/ed Рарет

З'd р, inting March 2003

 N()TI(:E

ТЬе J)epartment 01 Ecology isапЕqшi10рр()rtul1ityаПdA.f~r:mаЧr# A(:til);Il€~lJlj)lriyer

and shaII not discriminate оп the basisofrace, че~, co)ol';nati()nal~
status, sеХШtlоriеntаtiоп, age, religion,ordisability !lsdetine.dJ!
state Md/orfederal regulationsor stJl,..щ~s.Iftli():s~~()Пi~])-,iП.g '1'i~~ ..
the Environmental Laboratory Accr;.~itation Progralll ,.,ауе г •

contact theDepartlIlent.,f' Ecology.t!tb~ccl'Cditat:i()l~ s~ttiОП;Jl(360).;.~~.~.

Ecology's telei;ommunicllti()ns~evicef()itl1~ d~аf.(Т'рD)I1~~е:r;i~~Х~~Ю;··
_.,<.-:(-:
 ТаЫе ofContents

1 Backgгound 1

2 Аррюved Methads 3

3 Sampling, Sample Preservation, Holding Times 4

а Sampling Location 4
Ь Sample Size 4
с. Preservation and Holding Times 5

4 Apparatu5 5

а Меавшетеп! ofDissolved Oxygen (DO) 5

(1) Electrode 5
(2) Wink1er. 6
Ь Thermometers 6
с Incubator о! Water Bath 6
d Dilution water container' 6
е. Incubation (ВОD) Bottle5 . 7

5 Reagents 7

а Buffer" 7
Ь NutIients 7
с Standards 7

6 Pretreatment ofSamples. 8

а SamplePrep 8
а 1 empeIature 9
Ь Adjustment of рН 9
с. Dechlorination 9
d Other' Toxic Substances , 10
е Supersaturated Samples 10
f' NitIification Inhibition 11

7 Procedure 12
а P!eparation of'Dilution Water' 12
(1) Source of Water. 12
(2) CBOD and BOD Dilutian Water 12
(а) Dilutian Water far CBOD 5 12
(Ь) Dilution WateI fOI BOD s 13
 (3) Dilution Water Check (Blank) 13
Ь Estimating BOD 5 14
е Seeding 14
О) Requirement to Seed 14
(2) Sources ofSeed 14
(а) Domestic Wastewater Treatment Plant 14
(ь) Соmmегеial Labolatory 15
(е) Othe! Labs 15
(3) Seed Check 15
(а) Cheek Standaгd 15
1 Glucose!Glutamie Acid (G!GA) Check 16
~ Potassium Hydrogen Phthalate (КНР) Cheek Standaгd 16
(Ь) Seed Control Check 17
(4) Choiceof'Seed 18
d Dilution of' SampJes 18
(1) Bottle Method 18
(2) Graduated Суlindе! Method 19
е Determination ofInitial DO ' 19
(1) Winkler Technique 20
(2) DO РтоЬе Teehnique 20
f Incubation 20
g Determination of'Final DO 20

8 CalculationslData Recording , 20

а DiJution Faetol . 21
Ь BOD - Not Seeded 21
е. BOD - Seeded . 22
d. Benchsheet. 23

9 Quality AssuIance/Quality Control 24

а Мinimиm criteria 24
ЬШankв ~
(1) Contamination 25
(2) Supersaturation 25
(3) Iemperature. 26
(4) Pressure 26
(5) Source (DistilledlDeionized) Water 26
(6) Меаsuгеmепt of'DO 26
с Check Standard 27
d Dup!icates 27

11
1О Method PerfOImance Summary ( Вiш, Precision, Detection Limit, Working Limits). 27

а Bias 27
Ь Precision (OI Imprecision) 27
О) Total Imprecision 27
(2) Within-batch Imprecision 27
(3) Between-batch Imprecision . 28
с. Detection Limit. 28
е Working Limits 28
(!) Мinimum 28
(2) Maximum 28

11 28

Appendices:

А - Preparation of·Solutions. A-l

В - Sources of·Reagent Grade Chemicals . В-l
С - Sample Benchsheet (Bottle Method) С-l
D - Sample Benchsheet (Graduated Cylinder Method) D-I
Е -Statistical Terms Used in BOD 1 esting Е-I
F -1 roubleshooting the BOD 1 est F-l
G - BOD/CBOD Checksheet G-I

ТаЫез:

lаЫе 1 - Comparison of·BOD s, CBOD s, and COD Values for WP Studies 2

ТаЫе 2 - Сотmоп BOD Ranges for VaIious Waste Samples 4
Table 3 - BOD Dilutions 9
TabJe 4 - Solubility of·Oxygen in Water Exposed to Wаtег-SаtUIаtеd Air. 11
Table 5 - Expected Values for КНР Standard 17
ТаЫе 6 -lypical Seed Control Check 18
lаЫе 7 - Evidence ofPossible Toxicity Interference 25

IU
(This page intentional1y left blank)

iv
 Supplementa! Guidance fOl the Determination of'
BIOCHEMICAL OXYGEN DEMAND (ROD)
and
CARВONACEOUS BOD (CBOD)
ш

Water and Wastewater

March ]998

1 BackgI'ound

а IЬе ршроsе ofthe biochemical oxygen demand (BOD s) test 1& 10 mеаsще the potent1al о!
wastewater and other waters to dep!ete the oxygen level ofrcceiving wateIs Wastcwatcrtreatment
plant ореratшs a180 use thc BOD s tC5t to determine the cfficiency oftheir plant Ьу measuring BOD s of
the influent, and ofthe eff!uent, and calculating the percent removal, IЬе BODs {е81 тау aIso Ье used
to test wа1еп; other than wastewaters to dеtепniпе thei! oxygen-depleting potential

Ь
Oxygen i8 requiled (used ир, depleted, consumed, assimi1ated) Ьу miс!'Ошgапisms as they
assimilate various organic and inorganic matelials in wateI Зоте о! those matelials contain Icduced
forms оfпitшgеп Collectively these materials are called пitrоgепоus materia!s, and they юе
consumed Ьу nib:ifYing bacteria If those nib:ifYing bacteria ar'e inhiNted, usually using а pyridine
compound, {Ье test measules only сюЬопасеоus organic mater1al, and 18 rеfепеd to аБ сюЬоnасеоus
BODj , 01 CBOD, 11 СахЬопасеоиБ material i8 that consumable materiaI which does not contain
nitrogen

Ь\Vhile the telm "BOD" technically lefexs (о the oxygen uptake demand of contaminated
wateI, the term 18 comrnonly used to lefer to the organic and inOl ganic mater1als consumed Ьу
bacteria, аБ if"BOD" i8 а contaminant in the watcr FOI example, wastewater treatment plants Ieport
"percent хешоуаl ofBOD" аБ an indication ofhow well (Ье plant i8 perform1ng. IШБ docuтent
occasiona11y иБСБ BOD and CBOD in БисЬ а таnnет.

с. Мanу
biological treatment рlап! eilluents contain enough niuifying bactelia that their
consumption of llitrogenous materiaI (material containing пitrоgсп) i8 significant ВесаиБе
nitюgепоus demand has histOlically been considcred ап interference (i с, the primary риХРОБе ofthc
BOD; test i8 to measuI'C Са! ЬоnaсеоиБ matcrial . ,nitrogenous material сап Ье теаsшеd Ьу other' tests
such as ашmоniа, nitIate-пiЬ:itе, and total Kjeldahl nitrogen), (Ье CBOD$ !е5! i8 рrеfепеd [Оl samples
ofsecondary cff!uent. Fo! regulatory programs, hO\VeVeI, the bottom line i8 that the te5! 8pecified Ьу
the I'egulato! тиы ье .тип. In Боте саБеБ, the regulator тау require both BOD s аnд CBOD 5 data

1 Reduced forms 01 other inorganic materials ,исЬ а, su1fides anд fcrтous iron are al80 oxidized dшing both BODs and
СВОО5 tests and wouJd c!cate а po,'itive Ыа. if present in tl.c sampJe 01 dilution water Other inol'ganic materials,
such а, hexavalent cmomium, сорре! , апд frec chlorine тау crcate а (ох,с environment for the bactcria and саш;е а
negative blas. If these typcs 01 intelferrents саnnо! Ье eJiminated рпor 10 тunning (Ье ВОО, or СВОО, (е51, Iheir
prescnce сап Ье menlioned in te51 rерш!s,
 d. It would take the bacteIia in а wastewater saтple 20-plus days 10 assimilate аН the
consumable materia! in а water sample ВесаиБе it i8 impractical to spend 20 days analyzing а
BOD Батрlе, а 5-day test has Ьееп established а;; the standard (hence, BOD5) Depending оп the
БресШс waste stream, 60-70% of' the availablc material 18 consumed in а 5-day period. Another
te8t that meaSUIes the oxygen requirements ofwateI' 18 chemical oxygen demand (COD) wblch, in
а relatively Sh01t time, measUIes essentially 100% о! the chemically oxidizable material.. Onе сап
expect BOD, and COD resu1ts f01 the Бате saтple 10 Ье relaled, thc BOD, value being регЬарв
60-70% ofthe COD value depending оп the natUIe of'the sample. Al50, Ьесаше BOD j i8 а
теавше of nitrogenous and carbonaceous malerial, and CBOD s i8 а теаБше оУ only the
сщЬоnасеоus material, one would ШБО expect а сопеlаtiоп [01 BOD5 and CBODs [О! а given
samp1ing site. Indccd, ТаЫе 1 shOW8 therc i8 а уегу consislent ratio of BOD j to COD, and BODs
to CBOD5 f01 а givcn type of saтple (in this саБе, реrfоrшаnсе evaluation, 01 РЕ saтples fiom
ЕРА'Б Water Pollution, 01 WP Studies). Опе should Ье аЫе 10 determine BODs/COD s and
BODs/CBOD 5 ratios f01 а given waste stream, although the шtiоs тау not Ье аБ cOn8tant, 01 о! the
Бате magnitude as those shown in ТаЬ!е 1. ТаЫе 1 а1в0 shows а consistent ratio between BODs
and tota! 01ganic calbon (ТОС) fOI а given sщnр1ing site И'а reJatively consistent BODsfCOD
111tio сап Ье established fO! а given saтpling point, the COD test сап Ье used as а screening
рюсеss f01 estimating the BOD 5 of the вате sample When used for this РШРОБе, the Hach
СотрanУ'Б lecently developed (but not уе! EPA-арРIOvеd) mаngапеsе-Ш COD method сan Ье
used rather than ап appIOved method, аН of'which gene111te considerable contaminated ,vaste.

ТаЫе 1 - Comparison оС ВОО5, CBODs, COD, and ТОС Values for WP Studies* .

Study BODs CBOD; BODs!CBOD s COD BODs/COD ТОС BODslTOC

.!!!,g[L .!!!,g[L Ratio mglI~ Rlltio mglI, Ratio

WP040 119 100 119 19.2 0.62 76 157

\VРОЗ9 37.6 31.9 1.18 60.7 062 24 1.57
WP038 50.3 43 1.17 81 0.62 32 1.57
WP037 93.1 80 1.16 152 0.61 60 155
\УРО36 13 11 3 1.15 20.8 063 8.2 159
WP035-1 141 117 121 235 0.60 93.1 1.51
WРОЗ5-2 625 516 121 101 062 40 156
WP034-1 302 262 1.15 481 0.63 19 1.59
WРОЗ4-2 999 8.7 1 15 159 0 . 63 63 1..59
WP03З-l 121 107 113 19.5 0.62 77 1.57
WP033-2 547 46.7 1 17 88.6 062 35 156
WP032-1 70..9 64.7 1.10 111 0.64 44 1 61
WPОЗ2-2 152 137 1.11 24.3 063 9.6 1.58
WP031-1 44 . 9 385 1.17 708 0 . 63 281 160
WP031-2 131 112 117 207 0.63 82 160
ViP030-1 14 12 1.17 21.8 064 861 1.63
WP030-2 21.8 195 1 12 35.4 062 14 1.56

Avelage 0,62 1.58

* Demands sэmрlеs in ЕР А'> discontinued Water Pollution Studies were а 50:50 шiхtUI'е of· g1l1соseJglutaшiс acid.

2
 е. ТЬе BOD 5 test ig V'/idely used 10 determine the degree to which а waste stream wiП
contribute 10 pollulion of receiving walers Ьу depriving organisms in those waters (e.g, fish) of
their вощсе ofoxygen. ТЬе BOD5 test is ofpIime impOItance in regu!atory рюgrаms and in
determining the overall health ofreceiving waters оп the other hand, ofa11 the tests done Ьу and
[or wastewater dischargers, BODs is arguably Ihe тов! Jikely 10 result in invalid data if adequate
pIecautions are по! taken 10 аВБШС quality of test results, ТЬе РШРОБе о{ this document is 10
рюvidе supplemental gиidance 10 assist lаЬБ in foJiowing quaJity control рюссdmеs in published
analytiea! methods If'used properly, those methods and pI'Ocedmes will рюvidе evidence that the
measurement of BOD s i8 in contlO!, and that the results meet established data quality objectives,

f Seve1a! technical terms used in this document тау по! Ье familiaJ to some гсООе!Б.
When in doubt аЬои! the meaning of а technical term for the рщроsеs ofthis document, check
Appendix 1 of'Eco!ogy's Prvcedural ManualIor the Environmeпtal Laboratory Accreditation
Program (G/ossary oIQA/QC Terms), Refercnce F 10 lhis docllment.

2 Approved Methods

а. ТЬе BOD 5 test is пorтаНу required Ьу а regu!atory рюgram that i8 governed either
directlyor indiIectIyby Chapter 40, Code ofFederal Regulations, Part 136 (40 CFR 136) which
сопсешs {Ье National Pollutant Dischat'ge E1imination System (NPDES). 40 CFR 136 allows {Ье
иве offour analytical methods fo! reporting BOD5 andior CBOD s: ЕРА Method 405 ..1; Standard
Methods 521ОВ; USGS Method I-1578; and АОАС Method 97344 ЕРА Method 405,.1 refcrs the
иве! to Standard Methods 521 ОБ [от specifics оп conducting the test. It i8 Standard J'vfethods
5210В 01 its derivative tOOt i8 most commonly used in епviюnmепtа1lаЬоrаtО!iеs. It must Ье
emphasized that this guidance dОСllШепt i8 not an aJ1alyticaI method, and that use ofthis guidance
dОСllШепt 01 апу PaJt of' it 18 not requ1red Ьу Eco!ogy, Тhiз sllpplement was written v.>ith the intent
that implementation of its suggestions would тее! 1equirements f01 NPDES and other discharge
monitOling pt'ogrmns. l! i8 not intended to Ье used ав а stand-alone РI'осеdше, but тathe! ав а
supplement {о опе ofthe EPA-арр1'Оvеd BODs methods На simplified BOD 5 method i8 needed,
the 1'еООе! i8 епсошаgеd to review References l1с and l1д оп page 28 ofthis document Both
satisfY the 1equirements of 40 CFR 136 since they follow Standard Methods 5210В,

Ь. АН
BOD5 methods аррюvеd Ьу ЕРА те based оп determinat10rr of oxygen depleted Ьу
bactelia in samples during а 5-day period, in {Ье daJk, а! 20 ± ! о С ав those bacleria сопвuше
organic materials in water samples BODs samples ш'е по!та11у incubated in 300-mL BOD
bott!es, and а1е diluted ifnecessaJY to allow at least 1.0 mglL о! dissolved oxygen to remain at the
end of the 5-day incubation Т о Ье а va1id test, а! least 2 О mglL of oxygen mшt Ье dep!eted
dщiпg the incubation, and а! least 1. 0 mglL must Ье retained at the end ofthe incubation peliod
If necessaJY, samples тиБ! Ье pI'etreated to аssще рюреr рН, temрешtur'е, and absence of toxic
materia1s (e . g." chloline) thus cleating а suitable erтvironment for sщvivаl of the BOD-consuming
bacteria . The foregoing ale Iequirements ofall ЕРА-аррюvеd BODs methods,

- The incubation period is generaJly considered to Ье 5 days ± 2 hours, Ьи! the method does not specify the
tоlеrэn.:-.е

3
 3 Sampling, Sample Pr'eservation, Holding Times

а, Sampling Location. DischaI'ge репшtз о! other Iegu1atory dосшnспts ивиа1lу specify

sampling lосаtiопs, СатС тив! Ье takеп 10 make зш'е the sampJe i8 rcplesentalive of the watel'
Ьоду fiom wшсh it was taken WhetheI taking а grab 01 composiled 8aI1Iple, il i8 very impoltant
to thш'оugh1у mix the ватр!е before \vithdra\ving ап aliquot (SUb-SaI1IрJе) fOl analysis Failure 10
do this fO! а wastewatcI t1'eatment plant iпfluепt, fca ехаmрlе, сап easi1y doublc BOD (and tota!
suspended solids) valucs fO! that samplc

Ь Sample Size" SaI1Iples should Ье takerl in а clean plastic or glass containe1 of sufficiellt
sizc to p!ovide enough Баmрlе fOl the питЬС! ofBOD bottles that will Ье incubated for that
sатрliпg site Ifthe sаmрliпg sile пormа11у rcsults iп а low and predictable BOD value (e,.g,
тапу secondary cff1uents consistcntlyhave BODs's les;; than 10 mglL), il тауЬе песеввату то
iпсuЬаte only опе bott1e, Ьит il would Ье primari1y sample (} с, little ifany dilution waler) 3 Some
ana1ysts prefer 10 a1ways иве а! least two dilutions 1!,"gaтd1ess ofhow ptedictable the BODs Уа1ие
i8 ТЬе tequired 8aI1Iple could therefoтc Ье as Бтаll ав 300 miШlitСIS (Н only опе 300-mL bottle i8
to Ьс incubated). Iftlle sampling site поттаllу rcsults in high and varied BODs values (e.g , raw
inflllent) it тау Ье песе8вауу to incubate ав тапу ав three от four bottles, Ьи! еасЬ would contain
only а fiaction of saI1Iple, with dilution water added to fill the BOD bottle. Again, thc tota1
I'equired saI1Iple might Ье ав втаll ав 300 mL AnotheI considcration in determining saI1Iple size,
howcver, is that the sample mus! Ье I'eplesentative ofthe waste strCaI1I, and the втаllсу the
saI1Iple, the more difficul! il is !о тэkе sше tlш! it is rcpresentative . Also, if saI1Iples тив! Ье
decblorinated (вее paragraph 6с below), от iflab stапdщd operating ршсеdшеs (SOPs) I'cquiIC
апа1уziпg duplicate SaI1Iples, ш analyzing аН samples а! mше than опе dilution, extra saI1Iple must
Ьс {эkеп.

ТаЫе 2- Соmmоп BODs Ranges COI' Various Waste Saroples

Samp1e Соттоп BOD Range (mg/Ll

Influent, domestic ,vaste 150 400

Primary еffluепt 60 160
Secondary Effiuent 5 60
Digester supcrnatant 1000 4000+
Industria1 waste 100 3000+

J IЬе que$rion Qften соте$ ир, "What i$ (Ье maximum yolume of sample сап Ье used in а 300-ш bottle" IЬе 20"
Edition 01 StаndШ'd Methods бnallу clears (Ыз ир Ьу stating сап add almost 300 mL, Ьи! leaving room for seed, and
0.33 mL. ofpho$phate buffer, and 0.33 mL еасЬ оПvlgS04, CaCI2, and FeCJ3 solurion, or, ifthe buffet and nutIients
are already mixed, 1 3 mL ofthe mixtuт·c Anу remaining volume is taken ир Ьу adding Бопt'се water, по! dilurion
water IЬе purpose !о! doing this is 10 make sure (Ье ЬоЩе is properly buffered, and has sufficient seed and nutIients
(о support the support the 5-ооу incubation
4 ТЬеБе BOD ranges are fiom Opetation о) Wastewate, Тreatment Plants, VollI, З'd Edition, ЕРА, 1991 Actual
ВООБ тау Ье outside these ranges (eg, Боmе industrial dischargers ol'BOD, yalues of<2 О mgfL)

4
 АН things conside:red, опе liter of sample is а good planning sizc fш most sampling sites
Expericnee тау show Ша! Боте other size is bettc! .For exaтple, Eeology's ManchesteI Lab and
Боте commcrciallabs require Iшgе! Батрlе;; (опе gallon fш Manchcster Lab)

с Preservation апд Holding Times

(1) ln тОБ! environmental saтples, bacteria паtшаllу РIеБеп! in а waste stream ше

consuming BOD, and tl1еrсfшс depleting oxygen levels, befoIe, dшiпg, and after the Батрlе is
taken It is thercfole very important to begin the analysis а;; БООП ав possibIe afteI the sample is
taken If sto!age о! saтples before analysis еatШоt Ье avoided, they тиБ! Ье kept а! 4 С Ollower
0

(but по! fIeezing) 10 slow (Ье oxygen-depleting activity ofthe bacteria 40 CFR 136 estabJishes
ше maximum storage (holding) time а;; 48 hощs S/andard Method5 5210В recommends that ше
holding time по! exceed six hоmз, and ifanalysis саnnо! sta!t within 6 hоmз, (Ье Батрlе тау Ье
held а! 4 С f01 ир 10 24 Ьош;; Standard Methods also says (Ьа! if analysis еan start оп а grab
0

sample within two hоmз of saтpling, p1eselvation а! 4 С is по! песеББауУ. Composite ватрlеэ
0

must Ье kept а! 4 С dming the entiIe compositiug period, which should not exceed 24 Ьошв
0

0
Аf'tегrетоvаl fiom the oompositor, samples should Ье stored а! 4 С j{analysis сatШоt statt within
two hощs, and just like gтab samples, Standard Methods ваув they should по! Ье held longer than
six hours after retrieva! fiom {Ье compositOI. DischaIge pelillits specify whether saтples should
Ье gтab ог composite .

(2) Some labs operated Ьу permitted wastewater dischatgers have difticulty

meeting even the 48-hour holding time requirement, especially when required (о do BODss аН five
working даув ofthe week 01 тоте . Relieffrom the requircment 18 allowed in 40 CFR 136 (Note 4,
ТаЫе Il) which statcs that "saтples mау Ье ЬеЫ fOI longcr periods only if the pe1mittee, о!
monitoring labO!atory, has data оп filе (о show that (Ье specific types of' ватрlев undCI studyatc
stable for the longer time, and Ьа;; received а vюiапсс." That vatiance would соте fiom the data
ивег Мов! often, fш the UБетв of this docIm1cnt, the data ивет would Ье the Depattment of
Ecology It is unlikely Ша! Ecology would al10w а vatiance exceeding 24 Ьош;; (72 homs tctal)
That period should allow sufficient flexibility f01 (Ье situation at anу sampling site. Т Ье study
suppO!ting а request f01 vшianсе should include analysis о{ several BODs samples r'Ul1 within the
Iegulatory 48-Ьоиг period compatcd to splits of the вате samples storcd at 4 0 С and analyzed just
after 72 h'Ошs. Eeology's Lab AccI'editation Secti'On (360 895-6149) сап assist in determining if
Шете is а statistical diffcr·enee between the two sets о{ data,

4. Apparatus

а. Measurement of<Dissolved Oxygen (DO). 40 CFR 136 allows measurement ofDO

using either an еlесtюdе and meter, '01· the azide ill'Odification of а Wink1eI titration Applicable
methods ше ЕРА Method 360.1 01 Standard Methods 4500-0 G (еlесtюdе), 013602/4500-0 С
(\Vinkler) .

(1) Electrode, Ап oxygen-sensitive теmЬ1anе clectrcde, p'OlaI'Ographic о!

galvanic, with apP!opliate meter meets EPA-аррI'Ovеd method lequirements The YSI 50-series,
anд Orion 800-series instruments юе exaтples of< commelcial meters meeting th'Ose requilcments

5
 fOl measming DO. ТЬе method саllБ for calib!ating (Ье meter using охуgеп-satшаtеd wateI,
wаtеr-sаtшаtеd air, or а Winkler titratioo befO!e еасЬ иве. IГ DO is routinely measured using ап
electrode and mete!, checking calibration ofthc mcte! periodical1y with а Winkler titration wШ
help make sше (Ье meter is functiooing properJy Standard Methods 4500-0 G is а commonly-
used electtode method fO! measming DO.

(2) Winkler·. Bmettes capable of measming accmately to О 1 mL ате sufficient [or·

the Winkler titгation . Standard Melhods 4500-0 D i8 а commonly-used method for measmement
о! DO using а modified Winkler tittation

Ь. ТbcI·mometeIs, TemperalUl·e in the BOD incubator is теавUlед 10 ± 1ос ассшасу

using а thermometer that ig еithегNISТ-сеrtifiеd (Nationallnstitute of Standards апд Teclmology,
fO!merly NBS), 01 Ьав а calibration certificate 01 other· documented evidence showing it is
traceable 10 а NIS Т-certified thermometcr. Ecology' s Lab Асс! cditation Section сап assist
Ecology-accr·edited lаЬБ having а good-quality thermometer Ьу calibtating it against а NIS Т­
certified thermometer (сэll the Lab Accreditation Section а! 360 895-6149) Ifusing ап Ш1
incubat01, the thermoтeter· should Ье immer·sed to the immersion liпе in а suitable containel (е g ,
Ьеakш, ЕщIeтnеуеr flask) filled with water OI glycol solution which acts аБ а heat sink IГusiпg а
water bath, siтply imтelse (Ье thCImometer to the immersion line. If the lab Ьав an expensive
KIST -ce!tified thermometer, to minimize risk ofbreakage it should not Ье used in the BOD
incubato! Rather, it shou1d Ье used to calibrate а lеВБ expensive thcrmometer for иве in the
incubatoI.

с Incubator о! WatcI· Bath. Either an air incubatoI 01· water bath тау Ье used (о
incubate BOD Ьоttlев. Тhe apparatus тиБ! Ье аы1e {о maintain а tеmРCIаtше of20 ± 1ос for the
епНуе 5-day incubation period It тиБ! Ье о! sufficient size to hold аН BOD bottles foт а given
batch (; с, enviIonmental samples, bIanks, Беед сопtюls, glucoseiglutamic acid starldards).. Fo!
Ieasons explained later, it is эlво advantageous foт ап ап incubatoт to Ье large enough to hold the
di!ution water container used for the BOD; detelmination. 111е iпсuЬаtоr/wаtш bath тив! exclude
aHlight to prevent photosynthesis (which would result in а positive contribution to dissolved
oxygen in the incubation bottles, resulting in а negative Ыав in the BOD test). Incubators
specifically designed for BODs ше can Ье pmchased fiom scientific supply vendors (эиеЬ аБ НасЬ
Сотрапу'Б lncuttol®). Less expensive ау·е household refiigelators \vhich have been modified (о
meet BODs test needs Опе required modification is installation of а small fan to create ап airflo\v
aпd епsш·е an еуеп temperature tЩ·оughоut the lefrigeratoT ТЬе thermometer used to monitor
temperature in the inсиЬаtoI should Ье pJaced in the vicinity of the majority of the BOD bottles.

d. Dilution water container. А glass 01 plastic container oflaboratory grade (е .g, по! ап
old ЫеасЬ bottle), large enough 10 supply аН dilution watCI to Ье used in а given batch of samples
should Ье ивед ав the di1ution water containeI. Although по! requiTed, а convenienl way to
iпtюduсе dilution water into BOD bottles without cIeating air bubbles is to siphon the water fiom
the dilution water container. Ifthis teclmique is used, the siphon Ьове should terminate with а 6-
inch section ofgiass tube висЬ that а BOD bottle сап Ье fil1ed fiom the bottom ,vithout
submerging the Ьове. ТЬе dilution water container aпd аН associated equipment must Ье kept
clean (washing with deter·gent and rinsing with disti11ed water should Ье sufficient. . see пех!

6
 suЬрашgrарh) Ifusing а siphon шЬе, the first few mШШtеIS of dilution water should Ье
discarded down the drain.

с. Incubation (BOD) Bottles. Bottles u8ed fш BODs tests вЬои!д пеуе! Ье lIзед [OI апу
tcst othc! thап BOD5 . Ihey сап Ье either 75-,250-, о! 300-mL (300-mL i8 (Ье пюst widely lIsed).
Standar d Methods does not Iequ1re the bottles to Ье made о! апу вресШс material. Аз tms тапиа1
15 being \vri!ten (spring 2003), glass i8 the тов! соттоп material, Ьи! disposablc, plastic bottles
аУС beginning to show ир in thc maIket. Just like the di1ution wate! containc!, BOD bottlcs must
Ье kept clcan Wash aftcl еасЬ изе with detergent, rinse with distilled water, d!ain, and stOIe зисЬ
that the Ьо!йез are по! exposed to dust OI other contam1nants in the lab If Ыаnkз uncxpected1y
Ьесоте higher· (Ьап normal, 1t mау теап (Ье BOD bottles need acid washing. This сап Ьс donc Ьу
first washing, rinsing, and draining аз аЬоуе, aпd (Ьсп rinsing with 1: 1 mшеIаl acid (ршсhаsсd
rcady-madc OI preparcd Ьу slowly adding concentrated hydrochloric OI nitric асЫ to ап equal
volumc of distilled \vater) Rinsing is accomplished Ьу carefully swirIing 1О (о 20 mL оНЬе dilute
acid until аН inner sшfасеs ofthc bott1e ше wetted . Allow the bottle 10 81t [о! а few minutes
before properly discarding the асЫ ТЬеп rinse the bott!e with distilled water and drain. Acid
rinsing i8 по! necessary evelY time (Ье БОD bottJes щ·е used and might neveI Ье required as Jong
ав Ыаnkв ше less (Ьап 0 . 1 mg/L . БОD bottles used [OI Ыankв should Ье сЬозеп rarrdomly to avoid
checking only (Ье "cleaпest" bottle.

5 Reagents

а.. BuHer. То provide the optimurn enviIOnmen1 [or suтyjval оfЪасtеriа in the шсuЬаtеd
sample, it i8 песевваУУ to buffer the ватрlе висЬ (Ьа! it maintains а рН of 6.5 to 7 5. IЬе buffer
carr Ье prepared with vaIious phosphate compounds ав described in Аррепшх А, or il сап Ье
purchased ready-made (вее Аррепшх В). If stOIed, the buffcr solution shou!d Ье refrigcrated а! 40
С to pr·eclude biologica! growth. It must Ье discarded if висЬ growth аррсаУВ Ьесаизе (Ье growth
Ьав arr oxygen demaпd \уЫсЬ would introduce а positive Ыав into аl1 ВОО 5 measuтcments.. ТЬе
рН of the buffcг solution should Ье 7.2..

Ь. NutIients In addition to the nutrient уаlие оНЬе phosphate buffer, nutrients in the
fщm of ammoniurn chJOJide, апd trace mctaIs in the [orт о! fепiс chloride, magnesium sulfate,
and ca1cium chIoride ше added to the dilution water. ТЬеве solutions сап Ье puтchased ready
made (а11 comblned in опе packet), or thcy сап Ье prepared individua!ly as described in Appendix
А НасЬ Соmрапу, North Centra! Labs, aпd ретЬарв others веН packets (рШоws) containing both
buffers апd nutrienls (вее Appendix В).

с Standards То pIOvide а check оп efficiency of tbe seed aпd effcctiveness of dilution

watel, Standard Methods 521 ОБ impIies that а stапdard solution should Ье analyzed with each
batch ofBODs samp]es ТЬе тов! common standard i8 а solution containing 150 mgIL glucose
(dextlose) and 150 mg/L of glutamic acid, commonly referred to ав the "G/GA" solution ТЫв
solution сап Ье prepaIed as described in Appendix А, 01 ршсhаsеd ав а solution fюm а commc!cial

7
 vendO! эисЬ аэ Hach Сотрапу5 о! NOIth Сепиаl Labs In ап interlaboтatolY study, sevela! labs
J'epOIted Iesults f01 the аЮА !es! that averaged 198 mg/L BOD5 , with а inteIJaboratory standaI°d
devialion оВО.5 mglL (эее Appendix Е [ш а discussion of stalistica! terms)о Аэ discussed in
Standard Met}lOds, а sing1e lab should ехрес! 10 achieve an аvешgе ove! severa! tesls of!he аЮА
standard in the vicinity of 198 mg/L (lesults fiom а! 'еаэ! 20 tests, sproead ои! оуе! as тапу
batches, pr-ovide statistically significant data) If а lab' s average is considelabJy 1евэ than 198, а
suonger seed should Ье tried vice versa ifthe average is considerably тоте than 198. А single
1аЬ should Ье аЫе 10 achieve а standa!d deviation шисЬ loweг than 30.5 mglL. А typical single
lab standard deviation would Ье in the mid- to low-teens А standarod deviation of less than ] О
mglL would indicale eXITaOJdinary precision А standard deviation approaching OI exceeding 30о 5
mglI~ indicates excessive шпdоm енот caused Ьу differences in the рroсеdше fiom test to !ев!
ТЪе 20'" Edition оГStаndard MethodJ ваув {Ье 305 mg/L should Ье considered to Ье ап action
limit, or tlu°ee standard deviations Тhiэ wou1d шеan they ехрес! the standard deviation [ш а
single lab 10 Ье 10 mgIL о! less Ecology consideros this goa! to Ье overly optimistic (a!though
асЫеуаЫе) and suggests that а lab should stlive to get а standard deviation in the mid-teens ог
10wer.

б PJoetroeatment of Samples

а Sample Рrоершоаtiоп. After rcmoving апу items 1hat are obviously по! representative of
the sarnpled water (e.g., sticks, othernon-representative solids), the sarnple сап Ье homogenized if
necessary to шзkе sше that а representative aliquot is used for t11e BOD5 tes!. lhis сап Ье done
wi1h а [ood Ь1епдет оп а slow speed, an aggxessive stiпiпg Ьш, от other device that provides
thorougb mixing without being overly disrup!ive to micIOorganisms in the sашрlе а" migb! оссш
iГ, [от example, а high-speed blender is used. Ав pleviously mentioned, it is very important 10
шзkе sше the sarnple is tho!Oughly mixed Ье[orе withdra\ving aliquots (sub-sarnples) [or testing.
If BOD, is expecled (о Ье 80 higb tl131 а уеlУ вшаll aliquot шust Ье laken (вее I аЫе 3, next page),
the entire sarnple сап Ье diluted such that а larger, шоге readily mеasшоеd, aliquot шау Ье tзkеп 6
If а pipette is used (о теавиге the sample aliquot, use of а wide·otip variety is beneficial (ieo, inside
diameler oftip 1/16" to 1/8")

5 НасЬ'в standard, preparoed according to theiT instructions, Ьав а conccntration 01 .300 тgll. .асЬ 01 g!ucosc апд
glutamic acid, twice {Ье concenltation 01 the standatd rеfспед to in Standard Methods НасЬ impJies that rcpeated
"nalyses ОГ Ims solution should result in " mean v"lne пеа! 396 mg!L, ,vith а standatd deviation of 61 О mg/L 01 less
(! е , twice ше 198 mg!l mean апd 30..5 mglL standard deviation reported in Slandafd Methods) о Without supporting
analytica! data, these statistics should Ье иэед with сате since ассш"су is concentrntion dependent When using НасЬ
Сотрапу's standaId, it is Ьеэ! 10 dilute Ше ваmрlе 10 haJllhe strength specified in the inst!Uctions (е go, Ьу
withdгawing halHhe volume [гот (Ье НасЬ vial, or Ьу diluting jt 10 I\vice гЬе volume specified Ьу НасЬ). ты. wШ
resu]t in а solution that;s 150 mgIL ofeach ingredient а. suggostcd ill Standatd Methods.

6 F01 ехаmр]е, iIBOD; is expoeted 10 Ье 1200 mg!L 01 higher, а, shown in ТаЫе , (пех! page) (Ье aliquol (зkеп
wouJd Ье 05 mL, а difficult volume to measure accurately when ,vorking with samples containing solids IIше
sample is diluted to one-tenth its otiginal concentration (е go, 10 тl. 01 sample diluted (о 100 mL) {Ье aliquot taken
would Ье 5 О mL, а уо!ите which сап Ье mеаsuп)d with sulficient ассщаеу Care must Ье taken to thoroughly шiх
such dilutionso

8
 ТаЫе.3 - BODs Dilutions

SampJe (mL) added (о Expected ВО0 5 Range Cmg/L) DiJution Factor

300-mL ВОО Bott1e Minimum Maximum

05 1200 3400 БОО

1 б30 1800 300
3 210 560 100
6 105 280 50
9 70 187 33 ..3
12 53 140 25
15 42 112 20
18 35 94 16.7
24 26 70 125
30 21 56 10
45 14 37 667
60 J1 28 5
75 8 22 4
150 4 12 2
300 2 6 1

Ь.. Теmреrаtш·е. Samples should Ье at 20 ± 1ос before initial ОО i8 read.. Ihis сап Ье
donc byplacing зашрlе containers in cold wateI in а sink ifthey юе too warm, ог in warm water if
they ще too cold. Ношу а зта1l volume of sample i8 going to Ье diluted, it is 110! necessary to
соо! or warm the sample Ьесаизе, when diluted, it wil1 по! significantly affect the tеmрerаtше of
the дНиНоп wate! which should already Ье 20 ± 1ос if· зошсе ,vater i8 stored in the incubator

с. Adjustment оС рН .. Sample рН тпs! Ье in the Iange о! 65 to 75 рН units Add

sulfuric асЫ (H2S04) or ЗОФит hydIoxide (NaOH) о{ sufficient concentration that the quantity of
acid 01 Ьазе added does not dilute the sample Ьу тorе than 0.5%.. FOI example, if the sample i8
опе liter (1,000 mL), the acid 01 base should Ье strong enough that по тorе than 5 mL would Ье
added 10 the sample to bring it into the range of 65 - 75 рН units. (CAUTION! Use extreme
car·e when using strong acids and bases!)

d . Dechlorination. If allowed Ьу the discharge permit and ifpossible given the design of
the treatment plant, ВО05 samples taken а! \vastewater· treatment plants using chlorine to disinfect
the final еШuепt should Ье taken ahead of the cblorination point If this is not possib!e,
decblorination 1В reqU1red, and following decblolination, samples must ье seeded (Ьесаизе the
chlorination рюсеS8 kШs the bacter1a that otherwise would сопзиmе the ВОО in the waste
sample).

9
 (1) If the ватрlе i8 по! highly cblO!inated, dech1O!ination тау оссш пашта1!у if
samples ате allowed 10 8it in the light [or опе 10 two hows. Samples taken fiom waste виеат8
where the fina! етиеп! i8 dechlOIinated ивиаIlу do по! need finther dechlOIi11ation in the lab

(2) Ifnecessary, residual cblO!ine i8 dеstюуеd Ьу adding sodium sп1fitе (Nа,sоз)

so]ution prepared according to (Ье instructions 1П Appendix А Detelmining how тисЬ sоdiшп
sulfitc 18 required to dech10rinate а given атоun! of sample requircs acidification of thc sample,
addition of po!assium iodide, and titшtiоп with standaтd sodium su1fite. Sincc this епйте
рюсеdше само! Ье done оп the ватр]ев that ате later incubated, 1! тив! Ье done оп а sample
dedicaled to that ршрове. This сan Ье done ав [ollows:

То а mеаsшсd атоип! о! sample neutralized 10 рН 6.5 - 7. 5, add 1 О mL оП:1 acetie acid

01 1:50 sulfuric асЫ, and 1О mL of 10% potassium iodide (KI) solution (1 О g КIJI00 mL ав
indicaled in Appendix А) ре! 1,000 mL о[ватрlе . FOI example, if500 mL ofsamp!e i& 10
Ье dech1ori11ated, use 5.0 mL о! aeid, and 50 mL oCКI solution. Add 0.5 ml ofslaтch
indicator solution and titrate with standard sodium sulfite to the stalch-iodine end point [О!
Icsidua! сЫorinе 1 о thc neutralized sample to Ье incubated latcI, add ап equiva1ent
amount оГsоdium sulfite аБ dctcrmined Ьу the above test, wait [0120 minutes, and check
76

[or chlorine using, [от example, а DPD colOJimetric technique Сатс must Ье taken not to
add excess sodium sulfite. Having it8 оwn oxygen demand, excess sodium sulfite would
rcsult in а Ыав оп the high side ofthe actual BOD concentration (i.e., а positive ЫаБ)

е. Other Тоыс Substances. Some wastes, particularly industrial wastes, contain metals
which аге toxic to the orgЭl1isms responsible [о! oxygcn depletion dшiпg the BODs incubatio11
Such toxic materials would rcsult in а negative ЫаБ (i . c, lower than the actua! BOD 5
concentration) IЬе presence oftoxic substances can Ье confirmed bytesting а sct ofserial
di!utions Ifthe mеаsшеd BOD5 fOl а given sample inСlеаБеБ significantlyas the sarnple i8
increasingly di!uted, а toxic виЬБшпсе in the sample (! е., matIix interfelence) is the тов! likely
саше If that toxic substance carmot Ье avoided, it8 presence should Ье leported with lеsпlts
submitted Ьу the lаЬ. Commerciallabs тау know nothing аЬои! possiblc toxicity of samples
received from most clients In БисЬ еаБеБ, the lab might consider doing а ве! о! scria! dilutions .
Although it wШ Ье 100 latc to do anything аЬои! toxicity а! the end ofthe five-day incubation
period, the ртеБenсе of а matrix interference сan а! least Ье Iepolted to the lаЬ client I{ а series of·
dilutions leaves по doubt that interfeI·ence is OCCWling, the highest BOD valuein the
selies .usua11ythe value [orthe most dilute sample ... should Ье !eported.

f Supersaturated Sarnples. If initial dissolved oxygen (DO) readings with а рюреrlу

ca!ibrated DO meter (от аэ mеasшеd with а Winkler titration) indicate the sample contains тorе
DO than it should [or the baт·ometric рrеssше and sarnple temperature а! the time (see Т аЫе 4 оп
the пех! page), the sarnple i8 suреr·sаtшаtеd with DO . Suреrsatшаtiоп might result when the
sample Ьав Ьееп vigorously agitated just prio! to thc DO reading without giving aiT ЬиЬЫев in the
sample а сhanсе to евсаре, OI when the sample 18 undergoing photosynthesis. Supersaturation at

'!
) Fo! example, if а J00 mL ,атр!е i5 u5ed for dete!Jnining how тисЬ sodium sulfite " required, and required 2.30
mL of standaId КI 10 neutraJize the residual chlorine, а ! ,000 mL. sample пот which aliquots ате to Ье taken for
incubation would Ье deehlOlinated with 23 О mL ofthe standard КI (i.e, 100011 00 * 2 30 = 23 о).

10
 ТаЫе 4 - SоluЬШty of'Oxygen iu Water Exposed to Water-Saturated Air
(in mglL at various atmospheric pressures and temperatures)

1 еmреrаtше (OC/OF)
Pressure 170° С 1800 С ]900 С О
20.0 С 2] 00 С 22 о" С 2300 С 24 00 С
(шmliпсhеs Hg)
62 Б F
О
б4 40 F 66.20 F 68 ..00 F 6980 F 7J 60 F 7340 F 7520 F

710/28 О 903 884 8.67 849 833 8 ]7 80] 786

715/281 909 891 8 .. 73 8..55 8 . 39 8.. 23 807 792

~ ~
720/283 9.16 897 8.79 8 . 61 813
725/28.5 9 ..22 8 .. 85 8.67 8.18 803
730/28.7 928 909 8.91 873 85б 840 824 809
7351289 9. 35 9.16 8.97 8.62 846 830 8.14
740129.1 94] 922 9 . 03 8.85 868 851 835 820
745/293 I 891 874 8.57 841 8.25
750/295 9.54 934 9.15 I 0.7' 880 863 847 831
755/297 960 941 9.21 9 . 03 8.86 8.69 8.52 8.. 36
760/299 967 9.47 9..28 9.09 892 8.74 8.58 8..42
765/30.1 9 . 73 9..53 9.34 915 8.97 880 8.63 8.47
770/303 979 9.59 9.40 9.21 903 886 869 8.53
775/30.5 9.86 9.66 946 9. 27 9.09 892 875 8..58
780/307 992 972 952 933 915 8.97 880 864
785/30.9 9.98 9..78 9.58 921 9..03 886 8.69
790/Зi I 10.05 9.84 964 9.45 927 909 892 8.75
795/Зi3 10.11 9.. 90 9.70 9..51 933 9..15 897 8.81

{Ье time of'initial DO l'eading would result in а positive bias. Suреrsаtшаtiоп сап Ье avoided Ьу
taking the initial DO reading опlу оп samples that аге very close to 20 С, and Ьу vigorously 0

agitating the sample and then allowing it 10 settle foт at least 30 minutes before taking the DO
reading, А рroЫет with stlреrsаtшаtiоп is иБиаllу indicated Ьу high bJank results (eg, b!anks
exceeding DO depletion ofO..2 mg/L might Ье due to suреrsаtшаtiоп) 1t is шво possible to Ьауе
suреlsаtшаtеd ватрlеБ without the di1ution watcl' being suреrsаtшаtеd, in which саве the
supersatuIation would not Ье levealed Ьу Ыankв exceeding а depletion о! 0.2 mg!L.

g. Nitrification Inhibltion. Мов! waste st!'eams contain bacteria {Ьа! сопsшnе nitrogen-
containing organic and inorganic matcrials. Glutamic асЫ in the G/GA standald i8 an example of
а nitrogen-containing Ol'ganic matcria! Ammonia is an examplc of an inorganic material
consumed Ьу nitrifying bacteria (! е, bacteria that convert nitrogen-containing materials to nitrite

11
 and nitrate) 1Ье
materials they соnsите ат'е cal1ed nitrogenous matelials, 01 П1tюgепоus BOD 5 ,
СатЬопасеоив BOD! (CBOD;) 18 that рат! ofthe total BOD; that does not 1nclude nitrification" If
CBOD s i8 to Ье determined rather tharr BODs, it i& песеББауу to inhibit (Ье nitrifying bactcria
prcsent in the sample arrd!or seed, 1his 1& done Ьу adding 2-сhIОIо-б-(triсhlоro methy!) PYIidine
(1 СМР) If the lab i8 using the bottle method, 3 mШigrаms оГ Т смр should Ье added to еаеЬ
bottle, Ifthe lab i& using the graduated cylinder method, sufficient 1СМР should Ье added to
make (Ье fina! concentration in the dilution water 10 milligr-ams ТСМР рег litel ofdilution watcr
Рше IСМР dissoJves very slowly in watel" It тау Ье advarrtageous to иве commelcial1y рrершеd
rcagent (see Appendix В)

7, Pl'OceduJ'e

а Preparation of'Dilution Water

(1) Source Water', Water used [or preparing r'eagent solutions and dilution water,
hercaftC1 rcfC1rcd to as sошес water, must Ье of'the highest quality It must contain leSB than 0.1
mg/L сорре! or othel heavy metals, and must Ье completely free of' chlorinc, chloramines, OIganic
material, acids, and ЬаБев" Distilled water prepared using а glass stШ in the lab i8 sometimes
suitable, although water fi:om some виllв might contain t!ace metals, ch1orine, ammonia, 01
voIatile organic materials making il unsuitabIe DistiJled water coming from а сорре! still 18
8eldom suitabIe" Deionized (DI) water sometimes contains O!'ganic materials leached from {Ье
resin bed, Some ригсЬавоо disti!led waters ш'С suitabIe, arrd Боте ате по!, If water is pmchased,
тапу 'аЬв Ьауе fошrd "steam distilled"g water to provide the Ьев! rC8ults Distilled dr'inking water
is по! suitablc ав it geneIally contains chemicals (e"g, chJoline) that would adversely affect BOD s
results Ifunseeded BOD 5 bIanks always run high (i,e" they al\vays exceed 0..2 mg/L), the
distilled water used 10 ртерахе dilution watC1 should Ье опе ofthe first things to investigrrte in
Irying 10 find the саше, Some labs Ьаус had Биссевв using tap water, others spring water, {Ъс
bottom Нпе i8, ifit works (i с" ifthcIC i8 по evidence of'toxicity, ifthe averagc J'csult for
glucose/glutamic асИ analyses i8 по! substantially 1ess tharr 198 mg/L, and results for bIal1ks шс
cOl1sistently below 0.2 mg/L), use it, If it does not, try another watcr

(2) CBOD and BOD DiIution Water' Dilution water s!JouJd Ье рrершеd Ьу
differcnt рюсеdше8 depending оп whether it i8 to Ье used fш' CBOD s OI fш BOD;

(а)
Dilution Water Со! CBOD. Ре! liter of вош'се water, add 1 О mL cach
of the phosphate buffer, magrresium sulfatc, calcium chloride, and fепiс chloride solutions (see
Appendix А) OI add the contents ofprc-measmcd packets (sec Appendix В), Aerate (1 е, sаtшаtе
{Ье solution with air) Ьу shaking а ршtiаllу filled container О! Ьу bubbling filtered Ю! though the
solution" Use а plastic от glass carboy OI other container о! а sufficient volume that it wШ provide
sufficient water for ап entire batch, but втаll enough that it СШ Ье еаБilу shaken (if sl1aking i8

8 АII distiПеd water could Ье thought 01 а. steam distilled because the water goes through а steam рЬаэе Howeve!
"steam distilled" water, wheIe tbat designation is advertised оп the containel's in large letlering, goes through а special
process that results in а particuJarly suitabIe water ГОС иве in the BOD test, If the lab cannot find this type of water in
local supermaIkets, (Ье i,ab ACCIeditation Section сап Ье contacted ТО determine Ьтапд пате, and potential SOUI'ces
(360895-6149)

12
 used to aerate). 1о minimize the plObabi!ity that dilution \vater will Ье supersaturated with
dissolved oxygen when used, store this water foт at least 24 hошs in the dшk at 20 ± 1 о С
(storage in the BOD air incubatoI 15 ideal) То alIow ftee transport of oxygen to and ftom the
containcr during storage, thc top of the containcI· should Ье loosely fitted, от rcplaccd with а
looscly-packcd wad of соНоп Check stшсd dilution watcr to determine if sufficicnt шnmопiа
rcmains aftcr stшаgе. Add ammonium chlOIide solution (Бее Appendix А) if necessary (о bring
the шnmопiа level to аррюхimаtеlу 0.45 mg/L ammonia а;; nitlOgen 9

(Ь)Dilution Water 101· BODs. Fш BODs, aerate а container ofsoUIce

water as fO! CBOD5 in 7а(2)1 аЬоус, Ьu! without the buffer 01 nutrient solutions It is
advantageous to do this по later (Ьап the day befoтe the BODs test Place the container in the BOD
incubatol to make вите the water is at 20 ± 1о С when leady foт иве. ApPlOximately опе Ьоur
beforc the BOD bottles ше (о Ье 5е! ир, add the buffer aJId nutrient reagents to (Ье di1ution \vater
Do this 80 а;; to minirnize introduction of· ш! bubbles into the dilution water eontainer. Опе way of
doing this is to dissoIve the nutrients aJId buffers in а втаll шnоипt of water (Hach Сотрму' S aJId
NCL's buffcr!nutrient pi1lows аге a1ready in solution), рош the solution down the inside \уаl1 ofthe

dilution water container, and gently swirl (по! shake, as in aerating) the container to mix the
so!utions Ihe reason fO! по! stO!ing BOD s dilution water with the nutrients/buffels aIready added
is (Ьа! а significant poplllation ofnitrifying bacteria тау deve!op 1fthe nutrients-buffers are
present in (Ье water Fш the CBOD s test, the nitrifYing baeteria are inhibited and thеr<:fше not а
probIem (вее ршаgrарh 4Ь, Standatd Methods 521ОВ)

(3) Dilution Water Check (Blank). Rеgшdlеss of how il is made, 1! is necessary

to check dilution water wilh every batch ofBODs ватр!ев to make ВШе it i8 по! causing епог in
the te8t .

(а) Опе
check is to make вше (Ье buffer has done its job and that the рН i8
in the vicinity о! 7 2. Although not mentioned in Standard Methods, the dilution water СМ Ье
checked aftcr buffers aJId nutrients Ьауе Ьесп added 10 determine ifthe butIers have stаЬШzеd рН
а! аррюхimаtеJу IЬе рН of the dilution water 10 б.5 10 8.5 рН unils (page 276, reference 11 е). 1f it
ig по!, sош·се water is рюЬаЫу the саиве and a!ternative waters вЬоиИ Ье investigated.

(Ь) Another check invoJves simрlуmеаsшiпg the initia! DO ofa BOD

bottle [иll of'dilution \vater, incubating it [от 5 даУ8, and reading the fina! DO. DO dep!etion
should not exceed 02 mg/L. IfDO depletion oUhe ЫЗIIk consistently exceeds О 2 mg/L, the
саиве might Ье the вошсе water, ав already mentioned.. О! it could Ье that (Ье dilution water 1В
suреr·sаtшаtеd with oxygen when tlle initial DO is read, indicating а need to let the water staJId fO!

9 НасЬ Company and NCL bllffeI-·пu!Iien! pillOW8 contain ammoruum chlo!ide 10 а8sш·е О 45 mgIL ammonia wi!! Ье
presen! Additiona! N!i, СI should по! Ье required. Because ассшасу " пot critical for (Ье test used to detmmine '{
ammonia concenttalion " а! leas! 0.45 mgfL, '! '" по! пеСС58агу 10 и50 ап EPA-approved !О5! method Inexpensive
00101 oomp",ison kits Се g., disks and cllbes) made Ьу scveTa] сотрап,., (e.g., CHEMetrics, HF Scicntific, LaMotle,
0lbeco-Hellige) ате availabIe fO! scientific equipmen! supp1iers (eg., VWR Scien!ific Praducts). НасЬ Сатрапу also
рю,idеs а соl0Т disk (Са! #224 J-00) and а соl01 сиЬе (Са! #22669-100) fOl!ow-lаugе ammonia !е51в

13
 а longer pe!iod befo!e setting ир the BOD bott]es. IfDO dep]etion [OI Ihe Ыаnk i8 excessive only
periodically, contamination (e.g., ofthe BOD bottles, dilution wateI containeI, deliveIY hose, от
otherlabwarc), fаilше 10 take both initial and final DO Ieadings а! 20 ± I о С, от епотs in DO
meter calibration (e.g., not changing Ihe тетЬгапе often enough) might Ье the саиве Ifthc
ini!ial DO !eading is Iower than the final DO reading (! е , ав if oxygen was somehow generated
dшiпg the 5-day incubation pexiod), the р!ОЫст could Ье that the initial DO i8 !ead when Ihe
dilution wateI temреrаtше i8 considelably higher Ihan 20 ± ] Q С. Stoтing the di1ution wateI in
the BOD incubator а! 'еав! ove! night should solve this рroЫет Нighe! DO !evels afteI the 5-day
incubation could also result from photosynthesis if incubation i8 по! done in total darkness .

ь Еstiшаtiпg BODs, Ав pJeviously mel1tioned (paJagraph ld), а ргеliтiпюу COD teBt

сan рюvidе а estimate ofBOD s allowing the analyst to deteImine what dillltions are mos! likely to
yield valid BODs I'esultB Being аЫе 10 make an estimate i8 especially useful [о! wastewate!
txeatтent plant influents which might vary widely fi:om week (о week, and [от commerciallabs
which might have по idea ofthe аррюхimа!е BODs of Батрlев received in !he lаЬ. AnotheI tes!
that сап рюvidе ап estimate of" BODs f01 was!ewatc!O samples is (Ье total suspel1ded solids (Т SS)
tes!. КnO\ving !Ье аррюхimаtе BODs/TSS ra!io i8 especiallyuseful in а domestic wastewater
tl'еаtтепt plant where тисЬ ofthe BOD s in an influcnt 18 in (Ье [отт of8uspended soJids Fo! а
gjven waste streaт, а юugh BODs/TSS сопоеlаtiоп сап Ье estabJished, a1though il тау vщу
according to веавоп, flow, 01 otheI factors. If lnfluent TSS i8 used to estimate BODs, l! i8
impoтtant хо thOI oughly hоmоgепizе 1Ье saтple. ТЫБ сап Ье done with а homogenizero от high-
speed bJender

с Seeding

(1) Requirement to Seed. Saтples which do по! already contain enough of Ihc
рюре! bacteria сап Ье analyzed [OI BODs опlу afte! addition of"8eed." Seed 18 nothing тоге than
а soltltiol1 сопtainiпg а sufficient population of suitable bacte!ia. Inflllent to а domestie
\vastewater tJ:eatmenl plant generally contains sufficient bacteria and usuaJly does по! need 10 Ье
seeded.. А! the othero епд of the plant, final етиеn! which nas Ьееn disinfected (with chlOIine о!
иltravioJet 1igh!, fo! exaтple) ивиаlIу does по! have а sufficient bacterla population and тив! Ье
seeded . Other waters that usually require seeding are untJ:eated industrial or high,teтperature
wastes, о! those of extreme рН, Effillent [ют а domestic wastcwater treatment plant is (Ье
РI'еfепеd Beed for the detelmination ofBOD 5 in samples fiom that plant beeause (Ье tI'eated, Ьи!
по! disinfected effluent, cOl1tains bacte!ia that юе acclimated to (Ье waste. Perf01mance evaluation
samples (РЕ saтples, now rеfепеd (о аБ p!Oficiencytesting, о! РТ sampJes) and standard solutions
always need seeding since they contain по паtша! bacteria.

(2) SOUlCes of Seed

(а) Domestic Wastewater Тlеаtшепt Plants. Effiuent fiom а domestic

tIeatтent plant also mау Ье the ЬеБ! seed for' waste fiom ап indllStJial process OI f01 wateJ'S по!
expected to contain а sigr1ificant population оfЪасtсriа. Ifo еШисп! fr'om а secondary tJ:eatтent
process is deteImined to Ье too weak through eilheI the g!ucose/glutaтic acid (ев! 01 the seed
eontJ:a! te5t (вее the nех! slIbparagraph), етиеп! ft'om the primary tteatтent РlOсеВБ should Ье

14
tried In eitheI саве, иве supernatant ftom the treated effluent (decant 01 withdraw using а pipette)
afte1 settling а! 100т tеmреlаtше fш а! least опе Ьош but not longe!' that 36 hOUIS at apploximately
20°С (i е, шоm ш incubatoJ' temреl'аtш·е). Inf1uent to а domestic wastewatcI treatment plant i8
gene!a!ly not suitable Ьесаиве of problems with d!awing homogeneous aliquots of' consistent
quality. Ifinfluent is used fщ' seed, it should Ье thoroughlymixed and allowed to settle ав аЬоуе
Some Jabs Ьауе found аиссевв using settled matelia! ftom а c!a!ifiel.

(ь) Commercial Labs. Мanу сотте! cia!labs anд some industrial

discharger !аЬв pтefer' (о иве aгtificia! seed, аисЬ ав Polybac®,Po!yseed®, ог BioSystems®. If thcse
matC!ia!s аге used, spccial саге must Ье taken il1 рп::раliпg the seed in accoIdance with thc suppliels
iпstшсtiопs Failше to do 80 will Icsult in unacceptably low lesu!ts fш the seed control bottle(s)
(i.e, the seed contro! willlcsult in а depletion of <06 mg/l, DO ре! milliJiter о! seed) and fOT аН
samples that rcquirc secding, including РЕ ватр!ев.

(с) Other Labs.. Othel' lаЬв might со1lес! seed matelial pe!iodical!y fiom а
tlealment plant and keep it viable Ьу feeding it starch 01 воте other nutrient while stOIed in the
incubatoI 1Iial and епor will disclose how тuсЬ nuuient is needed, and how long (Ье seed сan
Ье maintained.. Effectiveness of висЬ sloI'Cd seeds ВЬОlllд Ье closely monitOled Ьу checking !CSllItS
fiom the G!GA and seed control tes!;; and monitOIing for tIends indicaling deteIiOIation of' the
seed Use ofprccision contro! cha!ts is the best mechanism for monitOIing perfolmance ofthe
веед and otheI aspects ofthe BOD s tes! Accledited labs сan Ieceive а nее Ехсеl spI'eadsheet
program the automates the contto! сhюtiпg Рl'Oсевв Ьу contacting the LзЬ Accreditation Section
(360895-6149).

(3) Seed Check. Ав aheady discussed, bacteIia added 10 samples containing few
viable bacteria (e . g , due 10 ch1orination, UV tIeatment, extreme рН, 01 the action of toxicants that
might Ье found in воте industlia! wastes), атс called "seed" arrd IЬе рroсевв is calIed "seeding" Seed
must Ье of sufficient microbio!ogica! activity (i.e, тшl Ье potent enough) 10 pIopeIly activate а BOD
bott!e (or g!aduated cylinder if using that рroсеdш'е) without adding а significant vо1шnе of seed
compaled 10 the уо1ите о! the solution 10 which it i8 added Т wo tests юе used 10 detelmine the
effectiveness of the seeding matelial: (а) а check standaг'd; and (ь) а seed control check

(а) Check Standard. ТЬе тов! \videlYllsed check starrdatd (i.e., а sO!lltion
ofknown concentration used 10 check the performance ofa tcSI) forthe BOD s tes! is а sol11lion of
150 mg/L glucose (аlзо called dехtюsе) and 150 mgiL g!utamic acid (i с, the ОIGА te8t) An
alte!'llative to ОIGА i8 а solu!ion оПОО mglL potassium hymogen phthalate (КНР). An advantage
ofusing GIGA оуе! КНР i8 thal ОIGА i8 more widcly lecognized ав the check standard fOl the
BODs test which has п:sultеd in а lюgе data basc fOI tcst Iesults Also, glutamic acid contains
пitюgеп which рюvidеs OIganic material fOl nitrifYing bacteria to сопвите. An advantage ofthc
КНР te8t 1в that the 300 mg/L so!ution сап Ье used ав а check standюd f01 seveIa! other tests ВllСЬ
as COD, ТОС, рН, acidity, total solids, volatile so!ids, and conductivity (вее ТаЫе 5). Тhe ОЮА
sollltion could likewise Ье used ав checks for those tests, but the expected values al'e not wel1
documented. Whichever solution i8 used ав а standard, notice that the solution i8 acidic and might
need to Ье treated with sodium hydlOxide (NaOR) {о bling рН into the range of6.5-7..5 рН units.

]5
 1, Glllcose/Gllltamic Acid (СIGА) Check Standard
J! А standard so!ution о! 150 mglL ofg!ucose and 150 mglL
glutamic acid is prep81ed in accordance with the iпstшсtiопs in Appendix А 1Ье standard сап
а!во ЬеpUI'chased ready-made (вее Appendix В).

.\:!' Standard Methods suggests а G/GA soJution shoald Ье

analyzed in each batch of BOD 5 ваmр!ев 1тв is а good idea if а lab is gathering initia! data оп
the analysis, Опсе the lab is confident with its abi!ity to do the BOD 5 test with acceptable Ыав and
good pIecision, fiequency of {Ье G/GA test тау Ье reduced to pelhaps оnе ре! week, 01 two per
month 1t should Ье remembered, however, that апу set of'data is mОlе еавilу defended
(scientifical1yand legalIy) if а check standard and othe! QC s8lllples аIе analyzed with еасЬ batch,

~, AfteI а! lеав! twenty (ten is suf'ficient if {Ье lab is just

getting staIted, but 20 is pIeferтed) G/GA so!utions have Ьееп analyzed ОУе!' а сошsе of several
days OI' weeks, the mеan (average) Iesult and standard deviation of' rcsu1ts а!е calcuJatcd (вее
Appendix Е [ш ап exp!anation ofstatistical telms) Standar'd Methods r'epOIts that in an
interlabotatory study (i..e , invo!ving many Jabs), (Ье теan геви!! was 198 mg!L, \vith а st811dard
deviation ofaJJ results of 305 mg!L With that in mind, the objective established Ьу тanу labs i8
that IЬеу Ье аЫе to acmeve а mearl of аррюхimаtе!у 198 mg!L, \vith а standard deviation 0005
mg!L OI Jess. Опе should realize, however, that the 30.5 mglL standald deviation was cslcu1ated
ftom data submitted Ьу severallabs, where веуега! analysts llsed several diffelCn! seeds, with
several vatiations in otheI' (ев! par8lllelers 1 тв \vou!d Ьс expected 10 IesuH in а higher 8tanda! d
deviation (ic, тоте imprecision) than would test rcsults [гот а single lab А better goal [ОI а
singJe lаЬ is 10 ехрес! а mеап of appгoximately 198 mg!L, \vith а standard deviation in the lеепв
(e.g , 15 mglL) 01 !ower А! least опе lab participating in EcoIogy' s Епviюnmепtа! Labotatory
Acc!editation P:rоgшm consistentIy analyzed (Ье GIGA solution with а stand81d deviation of
арршхimаtеlу 4,0 mg!L.. Whiie it i8 по! теавоnaЫе to ехрес! every iab (о астеУе висЬ p!ccision,
11 shouid Ье kepl in mind that such results 8lС possible.

l. Potassium Hydrogen Phthalate (КНР) Check Standar'd

~ А standard solut10n of300 mglL ofruP i8 prep81cd in
accordancc with the instructions in Appcndix А. ТЫв soIutiou can Ьс anaiyzed peliodicaI1y as а
suppJemcnt 10 thc GIGA tcst, о! it can Ieplace the GIGA tes! Bcforc rcplacing the GIGA teBt,
howevcI', consideration should Ье given 10 the facl (Ьа! G/GA lesults is most wideiy recognizcd ав
thc primary indicatoт ofpcrfoJmancc fO!' thc BOD tcst

Q. If'replacing the GIGA tcst, the кнр tCS! should Ье уип

with the same fiequency suggested [01 GIGA in p81agraph 7Ь(З)(а)lЬ аЬоуе

Q Only limited data 8lС ауаПаЫе ироп wmch to base quaiity

objectives гО!' the КНР tes! . In а seIics of' 159 tests of the КНР solution оуеI а pCliod of
аррюхimаtеlу five years, Ecology's ManchesteI I~ab achieved а теап value of 249 mg!L, with а
standard deYiation of 15 4 mglL. Пlеве wouJd Ьс rcasonable statistics 10 use ав initia! objectives

16
 until data gathered Ьу а lab indicates othe! statistics might Ье more appropliate. If the КНР
solution i8 used ав а standard fш othcr tcsts, Т аЫе 5 shows reasonabIe objectives [01 mean уаlиев
[01 the various tests ав achieved Ьу Ecology's Manchester Lab. PIecision data (e.g., standап:!
deviations [ш repeated analyses) Ьауе по! Ьеen determined (exccpt [О! BOD" as indicated аЬоуе)

ТаЫе 5 - Expected Values (о!' а 300 mg/L КНР Standard

Рашmеtеr Expected Уаlие

BOD, 249 mg/l

COD 343 mg!l

Т ota! Organic Carbon (Т ОС) 141 mgll

рН 44

Т otaJ Solids (1 S) 300mgil

VoJatiJe SoJid, (VS) 200mglL

Conductivity 169 тЬо.

Acidity 74 mglL

(Ь)
Seed Control Check. Another check оп seed effectiveness i8 the seed
control check. It is also used to detcrminc what contribtltion the seed itself will make to the DO
depletion о[ seeded samples . 1hat contIibution шust Ье subtIacted \vhen ca1cu1ating samplc
BOD,.. 10 do the check, set ир three BOD Ьоttlев with 3, 6, and 9 mil1iliters of seed (01 осЬеI
vоluшеs that will result in а DO depletion of а! least 2.0 mglL, and а retention of· at least 1.0 mgIL
after the 5-day incubation), and {i1l the bottles with dilution water IЬеве ше cal1ed "зеед contIol"
bottles, 01 sometimes "seed Ыank" bottles. 1t mау Ье necessary to do three dilutions fш· the seed
eontIol until the seed matelial i8 well-сhаIаеtеJizеd, at which time опе bottle mау give Ieliable
results. Меазшс initia1 DO, incubate fOJ five даув, mеаsше final DO, anд calculate the DO
depletion per· mШШtсr of seed 1o• An effective seed lesults in а depletion between О 6 and 1.0

10 Standard 11,fethod, 521 ОБ is nor entirely сlеат оп Iы;, I~quitement Paragraph 521 OB4d(2) states that "1Ье DO
uptake 01 seeded diJution water sbouJd Ье betlveen О 6 and 1.0 mglL" Ьиl il does по! associate Iы;, depJction with а
voJume of·seed In suЬра:шgraрh 5 under "Seeding оГsаmр!е" оп page 476 о! Operation о! If'a,tewatel Treatmenl
Plant, (Reference 11 d 10 Ims document), ЕРА IШу;, "Seed material shошd ртодисе а correction о! а! least 0.6 mglL
ре.· mL of seed" lЬе author interprets (Ьеве two stalemenls together to indicate thal по! only should the всед matcrial
result in а deple!ion ofO 6 - J.O mgIL ре. mL 01 sced, Ьи! aIso that (Ье amount 01· seed added 10 еасЬ seeded BOD
bottJe 0110 а graduated cyJindcl should cause а ОО depletiorJ of Об - I О mg!L in а seeded bottle

17
 mg!l for еасЬ mL of seed Опее ап effective seed Ьа,;; Ьееп idепtШеd and it consistent1y result;; in
а DO depletion of 0 . 6 - 1.0 mglL ре! mL о! seed, it is по 10ngeI necessaIY 10 incubate tlrree ЬоШеs
with еаеЬ batch and onJy опе need Ье done. I аЫе 6 shows data f01 ап effective seed. ВоШе 3 did
по! !esult in va1id data Ьесаиве Фе final ОО was less than 1.0 mgIL

(4) Choice of Seed. ТЬе hottom Нпе in choosing а seed materiaJ is (о pick опе that
results in G!GA tests in the vicinity о! 198 mg/L, апd in consistent seed contm! test results
between О 6 - 10 mg!L DO depletion ре! mL of seed Paragtaph 7с(2) оп page ]4 discusses
possibIe SОШ'сеs of such а seed

ТаЫе 6 - Typical Seed Contr'ol Check

ВоШе 1 Bottle 2 Bottle 3

Sample Volume (mL) О О О

Seed Vo)ume (mL) 3 6 9
Initial DO (mgIL) 8.9 89 8.9
Final (5-day) DO (mg/L) 6.2 33 0. 8
Depletion (тg/L) 27 5.6 81
Depletion pcr' mL of' seed 0..9 0.93 (по!valid '
(mg/L DO ре! mL) <1.0mglL
,esidual ОО)

d. Dilution 01' Samples. То тее! the method requirement that а! least 1.0 mg!L ofDO
гemain in the sample after the 5-day ineubation peliod, some sarnpJes тив! Ье diJuted (вее 1able
3).. ТЬеу тив! по! Ье diluted 10 such ап eXlent, however, Фа! DO depletion du:ring the 5-ооу
incubation i8 less than 2.0 mglL, the method-imposed minimum DO depletion fш а va1id bottle .
Ihele аге two alternative plOcedures to follow fo! diJution о{ sarnples p!ior to incubation One
involves dilution оНЬе sarnple in lЬе BOD bottle, refeIIed to as the bottle method. In the otheI,
the Бarnр!е is diluted in а gtaduated cylindcr апd then ап aliquot is ttansfelIOd to а BOD bott1e f01
incubation. ТЫБ is rсfеп'еd to as the graduated cylinder method. Choiee о! а method is а mat!er
оГ ренюпа! prefercnce and either сап consistently produce teliable and accmate Icsults.

(1) ВоШе Method

(а) Refin if neeessary 10 1 аЫе 3 10 determine how тисЬ sampJe 10 add 10

each BOD bottJe If the dilution would Ье gtcateI thап 1: 100 (! с, jf lеБВ lhan 3 mL of вarnрlе
\vou1d Ье added to а 300-mL BOD bottle), as а prelirninary step, dilute the entirc sarnpJe taken
ftom Фе waste st!earn with reagent gtade wateI before adding it 10 thc ВОО bottJe. Оо по! forgel
10 multiply the result Ьу the additiol1aJ dilution fасtш. If Фе sampJc being апаlyzеd i8 wеП
charactcrized (that is, it 18 well known to Фе Jab, having been analyzed mапу times, with results
always being w1thin а Ie!atively smаП range), and ifprccision f01 thc test is УClУ good (that is, Фе

18
 standaId deviation [о! (Ье glucose/glutamic асИ (ев! is relalively smaJl), it тау Ье sufticient 10 set
пр only опе BOD bottle [О! incubation ре! saтpling site (NOIE: Some discharge permit
maпagers тау по! al10w incubation оГ only опе bottle, even [о! а well-characterized saтpling
site) Ifthe watel being anaJyzed is по! well-chalaetelized, ifprecision is onJy marginally
acceptable, 01' if lequired Ьу а regulat01Y prograт, ШО1С than опе bottJe, еасЬ containing sample а!
а different dilution, should Ье analyzed" ТЬе goal i8 that а! least опе ofthe bottles rcsults in а DO
depletion ofat lcast 20 mg/I" with а! least 1 ,О mglL retained а! the end ог 5 days,

(Ь)
After tho!Oughly mixing the buJk sample, иве а wide-tip pipette (i,e",
1/32 to 1/8 inch inside diametCI) 10 transfe! the desired volume of sample 10 individual BOD
bottles, Illе вате pipette сап Ье used 10 tпшsfСI' аН samples if'bottles ате ве! up staIting with IЬе
sample expected to {евш! in the 10west BODs, ploceeding eventually to that expected 10 Ьауе the
highest BOD ав the last ватр1е, Using а diffeIent pipette, transfer into those sample bott1es
needing it, ап атоun! о! seed mate1iaJ sufficient 10 рюdисе а DO dep!etion of'06 - 1. 0 mglL
during (Ье 5-dэу ineubation (ав determined Ьу previous tests ofthe вате 01 similar seed)..

(е) FilI еасЬ BOD bottle with dilution \vater to а level where insertion of
the glass stopper displaees аН air, leaving попе trapped in (Ье neck ofthe bottlc Fi11 the врасе
аюund те bott1e stopper with distШеd water 10 form а water веа!. If tIsing ап air ineubator, рlасе
а plastic сар оуе! the stoppeI' 10 prevenl evaporation of (Ье water вса] while the ватр!е i8 being
incubated.

(2) Grаdпаtеd CylindeI Method

(а) Seed (Ье entile container of' dilution water ав necessa1Y to rcsu1t in а 5-
day DO depletion in а BOD bottle о! О 6 - 10 mg/L. Carefully siphon dilution wate1 into а cleaп
graduated cylindeI, usnaJIy either of' l-L от 2-L capacity depending on how many dilutions will Ье
lUn [О! еасЬ ватрlе. Fill (Ье g!'aduated cylinde1 approximalely ha!ffu1l, being cazeful по! 10 пар
aiI in the water (i.c., do not f01m ЬиЬЫев) Add the desired volume о! homogenized sample, and
fill 10 Ihe desired level with dilution \Vate1 Mix carefully with а plunger-type mixing юd, again
being cmeful по! to I!'аР ai1,

(ь) FilI еасЬ BOD bottle with dilution waler containing seed and ватр!е
(i..e, fiom the graduated cylindet) 10 а leve! where insertion of те glass stopper wШ displaee аН
air, !eaving попе trapped in the neck of the bottlc. FШ the врасе around the bottle stopper with
distilled watcr (о f'orm а water seaJ.. If using ап а11 incubator, place а plastic сар oveI' те stopper
to prevent evapO!ation of (Ье water веа! \vhile the saтp!e 18 being incubated.

е. DеtеIщiпаtiоп ofInitial DO. Mea8ule initial DO using eitheI (Ье Winkler titration 01
DO рюЬе technique. If а DO mеtеI/рюЬе is used to mеasше DO, it should Ье ea\ibrated
immediately pIior 10 anaJysis of 8anlples using the caJibIation technique гесоmmспdеd Ьу the
mапufасtшсг ofthe mеtеr/рюЬе. Additionally, the DO metel/probe should Ье cheeked
pCliodicaJly Ьу first taking а DO readil1g of dilutiol1 wate1 in а BOD bottle usil1g the probe, and
thel1 doing а Wink!el titratiol1 оп the saтc bottle.. If the two DO Ieadings differ Ьу mще (Ьап 0.2
mglL, the саиве ofthe difference should Ье sought and eliminaled

19
 (1) Winkler' Method. If the Winkler (iodometric) method i8 used, two bottJe8
тив! Ье Бе! ир for еасЬ dilution, Ыank, standard, and seed control, опе of which i8 dedicated solely
to detelmination of the initial ОО, (Ье other being incubated Initial ОО of' the incubated bottle i8
аБвитед (о Ье identical (о ОО ofthe titrated bo!tle,

(2) DO Probe Method. If IЬе DO probe method 18 used, а! least опе bottJe тив!
Ье ве! ир ре! dilution (additionaJ bottles а! the вате dHution would Ьс Icplicates which юе
norma11 у used only 10 check within-batch pIecision IЬе ОО РIоЬе \уiII displace а relativel у smаП
атоип! ofwater which сап Ье replaced with di!ution water without introdncing а significant еrЮI
in (Ье рroсеББ, 11 Alternatively, inscrts Эlс available which сап Ье used 10 rеtшп displaced water 10
the bottle, lnitia! DO should Ьс read starting with the Ыаnk, (Ьеп the glucose!glutarnic acid
standап:l, effluent (in а wastewateI treatment plant lab), seed eontrol, and fina1ly progressing
thlOtlgh othcr samplcs, starting with that expected 10 Ьауе (Ье lowest BOD s tlnough the highest,
IЬе DO рroЬе shou!d Ье rinsed after еасЬ reading, If'this рroсеdше i8 folIowed faitl1fully, thcte i8
по need (о fill two bottles ре! dilution, one for measuring initia1 DO and (Ъе othe! for incubation,

f, Incubation. AfteI' filling BOD bott!es as described аЬоуе, stoppe.! а1l

bottJes tightly
Apply а wate!' Беal Ьу adding sошее wale! (e,g, distilled watcr) arollnd the stopper' ifnecessary,
place а plastic сар (от other' device 10 prcvent evaporation оНЪе watcl' 8eal) оп еасЬ Ьоttlе, and
incubate сасЬ bottJe а! 20 ± I о С, in the dark, fOI' 5 days, ± 2 hошs, АН Ьоп!еБ (blank, seed
control, glucose/glutamic acid standard, епviюшnспtа! Батр!ев, performanee evaluation sarnples,
ifany) тиБ! Ье incubated togetheI ав а batc1l

g Dеtеrшiпаtiоп 01 Final DO. AftCI 5 days, Iead (Ьс final DO as in paragгaph 7с аЬоуе

8, CalculationsJData Recor'ding. Since BOD i8 defined ав (Ье miШgrllms of dissolved oxygen

сопsшnеd Ьу bacteria per' ]itel of sample оуе! а 5-day incubation, (Ье BOD s of' ап ulldiluted
sampJe еап Ьс dete!mined merely Ьу Ieading the initia! DO in mglL, (DOo ) and subtlllcting thc
final DO (D05) , ТЪив, if' ап undiluted sarnple ЬаБ а DOo of80 mg/L, and а DO 5 of 3 О mgJL, the
BOD ofthe sarnple is 8 О - 3.,0 = 50 mglL, Мов! sarnples will require di1ulion, howevcr (Бсе
т аЫе 3), which wil1 require 11Iа! а fi!CtOI' Ьс applied 10 compensatc for the fact that the incubated
sarnple i8 not рше sarnple, ln paгagraph 5 of Standard Methods 521 ОБ, (Ыв factor is given the
designation "Р" and is cqua! to the уо!иmе of (Ье sample divided Ьу (Ье vоlпmе of the BOD bottle
01 graduated cy1inder containing the sample, 1his factor, "Р," is then divided into the DO

j 1 Some ana1ysts prefer using two ЬоШе. ре, dilution еуen when а probe is used 10 mеаsш'е DO lЬе зrgпmеПI fer

using twe bott!es is (Ьаl insertion of ,Ье ОО рroЬе dislocates а 8та!! amount 01 sample fiom (Ье bott!e used 10 measure
initial ОО, anд (Ьа! replacing Ihal displaced volurne ,vith di!ution water fшthег di!utes IЬе ,атрlе, introducing епщ /1
,ЬоиЫ Ье r'ealized, however, that (Ье amonnt 01 water displaced is only approximately 1%
of the tota! volume, апд that (Ье maximum епоr IЬаl could Ье introduced is thеrеfше only ! % Such еПОr is minor
compared 10 olher potential sources 01 еrтor for the BODs t.sl. Another aтgument for using two bottles ре! di!ution is
to avoid contamination 01 incubated ЬоШе. Ьу using а probe IЬаl Ьа. just Ьееп пsед оп а bottle potentiaJly containing
BOD 1f measurements are taken {гот the leas! cOnlaminated bottle (eg, Ыаnk) thтough (Ье то,! contaminated (е ,g ,
raw eft1uCII!), and the probe is rinsed propelly bef:\veen bottles, tms potcntia! СНО! is also minima! IЬе two potentia]
error, (slight di!ution апд possibIe contamination) tend 10 сanсеl еа<:Ь other, and the risk to да!а quaJity is БтаН
compared to the gain in cfficicncy

20
dep!elion 10 delcrmine the sample BOD s Some ana]ysts find it easier (о use the recipтoca! of"P"
(! е , the container vоlпmе divided Ьу the samp!e volume) апд multiply the DO depletion Ьу this
facto!, Both рюсеdurеs соте пр with the same ппmЬег, Ьпt for the sake of simplicity апд use of
mos! common]y used term, thiз documen! use5 the latter I'atio, wmch i5 identified in 8а below аs
the Dilution J;'actor, DF (! е, DF = l/P).,

а, Dilution Factor IЬе dilution factO!, DF, i5 the ratio ofthe fina! volume (е g, for the
bottle method, the volume оНЬе BOD bottle, usually 300 mL; fO! the graduated cylinder method,
the уо!ите of' Ihe cy!inder, usualIy 1,000 mL) 10 the volume of' sample therein, DF fO! the bott1e
method = Volume ofDilllted Мiхtше/Vоlumе of Samp!e in Мiхtшс, 01,

FOI а sample ехрее1ед 10 have а higher BODs \vhere а small amount of'sample i5 dilllted, the DF
would Ье higher, For а 300-mL miХlше containing only 6,0 mL of sample, the DF i5 " ,

DF = 300 mИ60 mL = 50
Reference 10 1 аЫе 3 confirms that the two dilution factors аЬоуе Ьауе Ьееп сопесtlу ca1culated,
Ifinstead оГа ВОО bottle having а volume of300 mL, а 1,000 mL (J-liter) grаdпаtеd cylinder is
used to prepare the sample, the DF 15 ca1culated 1П the same way, If 100 mL о! sample 15 diluted
in а 1,000-mL graduated cylindeI, the DF 1s.

DF=1000mLlI00mL 10
Ь. BODs ·, Not Seeded" When samples are not seeded, the BOD ofa sample 18 calculated
simply ав the dilution facto! (DF) times the DO depletion during the 5-дау incubation (DO[ -
DOs).

21
 А question often asked is ifthe fina1 DO rcadillg пееш 10 Ье "Ыаnk сопесtеd" и.е., should the
Ыаnk contJibution Ье subtracted fiom the differcnce between DO] aиd Dos) Ifы
nkss ale usually
zею as they should Ье, with аи occasional 0.1 mg!L depIetion, and а very infiequent 0..2 mglL,
random ellOI is causing the positive Ыаnk, aиd the саиБе of that random еПОI тау exist only in the
Ыаnk bottle Results should по! Ье сопесtеd to compensate fO! this landom ено!. НЫankв
usually !ип 0.1 OI 02 mglL, о! even higher, Iathel than Ыаnk сопесtiпg, the analyst should find
the саиве(в) ofthe high blanks and eliminate it (them) So rеgап:llеss ofthe cause ofhigh blanks,
it i8 not а good scientific аррюасh 10 cOlIect fOI Ыankв . AI80, StandaJd Methods 521 ОБ, in Ihe
last suЬрашgrарh ofparagraph 5 ваУБ по! 10 Ыank COIIect.

с. BOD - Seeded. If Батр!ев are seeded, the tota! DO depletion oveI the 5-дау incubation
i8 caused Ьу both the ватрlе and the seed. 1Ье equation f01 calculating BOD oft11e sample
ЬесотеБ а little тOIе complicated Ьесапве the DO depletion caused Ьу the seed must Ье
snbtracted fi:om the total DO depletion. Ihe seed contIol bottle is used to detclmine the
contribution 10 DO dcpletion made bythe seed Standard Methods uses "Bj" to denote DO оГfuе
seed control bott1e оп Day-l, and "В 2 , " the DO of the seed сопtю! bottle оп Day-5 То determine
the lota1 depletion eaused Ьу the seed in fue sample bottles, опе тиБI а180 consideI the ratio of
volume of seed in Ihc sample bottle, to the volume of seed in the seed contr·o! bottle. ТтБ Гасtor
is given fue desigrration 'Т' Ьу Standard Methods БисЬ that f01 the bottle method

and fo! the gr·aduated cylinder· method

22
То detelmine tl1e BOD s fOI' seeded samples, the contribution ofthe seed to tota1 DO deplction in
thc illcubated sample mllst Ьс taken into consideratioll. ТЫ" is dOllc Ьу mllltiplying (Ьс difference
between the initia1 DO ofthe seed сопtюl bottle (В] in Equation #5 below) and the fina! DO (В 2 )
Ьу the seed ratio factOI "f" BODs foг seeded samples is then calculated ав

d Benchsheet. Benchsheets used to r-ecold obseIvations made dШ11lg the BOD test
should inelude врасе fOI' recOIding the following ав а minimum

(1) Date and time the sample was taken

(2) ldentification of the sampleI and ana!yst

(3) ldentificatioll of'the ватрlе (e . g., raw influellt, oxidation ditch, fina1 effluent)

(4) Sample рН

(5) Sample tempelature when initia! DO is read

(6) Bott1e NumbeIs

(7) Vo!ume of seed in еасЬ BOD bottle о! in the graduated cyJinder

(8) Volume of ваmр!с in еасЬ BOD bottle 01 in (Ье graduated cylinder

(9) lnitial DO of еасЬ bottle

(10) FinaI DO ofeach bottle

23
 (11) Space fопеsults of calculations, висЬ а", DO depletion (dюр), dеtеш1iпаtiоп
оНЬе seed сопеctiоп factOI "f," dilu!ion factO!, and BOD5

(12) Ап indicato! о! whether results ауе BOD, о! CBOD$

(13) Date/time the initial DO and final DO were геад

(14) А "'расе to indicate (Ье ЬепсЬвЬее! Ьа", Ьееп reviewed with the date and
initials о! the reviewer

(15) Comments

А sample ЬепсЬвЬее! i8 shown а! Appendix С foт the bottle method, and Appendix D fOT the
graduated cylinder method. Note that these benchsheets indicate only опе bottle i5 incubated [оу
the blank, веед сопtюl, anд вtзпдагд. 1Ыз тау уату aeeor'ding 10 the nеедв of (Ье lab, experience
ofthe ana1yst, and desi!es оНЬе supelvisoт 11 also implies that as тanу ав three should Ье шп [OI
thc influent since BODs strength is variable fO! thз! sample lЬе питЬе! of bottles incubated fOI
effluent sзтрlев 15 а matter о[ choice, based оп experience of the aпalyst and variability of
samples with Iegard to BOD, slгепgth. lndividua1 wastewater discharge permits от other
regulato!Y requirements тау dictate the питЬе! ofbottles thз! тив! Ье incubated [OI each
samp1ing site .

9. Quзlity Аssшзпсе/Quality Contr 01

а.Minimnm сritеr'iз. 10 Ье а уаliд te8t [OI а given BOD bottle, incubation must Iestllt in
а DO depletion of' а! lеа8! 2 О mg/L [2, with at least 1.0 mg/C DO remaining at the епд of the
incubation period. Thc орНmит depJetion i8 halfthe available DO, от 3-4 mg!L [Ш' most
situations. Foт а sClies of di1utions, !'Csults [о! а11 bottles meeting these c!ite!ia ale ave!aged {о
соте пр with the finaJ BODs to Ье IepOIted13 I 1 I[none ofthe bottJe8 [OI а given sample тее! the
criteIia, the lab might report (eg, оп the Di8charge Monitoling RepOIt, DMR) а уаluе [or the
bottle that соте8 closest 10 meeting the critcria and add а note to the leport indicating that (Ье
уаluе 15 ап estimate and \vhy. lfthe test is being шn in а \vastewater trcatment pJant, 11 might Ье

12 Some analys!s in!erpl'e! this requirement in Standar'd Methods а, meaning that the 00 depletion caused Ьу the
sample plus the seed contribution must Ьс а! least 2.0 mg!L ТЬе author interprets the requir'ement 10 !nеan that the
sample alone must eontribute а depletion ofat least 2 О mg/L Ifthe author's interp!etation " по! correct, the method-
imposed minimum de!ection limit (МDL) fo! ВО05 wouId Ье 1 О mg/L (ап undiIuted bottle i.e , DF 1 where
the sample contributed а 10 mglL dep1etion, aHd (Ье sced contributed I О mg!L, the maximum suggested for lЬе seed)
Ви! the MOL is nо! 1 .0 mglL .11 is 2.0 mglL . meaning that the 2 О mglL minimum depletioH applies to the sample
\vithout consideration for the dep!etion caused Ьу the "еед

!3 Аn exception 10 the rule (Ьа! уои aveJ1!ge аН bottles meeting the depletioniretention cr1teria is when the sample
exhibits toxiei1y (ie , something in the sampIe inhibits activi1y 01 the bacteria AIthough Standaf'd Methods 52! ОВ
does по! speeify what is теап! Ьу "exhibits toxici1y," оnе indication is that the 00 depletion per milliliter of sample '"
greateI [от lЬе more dHute bottles. Jn sucb ca80S, thшс is justification [от reporting (Ье BOD, value for the most
dHute sample that те! the depletioniretention cnteria For example, [о! the data in Т аЫе 7 below, where tbe seed \vas
,n
contributing а depletion ofO 6 mgIL еасЬ bottle, the data justilies !'cporting 222 mg!l, BOD foт the most dilute
bottle

24
 possible to I'esample and stil1 тее! monitoring x-equkements, If'certain samples пеуеI эеет to
deplete а! least 2 О mg!L DO, such samples тив! Ье ruп with less dilution, If'they are already
being шп а! fulJ strength (е g , 298 miJ1Шtеrs of sample and 2 mL of seed) and stiП fai! 10 deplete
а! least 2 О mg!L DO, (Ьеу should Ье Iep01ted as "<2,0 mg!L BOD s" (i.,e" less than 2,,0 mg/L, the
method-imposed minimum detection limit) Ifcertain samples пеуе! эеет (о leave а! !еаэ! 1,0
mg!L remaining ШС! the incubation, they need to Ье fшthеr diluted If песеБва!у, the enti!e
sample сап Ье diluted аэ а pI'eliminary step beforc analysis,

т аЫе 7 - Evidence o!PossibIe Т oxicily Inte!terence

Bon]e Samp]e Seed !nitia] Final Oeple- Oilution ВОО,

уо], Уо!. 00 ОО tion Factor

2 89 3.9 50 150 750

2 5 89 4б 43 БОа 222
Fina]
ЕШиеп! 3 10 8.9 43 46 30,0 120

4 20 89 39 50 15 О 66,0

Ь, Blanks. А! least опе unseeded dilution water Ыаnk should Ьс 1Щl with еасЬ batch of
sarnples" 1 he ршроsе of the Ыаnk is 10 indicate аЬвепсе of: (1) contamination 01' suрегsаtшаtiоп
of dilution water with DO; (2) tетреlаtше рюЫстs; (3) atmospheric ргеssше РIOЫетэ, and; (4)
and othcr sошсеs of' епOI that тау по! Ье r'elated to (Ье samples themse!ves Тhe lab shou!d
attempt 10 keep аН Ыank;; below О 1 mg!L, Ьи! action need по! Ье taken unless they exceed О 2
mg!L. In the fina! BOD s calculation, rcsиlts shoиld NOT Ье сопесtеd for the Ыank value а;;
alreadyexplained. Ifan isolated Ыаnk exceeds а depletion of02 mg!L DO, the report to the data
user' (e.g, оп thc Dischargc Monitoring RepOIt, OI DMR, 10 Ecology in some савев) should note
thc facl that the bIank exceeded method allowances If Ыаnk;; typically run higher than 02 mg!L,
the following potential саиве;; should Ье investigated, preferabIy опе а! а time 80 а;; to isolate (Ье
actual рroЫет aпd eliminate it

(1) Сопtапriпаtiоп. Contaminated labware maycontribute to BOD\ in the blank

Try thOI'oughly washing and rinsing аlllаЬwащ possibly using different techniques, and lU!U1ing а
Ыаnk оп several bottles in а single batch. Н' опе technique веет;; to give better resu!ts (i.e , lowe!
Ыankв), stick with it.

(2) SupeI'satulation. Ifthe initial DO is read in BOD bott!es when oxygen is

supcrsaturated, а positive Ыаnk wil1 result, and а1l висЬ bottles incubated will have I'esults tbat юе
biased high. Лlе Ьев! way 10 avoid sиреrsаturзtiоп is иве dilution water which is а! 20 ± 10 С, and
to avoid aerating di1ution wate!', sample, 01 seed immediately pri01 to taking the initial DO
reading. Dilution watcr сап Ье aerated the day before setting ир the BOD bottles (or еатНет), and
stOIed in а container protected Ьу having а cotton plug in, 01 а loose сар оп its opening to allow
евсарс of ennapped air, от cnny о! ail' if the water was not all'eady saturated. When
nutIientslbuffers ате added to the dilution wate! оп the day of the test (i . e, \уЬеп BODs and not

25
 CBOD5 is being IUn), and when ватр!ев and dilution water юе added to BOD bottles, care must
Ьеtaken to avoid f01ming air ЬиЬЫев.

(3) Temper'atuJe. rhe sаtшаtiоn level ofDO in \vateJ' depends оп atmospheric

pressure and temperaturc (ав weJl ав оп other factors such ав salinity which nоrmаПу ше not
encountercd Ьу mos! labs doing BOD" testing). The coldeI the water, the more dissolved oxygen it
сan contain at the sаtшаtiОll point. Effluent samples (01 other ватрlев expected 10 Ьауе а low
BOD s, alld thus а requilement fOl little dilution) тив! Ье wшmеd or cooled (о within ±1 о of 20 0 С
when initial DO i8 r'ead. F01 samples expected to Ьауе highel BOD, suengths and thus lequiring
dilution, it i8 тorе important that (Ье d:ilution water Ье at 20 ± 1ос since the diluted samp1e i8
primari!y dilution \vater. А good way (о аБ8ШС that temperature i8 to keep aerated Бошее water [01
BOD", 01 aerated dilution wate! fo! CBOD 5, at least оvешight in (Ье BOD incubator. If Боmе
bIanks for BOD5 ауе positive, Ьи! others Ш'е negativc (i.c., as if DO Ьад incr'eased during
incubation), tempcratIrre might Ье the probIem Another РlОЫет that could саиве negative bIanks
would Ье photosynthesis dшiпg incubation, Exclusion оПight dшing incubation precludes
photosynthes18

(4) Pressure. If'blanks ше ровШуе [01 воте batches, and negative for othe1S, the
problcm might Ье lemperatIrl с (ав ind:icated аЬоуе), or 1t might Ье а рroЫет with compcnsating
[ог atmosphc!ic ргсввше changes betwecn the initial DO and final DO I'cadings. оо meter8 are
often caliblated Ьу compensating only for' the elevation of the lab, and по! the actIral atmosphClic
pressuтe. А8 any Washingtonian kuows, atmospheric РГ'еввше сan cllange dramatically during а
five day period (еуеп though elevat10n ofthe !аЬ remains cOn8tan!) Calibtat10n Ьавед оп
atmosphcIic ртеввшс as measuTed with а baIOmcter 18 РГ'сfепеd 10 basing calibIation оп
elevations. Major manufactIrl'ers of DO meters веll top-end meters that Ьауе built-in Ьшоmеtегs
and automatically сопесt for atmosphcric plessurc (ав they до temperatIrIe)

(5) SOUI'ce (DistillеdIDеiопizеd) \Vater. МOIе often than по!, labs having а
problem with high Ыankв check for the pIOblerns 1n subparagraphs b(l) through Ь(4) аЬоуе, and
find попе i8 causing the рroЫет. ТЬе watcr ивед 10 ргерше dilution water i8 often the cu!prit.
\Vater distil1ed in а lab often contIrin8 ammonia, amines, аnд possibly othcl' materia!s that
contribute 10 BODs (i.e., саиве high blank8) OtheI contaminants such ав еоррег might inhibit
bactcria! activity Although such contaminated water would по! Ье detected Ьу Ыank resuJts, they
might саиве а negative Ыав in the glucose/g1utamic асИ determination and in envi!Onmental
samples. Even if distШеd water i8 рu! through а deionization (Ш) column, Ihe waler тау still
contain organic materia1leached fiom the column, In add:ition to keeping the distillation apparatus
сlеan anд leplacing DI columns ficquently, it тау Ье песеВБШУ 10 add au activated charcoal
column ав the fina! БсшЬЬег in (Ье water sошсе. An alternative, anд а тисЬ eheaper option when
trying to locate the Бошее ofhigh ЫankБ, i8 to (гу using distil1ed wate! ршсЬаБед fiom а 10ca1
store. If one b!and stillresults in high blanks, t!yanother Steam distilled water (Бее paragraph
7a(I)) secms to work ЬеБ! (when i1 сan Ье found) . If it сan Ье shown that water i8 the 80шсе of (Ье
high blanks, then it might Ье worthwhiJe in the long шп to upgrade the
distilledldeionized!scrubbed \vateI system 1П the lab .

26
 (6) MeaSUI'ement of' DO. If either the initial, final, or both DO measurements ах'е
not made ргорехlу, excessive bIanks could IсвиН When possibIe, the апа1ув! who теasшеs initia!
DO ShOllld а180 теазшс final DO.

с Check Standard. ТЬе primary ршроsе of analyzing а BOD s check standald, whethel it
Ье glucose/gllltaтic асЫ, potassillffi hydгogen phthalate (кнр), ог воте otheI material, i8 to
detellnine ifthe seed used Ьу the lab i5 sufficiently potent fOl the BOD 5 test,. If the теап (average)
value fol1o\ving seveIa! analyses ofthe glllcose/glutamic асЫ 8tandard 18 significantly lowelthan
198 mgjL (the gnidance given in Stаndшd Methods). а stгongcr secd i8 needed . If'the теап i8
considerabIy higlrcr than 198 mg/L, а weakcr secd i8 indicated The standard deviation f01 results
ofthe repeated analysis ofthe glucose/glutamic асЫ standard indicates thc total prccision о!
analysis . Standard Methods indicates the standard dcviation should Ье 30.5 mgjL 01 !owcr, but, ав
previous1y mentioned, а good single lаЬ should Ье аЫе (о шп the tes! consistently such that the
standard deviation is in thc tecns (with less than 1О mgjL indicating extraordinary precision)
Мапу (Ыпgз сап саuзе imprccision such ав variable seed, temperature and рrеssше рroЫетв,
contamination, DO measurement рroЫетв, inattention о! ana]ysts, and count1ess other вощсев of
vаriаЬШtу. It i8 uвиаllу much easier to di8cover and eliminate а рroЫет causing а ]ow (о! high)
теап value f01 the glucoseiglutaтic асИ test than it i8 (о do thc вате f01 а higlr standard deviation
indicating excessive implecision .

d Duplicates" The ршроsе ofrU!UIing occasiona! duplicates is to determine ifwithin-

batch precision i8 а ртоЫет.. lmprecision lП BODs testing (01 in anу measurement system) 18
caused Ьу within-batch imprecision, and behveen-batch imprecision See Appendix Е for а
тО1е thоюuglr discussion оп UБС ofstatistics il1 BODs testing

10 Method Performance Summary (Bias, Precision, Detection Limit, Working Limits)

а, Bias. Аgood lаЬ should Ье аЫе to achieve а тсan value in the vicinity of 198 mgjL f01
1epeated anа1узев of the 150 mgjL glucose plus 150 mgjL glutamic acid standard

Ь. PI'ecision (01 Imprecision)

(1) TotalImprecision. А good lab should Ье аЫе to achieve а standard deviation

ofless than 20 mgjL fш Iepeated an81yses ofthe glucose/g!utamic acid standaId (remembeI that
the 30.5 mg/L standard deviation cited in SM 5210В is f01 severallabs doing the GIGA te8t) An
MS Ехсе! control charting filе i8 ауаilаЫе to accledited lаЬБ fiom Ecology's Lab Accreditat:ion
Section that calculates tlre total standard deviation of repeated rcsults f01 analysis оНЪе GGA
standard IЬе control charting pIOgraт also tracks perf01mance оп subsequent GGA analyses.

(2) Within-batch Imprecision. Весаuзе within-batch imprecision i8 expected to

Ье the minol contribut01 (о tot81 imprecision, опе miglrt expect the within-batch standaId deviation
f01 several duplicatc pairs to Ье less thaп the standard deviation ca1culated f01 bctween-batch
imprecision. The вате Ехсеl plOgraт mentioned above c81culates (Ье standaId deviation of the
diffeIences f01 duplicate measurements of similar sampJes and а]80 tшсks performance оп

27
 subsequenl duplicates. It is important (Ьа! the samples СЬОБеп 10 duplicate vary little in
concentIation from batch (о batch Се ,g., fina! еШuеп! thal geneIally runs in lЬе vicinity оГ 1О mg/L

BODs).. Dividing (Ье stапdюd deviation о! lЬе difference (\уЫсЬ is ca!culated Ьу the above-
mentioned Ехсе! РlOgтаm) bythe squa!e юоt оп gives an estimate ofthe within-bateh standard
devialion О·е., Swi ~Sdinl'l/2)

(3) Between-batcb Imprecision . ВесаиБе between-batch imprecision is expected

(о Ье (Ьеmajo! connibutoт (о total imprecision, опе might ехрес! (Ье between-batch standaI d
deviation estimate based оп calculation о! variance (Бес Appendix Е, Statistics) 10 Ьс somcwhat
тоте than (Ье standard deviation fO! within-batch plccision.

с Detection Limit. Весаuзе о! (Ье method I'equirement that а test is valid only if2.0
mglL о! mо!е DO is depleted, and assuтing that БиеЬ а depletion was achieved fo! ап undiluted
sample (i . e . , the DF = 1 О), the theo!ctical detection limit i8 2 О mg/L DO (i .е, 2 . 0 mglL dcpletion
times 10 = 20 mglL).

d 'Vorking Limits

О) Minimum. 1Ьс minimuт working limit i8 2 mglL, (Ье MDL addrcssed above

(2) МШmпт. Considcring that waste samples might contain significant

suspended solid8, {Ье minimum volume о! а waste sample that сап Ье mеаsшеd with апу dcgтce о!
accuracymight Ье 1. 0 mL . ТЬс maximum BOD5 WOIkillg x·ange [о! such а sample (Ьа! 18 actually
incubated might Ье 2400 mg/L, assuming that initial DO was 9.0 mglL and {Ье final DO 1. О mglL,
{Ье minimum foт а vaJid tes!. DO depletion о! 80 mglL times the dilution factor of 300 calculates
to Ье 2400 mglL BOD5 HoweveI, опе тU8! also consider that the incubated sample mау have
Ьееn prepaIed Ьу diluting the actua! enviIOnmental sample . ]f that dilution mctor were 1: 100, fOI
ехamрlе, the maximum BOD5 would 240,000 mg/L. In practice, the tes! itsclf does not limit the
maximuт mеаsшаblе BOD,

] 1, Refer'ences

а. Standard Methods 1т the Examination о} Wate! and Wastewatel, 19th Edition, 1995,
АРНА-А WWA- \VEF, page 5-2 ff

Ь. Маnuаl o/Methods {о! ChemicalAnalysis о} Water and Wastes, EPA-БОО 4-79-020,

1983, Method 405.1.

с Basic Laboratory Procedures/or Wastewater Examination, 4th Edition, 2002, \Vatcr

EllyilOnment Federation

d. Operation о} Wastewater Treatment Plants, Vol П, 3п1 Edition, ЕРА, 1991 .

28
 е Cheтiltry [оу Sanitary Engineer~,. 1967, McGIa1.v НШ, page 394 ff (Ihis reference has
Ьееп
revised in а later' edition titled Cheтistfy (ш Environтental Engmeering, 4th Edition, 1994,
McGraw НШ Ihe 4th Edition was по! ауаНаЫе to the author ofthis doeument,)

f Procedural Маnиаl {оу the Environтental LаЬшаtогу Accredllation Prograт, \Vashington

DepaJ1ment ofEcology, Document #02-03-055, November 2002,

Appendices:

А - PI'epalation 01' Solutions

В - Sources 01' Reagent Gr'ade Chemicals
С - Sample Benchsheet (Воttlе Method)
D - Sample ВеПСЬБЬее! (Gr'aduated Cylinder Method)
Е - Statistical Теrщs Used in BOD Testing
F - TroubIeshooting (Ье BOD Test
G BODlCBOD Checklist

29
lhis page intentionally left blank
 AppendixA

Pr'eparation of Solutions

]. АсЫ Solution (fO! neut!alizing causlic samples).. WШ1е stiпiпg, slowlyadd 28 mL of

conccntJ:ated sulfuric acid (H2 S04) 10 distilled water. Dilute 10 1,000 mL Сап Ье stOIed
indefinitcly (CAUТION: Do по! add watel to асЫ!)

2 Ammonium Chloride Solution (nuuient fol' nitrifying and other bacteria) Dissolve 1.15
grams of ammonium chlor'ide (NH4 CI) in approximately 500 mL of distilled watcl', adjust рН to
7.2 with NaOH solution, and dilute to 1000 mL. Сап Ье stored indefinitely in а wel1-sealed
container

3 Calcium Chloride Solution. Dissolve 27.5 grams of' anhydl'Ous calcium chloride (СаСЪ) in
clistilled watc! aпd dПutс 10 1000 mL. Сап Ьс storcd indcfinitcly

3. Base (Alkaline) Solution (fO! ncutralizing acidic samples). Dissolve 40 grams ofsodium
hydr'oxide (NaOH) in clisti1lcd watcr Di1ute to 1000 mL. Сап Ье stored indefinitely.

4 . Fer'ric Chloride Solution. Dis80lve 0.25 gr'ams offerтic chloride (hydrated) (FеС1з '6Н20)
in distШеd waler and dilute to 1000 шL, Сап Ье stOled indefinitely.

5 Glucose/Glutamic Acid (standard solution for the BOD tcst), Dissolve 150 mg of dried
glucose (also ca11cd dехtюsс) and 150 mg of dricd glutamic acid in 1000 mL of distillcd watcI .
PIcpalc ficsh daHy as nceded. Сап Ьс ршсhasеd as а preparcd воlиНоп fiom Hach (Cat. No .
14865-10), NOlth CcnlIa! Labs (Cat No. B-12D), and perhaps otheIs. If'Hach's solution is used,
theiI inst!Uctions саП for clilution 10 а solution that is 300 mglL glucosc aпd 300 mglL glutamic
acid, IЪis solution should Ьс furthcr diluted to 150 mglL of each Ьу adding ап equal volume of
distillcd watcI to Шс solution pI'cparcd using Ше Hach inslIuclions. 1

б.Magnesium SUШltе Solution. Dissolvc 22.5 glams ofmagnesium sulfate (hydrated)

(MgS04·7H20) in distillcd water aпd dilutc to 1000 mL. Сап Ьс stOIcd indcfinitcly.

7 Phosphate Buffer' Solution. Dissolvc 8.5 grams ofpotassium dihydr'ogen phosphate

(КН2РО4),
21.75 grams ofdibasic potassium phosphate (К2 НРО4), 33.4 grams ofdibasic

1 lnstructions accompanying (Ье НасЬ Сотрапу standard sау 'Ье 300 mglL solution сап Ье зnаlуzеd without furthcr
diIution and (Ьа! 'Ье expected result fOT' repeated аnаlувеБ should Ье double 'Ье 198 ± 30.5 mgfL ав suggesled in
Stаndш-d Methods 5210 В, о! 396 ± 61 О mglL When using НасЬ'в slandard, it is best 10 dПutе the sample 10 half (Ье
str<mgth specified in the instructions (е g , Ьу withdrnwing ha!f (Ье vо]шnе пот the НасЬ via!, or Ьу di!uting " \vi!h
,N
twice ав тисЬ wa!er). 1his ,viJl reBul! а so!ution that;s 150 rngIl of еасЬ ingr\Зdiеnt, tbe concentration suggested ,n
Stаndшd Method, НасЬ plans 10 provide а 150тgll solution in the future

A-l
sodium phosph:tte (hydr':tted) (Na2HP04'H20), and 1.7 gr:tms of :tmmonium chloride (NI-4Cl)
in appIOximateJy 500 mI_ of'disti11ed wateI and diJute to 1000 шL. Check рН .... should Ье 7 2,
Сan Ье stored in sampJe re[rigerator а! appIOximately 4 О С. Discard uроп anу sign ofbiological
growth,

8 Pot:tssium Hydrogen Phth:tl:tte (аltешаtivе standard [or BOD tes!).. Dry а thin Jayer of'
potassium hydr'ogen phthalate ClYSta1S in а beake! [01 опе Ьош а! 103 - 1100 С. CooJing in а
desiccatoI [01 30 minutes Weigh out 300 miШgr'аms ofthe dJied КНР and dissolve in 1000 mL
оУ distilled water.. ТЫв solution i8 biodegradabIe Ьu! should Ье stable fOl appIOximately six
months if kept well stoppeIed and lefiigerated at 40 С.

9. Potassium Iodide (fOI titIation (о detelmine уо!uте of sodium sulfite needed f01
dechIO!ination). Disso!ve 10,,0 grams ofrcagent grade potassium iodide (Кl) in 100 mL of
distiJled water . Сап Ье stored indefinitely.

10. Sodium Su1fite (f01 dech101ination) . Dissolve 1.575 grams 01' Nа,SОз in 1000 mL о!
di8til1ed wateI'. Sodium sulfite solution i8 по! stabIe and mиs! Ье prepaIed даПу ав needed.

11 . Star'ch Solution (fOl iodine titration in dech1OIination РIOсеdше) Make 10 grams of soluble
starch into а smooth paste with WaIШ water РОШ' into approximately 1000 mL оfЪоШпg water
Imз solution does по! stoI'e indefinite1y and shouJd Ье рп;раI1::d ftesh when detelioration is fiIst
noticed.,

А-2
 AppelldixB

Sources of' Reagent Grade Chemicals 1

VWR' Fischer' Sigma4 Сиrnп Matheson' NCL 6 Hach'

Sodium Sullite (Na,SO,) П3922·] 5447·500 S 8018 832-]5] 5-67 195-0

Potassium lodide VW5225-1 Р410-500 Р4286 831-718 Р-73 167-01

Glucose (Dcxtrose) ЕМ-ОХО 156-1 016-50007528 МDХОI5б-1 а-50 11251·53

Glutamic Acld ПМ75б-7 AI25-IOO 06904 423-287 0-60 23409-23

Potassium Dihydr'ogen Л3246-1 Р284-500 РО662 М5]О8-1 Р-92' 170-01'

Phosphate (КН 2РО.)

Potassium Hydrogen ЕМ·РХI571- MK-6704-125 Р3792 М4876-2 Р-6] .3 ]5-34

Phthalate (КНР) МВ-2

Potassium Phosphare П3254-] Р288-500 Р 828] 83]-761 Р-9]' 7080-34'

Dibasic (К,НРО4 )

Seed Capsules

Bioseed 52468-360 022-354

Polyseed 66130-430 13-297-200 24712-00
BioSystems В-боа

Sоdiпm Phosphate, ЛЗ824-1 537,3-500 S 9390 423-323

Dibasic (hy'b'ated)
(Nз,НРО•• 7Н,О)

Аmmопiпm Chloride П0660-11 А649-500 А 5666 830-154 А-l t 105-0]'

Magnesium Sull'ate ЕМ-МХОО70-1 М63-500 М 1880 MMXOO7Q.-] M-IO' 6088-34'

(hydrated)
(МgSО 4 "Ш2 О)

Caleium CbIoride ЕМ-СХО156-1 CIO-500 С4901 MCXOI56-1 С_58 7114-34'

В-l
Fепic Chlor'ide EM-fХО21О-1 188-500 F 2877 MFX0210-1 F-l0' 1772-26'
(hydrated)
(FeCI, '6Н2 О)

2-Cbloro,·(t.icbloro- N-50 2579-24

metbyl) Pyridine (ТСМР), 2,2%

Glucose!Glutamic Acid Standard B-12D 14865-10

I Ву including them in (Ыв list, Ecology доев по! endorne о' discourage !Ье purchase of ch.micals йОт any vendor,
2 VWR Seientific Products, Р,О Вох 3551, Seattle, WA 98124,(800) 932-5000
, Fischer Scien!ific, Р О, Вох 58056, Santa Сlащ СА 95050, (800) 76()" 7000
4 Sigma Cbemica1 Сотрапу, Р О Вох 14508,51, Loui., МО 63178, (800) 325-3010
5 Cu!!in Matheson Scientific, 822 Soutb 3ЗЗrd Stre.!, Federal \Vay, WA 98003-6343, (800) 323-3987
6 North Central Laboratorie., Р О Вох 8, Birnamwood, WI 54414, (800) 648-7836
1 НасЬ Campany, Р О, Вох 389, Loveland, СО 80539, (800) 2274224
8 PiIlows containing аП the chemicals marked Ьу IЫ. note ате ауаПаЫе fium NCL (Н-14161) and ВасЬ (14861-98
УОТ 3 Jjters оГ dilution water; other sizes те ауаilаЫе).

В-2
Date!Тime Samples Taken: ВОО BENCHSHEET (ВОТТLЕ МЕТНОО)
Page 1
Samples Такеп Ву: _ _ _ ВОО (Indicate which) of pages
_ _ _ СВОО

А в с D Е F G н J к L м N р Q

~
1
~.

(")

1
(1)

?.....
'-'

DilulimJ water Blank DO DepIeUon (should ье ,:!.О.2 mgIL) ,Comments: Samples analyzed ьу:

CIнк:k о, seed Slrengtll (s11ou1d ье 0.6 -1.0 mgIL per mL of seed) ОзtelТiте initial 00:

mL seed ·Ше!Тiте final DO

J ..,. F far seed Control "
-- mg/L .,.

= mglL DO per mL seed ecked byldate:

=
•• Seed Ratio Factor f" VoI Seed(samp)Nol Seed(seed control)
Datemme Samples Taken: ВОО BENCHSHEET (ВОПLЕ МЕТНОО)
Jan 29, 1998 7:30 a.m. Page 1
Samples Taken Ву: Х ВОО (Indicate which) of ~ pages
1. М. Labber СВОО

А В С О Е F G Н J К L М N Р Q

t
х'
("')

ig

DilullOn WaIer Bllnk DO Depletian Ishould ье 50.2 mgI!.) Comments: Samples analyzed Ьу:
Trylng new seed • Clarifser #1 1. М. Labber
Chec:k of Soed Strength (should ье 0.6· 1.0 mglL per mL of нedl Rained а" night 1/28·29 Datemme initlal 00:
1/28198,9 am
J ... F for seed СoлIrоl •

~ 0.66
--
3.3 mglL .,.

mgI!. DO permLнed
- 5 mL нed DatelТlme final 00
2/3198 8:30 am
Checked by/dale:
lona Mercedes
• Soed Ratlo Factor =f = VoI Soed(sampjNoI Seed(нed conlrol)
[Daterfl';;~ Sample~ Taken: BOD BENCHSHEET (ВОТТLЕ METHOD) Page _ _ __
(Continuation Sheet) of pages
А в с D Е F G н J к L м N р Q

Sample

,f;

l.
('")
1-tj
;
('")
~
'-'"

Comments:
 Use ofBOD ВenсЬвЬее! (БоШе Method)

Following is an explanation ofthe varioиs columns оп Ше BOD Benchsheet оп Ше preceding

pages Those columns which асе self explanatOlY асе not inc!uded below

Columo A... Sample. Identification ofthe sample being analyzed. Note ther'e is only опе Нпе
provided fOI tbe Ыаnk, seed control, and standard, Тhe fonn сап Ье modified to do I'eplicates
(e,g., duplicates) for anу ofthese samples., Also, there асе three Hnes provided for an "effluent"
sample in I'ccognition of the variаЬШty of such samples. Anу sample fOI' which the 1аЬ does not
have а good estimate of the BOD should Ье run in more than one dilution

Со'пто В ... рН. РН ofthe sample (before dilution, ifit is а diluted sample),

Columo С ... Тетр Deg С. ТЬе temperatUl'e ofthe solution in the bott!e (should Ье 20 ± 1о С)

Columo E ••• mLs Sample. МШШtеrs of sample, eithe! ft'om the environment, ос the glucosel
glutamic acid зtandзrd, in Ше BOD bottle,

Column F ••• mLs Seed. МiШ1itеПI ofseed in the BOD bott1e

Columo J •••DO Drop G - Н. DO depletion over 5-day period. Initial DO minus final DO

Column K ... Seerl Ratio Factor (t). Ratio of зеerl in the затр1е to seed in the seed control
Equa1s the volume of' seed in Ше sample дмдед Ьу Ше vo1ume оЕ зеед in the зеед conu'ol bottle

Columo L .••Seed Correction (J_) х С. ТЬе DO depletion саизed Ьу Ше зеед WШСЬ тизt ье
subuacted from the overall DO depletion. Calculated Ьу multiplying the Column J value Сот ше
зеед control bottle Ьу Ше seed cOIrection factor, "Р' (the Column К уа/ие ) foI' the bott1e being
analyzed

Column M ••• Corr. DO Drop J -, L. ТЬе DO depletion саизед Ьу the sample (oveI'all DO
depletion minus tOO зеед correction) ТЬе value in Column J minus tOO ушие in Column L ЕО!'
Ше Ьоttlе !>eing analyzed

Соluшо N•••DiI. Factor 300/Е. Тhe factor Ьу wblch ше DO depletion must Ье multiplied 10
account fOI dilution ofthe sample (cal1ed the дПиьоп factOl in this guidance document).
Calcu1ated Ьу dividingthe volume ofthe BOD bottle (300 in Ше сазе ofthe example) Ьу the
milliliteIs оЕ sample in: the bottle being analyzed.

Соluшп Р •••Воttlе BOD М х N. Тhe calculated BOD ofthe bottle being analyzed, which is the
CQIrected DO depletion (value in Column м) times Ше dilution faCtOI' (value in Column N).

Column Q •••.Reported BOD. TheBOD value to ье I'eported, IftheI'C was only one dilution, Р
anд Q will Ье equal Ifthere was тоте than опе dilution, this is the aver'age оС аН шозе meeting
the reporting cliteria of > 20 mgIL DO depletion with retention оС :::..1 о mgIL DO.

С-4
 I IШ
Dаtеrпmе Samples Taken: ВОD BENCHSHEET (CYLINDER МЕТНОD)
Page 1
Samples Такеп Ву: _ _ _ 80О (Indicate which) of pages
_ _ _ СВОО

А 8 С D Е F G н J к L м N Р Q

~
'<:1
[
х'
t:I

~t:I
I
......
'-'

* ас Test -Blank DO drop shoukl ье <0.2 mglL ommenls: Samples analyzed Ьу:

- &eed strenglh Cheek Islюuld ье 41.8 -1.0 mg/L ре. mL seed) DateIТime Inltial ОО;

J + F for seed Conlrol " mg/L - mL зеес! DateJТime final ОО

.. mg/L 00 рег mL seed Checked by/date:

*** Зеес! Ratlo Factor =f. % Seed(samp)/% Seed(seed control)

lo~;;m~;S;;;;;,"~Thken: J BOD BENCHSHEET (CYLINDER METHOD)
(Continuation Sheet)
page _ _ __
of pages

А в с D Е F G н J к L м N р Q

Sample

~
8-
х·
t1
-;::а
~
<1>
t1
,
N·
'-'

[. ,,,..-""""
 1; Ш
DateJТime samples Taken: BOD BENCHSHEET (CYlINDER METHOD)
1/29/98. 8:30 а.т. P8ge---1
Samples Таkеп Ву: Х ВОО (Iпdiсаtе which) of 1 __ pages
Апм .!::!!t СВОО

А В с D Е F G Н J К L М N Р а

.g;-
1;;:'
t:1
"i:J
~
t:1
(..,
'-'

• ас Test - Вlапk 00 drop should ье <0.2 mg/L СomтепЬ: samples ana!yzed ьу:
мпа Lvst
•• Check of leed strength (shollld ье 0.8-1.0 mglL per mL иed) Blank exceeded crlteria «0.2 mglL). Random DateIТime initial 00:
comamlnation sUlpected. Does not Invalidate 1/29198, 9:30 а.т.
J -+ F ЮГ ЗееО ConIrol " н..... mglL ... ~ mL seed bзtсЬ. Will acid-rinse all glassware after Отеmте final ОО
this batch. 213198. 10:30 а.т.
,. 0.6 mglL DO рег mL seed СЬесКоо by/dale:
З. Visor
- ЭееО ~ Factor " f" % Seed(samp)1% Seed(seed control}
 Use oCBOD Benchsheet (Graduated Cylinder Method)

Following is ап explanation of the various columns оп the BOD Benchsheet оп the preceding
pages Тhозе columns which ше self explanatory асе not included below

Column A •••Sample. Identification ofthe sarnple being analyzed. Notethe!'e is only опе Нпе
provided for the Ыank, seed control, and standard. ТЬе fOlm сап Ье modified to do !'eplicates
(е. g., duplicates) for anу of these samples. Alзо, there асе thJee liпез provided for an "effluent"
sample in recognition ofthe vaIiability of'such sarnples, Anу sample fo!' wblch the lab does not
Ьауе а good езtimте ofthe BOD should Ье ron in more than опе dilution.

Соlптп В ••• рН. рН о! the sample (before dilution, if it is а diluted sample).

Column С .•• Тетр Deg С. ТЬе temреlаtще oftbe solution in the bottle (should Ье 20 ± 1о С).

Column Е ••• тМ Sample. MilIilitels ofsampIe, either fi'om the environment, oI'the glucosel
glutamic acid standaI'd, in the graduated eylinder

Column F ••• mLs Seed. МilIШters ofseed in the gr'aduated cylinder

Column J ... DO Drop G - Н. DO depletion over 5-day period, Initia1 DO minus fina1 DO

Column K •••Seed Папо Factor (1). Ratio ofseed in the sample to seed in the seed control,
Equals the percentage seed in the sample divided Ьу the percentage seed in the зеоо control

Column L ••• Seed COfrection (J_) х (. ТЬе DO depletion caused Ьу the зеоо wblch тuз! Ье
subtracted ftom the ovесаll DO depletion, Calculated Ьу multiplying the Column J va1ue Со!' the
seed control bottle Ьу the зеоо correction factOl', "Р' (the Column К уа1ие ) for the Ьоttlе being
analyzed

Соlотп M ••• Corr. DO Drop J - L. ТЬе DO depletion саивоо Ьу the sarnple (ove!'all DO
depletion шiпиз the зеоо correction). ТЬе value in Column J minus the value in Column L Сог
the bottle being analyzed.

Column N ...Dil. Factor 1000/Е. ТЬе factor Ьу wblch the DO depletion must Ье multiplied to
account for dilution ofthe sample (called the di1ution factor in tbls guidance document).
Calculated Ьу dividing tbe volume ofthe graduated eylinder (1000 in the сазе ofthe example) Ьу
the milliliteIS о! sample in the graduated eylinder used to fill the bottle being ana!yzed.

Column Р .•• Bottle BOD М х N. ТЬе ca1culated BOD of the bottle being ana1yzed, which is the
corrected DO depletion (value in Column м) times the dilution factol' (value in Column N)

Column Q...Reported BOD. ТЬе BOD value to ье reported IftheI'e was only one dilution, Р
and Q мll Ье equal. Ifthere was mOI'e than one dilution, tbls is the average of аН those meeting
the I'eporting criteIia of .::::.2 о mgIL DO depletion with retention о! > 1. О mgIL DO

D-4
 AppendixE

Statistics in BOD/CBOD Testing ~

1. Most analysts Ш'е уеху faithful аЬои! running quality сопtюl tests such ав а blank, а eheck
standaId (e.g., glueose/glutarnic асЫ), and а duplicate in еуеху BOD test . Some analysts
conside! опlу the Iевиа fiom each indi"idual test (о determine if the рюсеdще i8 "in сопtюГ'
and mecting data quality objectivcs Ро! ехarnрlс, ап analyst might consideI' а glucose/glutamic
acid !csults to Ье асссрtэЫе as 1000g аз it i8 between 167 5 mg!L (i..e . , 198 - 30.5 mg!L) and
228.5 (i е, 198 + 30 5) Ьесаиве thcir' intcrpIetation of Standard Methods i8 that the expected
averagc result should Ьс within зо.5 mg!L of 198, Such analysts Ьаус not used statistics
рюреrlу in evaluating theil perfoImance.

2, ТЬе statistics needed to evaluate performance ofthe BOD/CBOD !est ше Ielatively simple .
ТЬе simpIest i8 the mеаn 01 average result of repeated analyses of the saтc saтple 01 type of'
sample.. Virtually аН analysts know that an average result i8 вiшрlу the Бит of aIlr'esu1ts divided
Ьу the питЬех oflesu1ts. Ifthe average [евu1! i8 close enough to the true от accepted value, thc
test i8 unbiased, ВiaB i8 опе of the components of ассшаеу (01 inaccuracy), the other being
pI'ecision (О! impreci8ion).

1 Another statistic needed to ful1y evaluate lab perf01mance, а little ШО1е complicated than the
average, i8 the standanJ deviation ТЬе standard deviat10n of rcpeated anа1УБе;; ofthe Бате
sample 18 an indication of' pr"ecision, the other' component of ассшасу.. Only if а рюсеduге i8
unЫавед AND precise сan il Ье said 1t is being done ассшаtеlу

4. Опе othcr statistie might Ье used то assi8t the analyst in diseovcring possibIe SOUJces of erю!,
and that 18 the relative percent dijference, or RPD Рor the РШРОБе8 ofthis gШdanсе document,
RPD 18 8imply the difference between two results for а duplicate pair, divided Ьу the average of
the two, and expressed as а percentage**. ТЬе RPD give8 the analyst an indication ofwithin-
batch precision, which i8 influenced Ьу differences between, for exaтple, two bott1e8 in а BOD
bateh Contaminated BOD bottle8, whеп: some ахе тorе contaтinated than оthеп;, might саиБе
random differences between two bott1es in а duplicate pair ТЫБ would contlibule 10 within-
batch impl'ecision

* Analysts necd по! feel eommitted to using а1l о! the statistical analY8is tool8 mentioned in this
appendix., Тhe 10018 ше provided to allow ап in-dcpth review of lab performance if such i8
desil'ed Ьу the analyst or lаЬ management.

** Some statisticians pI'efer 10 give RPD а sign . positive if тЬе first result i8 larger than the
second, and negative ifthe second result i8 larger than the first. For the ршроsеs ofthis
guidanee document, it i8 simp!erjust to think of the RPD а;; an indication о! how шисЬ the two
results differed without considering which was greate!

Е-I
 5 Wbilc thelc are many other statistics that сап Ьс applied {о analytical resu1ts, {Ьс threc аЬоуе
ш'с those easiest to apply to BOD testing ТЬе fol1owing is а discussion of how these
statistics carr Ье applied to еасЬ of the tm'ee typcs of QC tests mentioned аЬоуе, the blank,
the check standaJd, arrd the duplicate,

Blanks

6. Nопnal1у it is по! neccssaJY to аррlу statistics to Ыank Icsults., Eithcr' the Ыаnkв meet the
cIiterion that they Ье iess tharr 0.2 mg/L, OI they do not If onfy arr occasional Ыаnk exceeds 0..2
mg/L (Бау опс every ten batches), there is по саше fo1' alarm Оп the othC! hand, if Ыankв ше
normal1y 0,1 01 0,2 mg/L, even though they веЫот excced 0.2 mg/L, the саиве of the ровШуе
Ыanks should Ье sought and eliminated if possible.

Check StandaJd (Glucose/Glutamic Acid)

7. Check standaJds arc aI'guably the тов! useful QC test in that they give the analyst information
аЬои!bias AND precision, {Ье two components of ассшасу.

а, Avel'age, {Ье Bias Indicator. Тhe statistic associated with Ыав is the avetage, OI
теan valuc fo[ r'epeated ana1yses I о Ье statistically va1id, the aVCIage should Ье ca1culatcd
Ьавед on а! least 20 Icsults (Stаndшd MethQds 5210В suggests 25). 1Ье average, as stated
above, is simply the вит of а11 I'esults, divided Ьу the number of Iesults, 01 ..

Equation #1 - Аverage х

whcr'c "х" is еасЬ rcsult, "ЕХ" i8 the 8ит of аН I'csults, апд "п" i8 the питЬет о! Iesult8,. Тhe
avcrage i8" х ,"рroпоипсед "x-bar," Fопереаtеd analyses ofthe glucose/g1utamic acid
standard, the avcIagc BOD should Ье in the vicinity of 198 mg/L.

О) How close should 1t Ье to 198 mg/L? If it is considerabIy greatcT than 198,

say 240, аН seeded samples (eg, effluent f10m а wastewate! treatment plant) атс probably biased
оп the high 8ide. Ifthc plant i8 still able to meet its peImitted effluCIJt limit for BOD with {Ье
Iesults known to Ье biased оп thc high side, there i8 по significaпt risk to thc CIJviюnmепt, arrd
the Ыав could Ье acceptable Howcvcr, thc lab тау not Ье аЫе to successfully analyze
реrfоrшапсе evaluation samples, which \vould Ьауе an adveIse impact оп its accreditation.

(2) Н, оп thc other Ьапд, the аvещgе lesult i8 cOn8iderabIy lowerthan 198, эау
156 mglL, Icsults f01 эеедед samples would рюЬаblу Ье biascd оп the 10w side, meaning а plant
might Ье connibuting mOle BOD to the t'Cceiving wateI than its lаЬ [еэulИ indicatc Тhiз IS а
tisk to the environment, and an attempt should Ье made to incI'case the avclage Iesult, ивиаllу Ьу
using ап alternativc эесд which wottld then Ье used f01 аl1 seedcd samples., .

Е-2
 ь Standar'd Deviation, the Precision Indicator. А statistic associated ;,уНЬ pIecision is
the standaId deviation of repeated analyses, ТЫ;; statistic is еаsПу ca1culated using а scientific
ca1culator or compute!. О! if such а calculatO!/computer is not availabIe, it is not-so-easily
ca1culated using tlle fol1o\\ting equations \v!lere "Б" is the staпdаз:d deviation,

Equation #2 - Standard Deviation s =

n-I

Lx/-n{x)
s n-I

where "х, "is the ith result, " х" is the теап of the "Г' I'esults, and "п" is the питЬеу of Iesults
used to ealculate х

For Бтаl1еI labs that до not Ьауе а seientific ca1culator or computer, aпd Ьауе по desir'e to work
theiI' way thюugh the аЬоуе equations to dete!mine stапdаз:d deviation, analytical I'esults сап Ье
sent to Ecology's Quality AssUIance Section where the calculation сап Ье made Ьу computeI anд
IepOIted to the lab

8, DupJicate Samples

а Relative Percent Difference (RPD), the Within-batch Pr'ecision Indicator. Ву

analyzing duplicate samples, the analyst сап get а fecling fo! the plecision of analyses vvithin а
batch One statistic used to express the degl'ee ofwitbln-batch plecision indicated bythc
duplicates is I'elative peIcent difference (RPD). FOI the pU!poses of tms discussion, RPD i5
simply the absolute difference bet\veen the two rcsults f01 а duplicate pair, divided Ьу the
aveIage result, and multiplied Ьу 100 во as to express the statistic as а peI'cent. ТЬш, ..

Equation #3 - RPD RPD = 100 , D1..=.Iы

(D 1 + D 2)/2

= 200 , D j - D 2 '/(D j + D2)

WhCl'e "D j " is the fiIst Iesult in the dup1icate pair, "D2" the second, and ,,' D! - Dzl" is (Ье
absolute diffeIence between the two ("absolute" meaning without rеgаз:d to whether it i8 рluв 01

Е-З
 minus). For example, ifthe first result in а duplicate pair· is 12 mglL, and the second тсвu!! is 20
mglL, the relative регсеп! dif[ercl\cc is.

Example - RPD RPD 200112 - 20 1/(12 + 20)

- 200(8)/32 = 1600132 = 50%

11\ prcparing this guidance, the authOl could по! find а widely-rccognized data quality objective
[01 the RPD of· duplicate BOD samples. Рав! experiencc indicates that the 50% RPD given in the
example аЬоуе, 01 10wer, might Ье а rcasonable goal for effluent samples in the range of 10 to
20 mglL RPD should decrease ав concentration incrcases Fo! example, RPD [01 duplicate
glucose/glutamic асИ sampJes (accepted value о! 198 mglL) might Ье 5% or еуen lower

Ь Standard Deviation of· the Difier'ence, Another Within-batch Precision Iпdiсаtог.

If samples in а narrow concentr·ation range, ретЬарв еШиеп! fi:om а wastewater treatment pJant
that norma11y тunв in the 10- 20 mglL птgе, ате analyzed in duplicate ОУет веуета! batches, опе
сап ca1culate the standшd deviation of the difference Ьу pooling the duplicate di[[erences. ТЫв
statistic is discussed in Appendix J ofthe Procedural Маnиа! fOr the Envirvnmental LabvratOfY
Acaeditation Prograт апд wiI1 по! Ье further addressed here An Ехсеl spreadsheet progIaIn is
available gratis fi:um Eco!ogy (Ьа! constructs а сопtюl chart based оп the standard deviation of the
difference between dup!icate paiIs in а Ielative!y narIOW concentration Iange (вее the foIlo\ving
рашgrарh). Ап advantage ofusing duplicates (о estimate pI'ecision is that anаlувев ате done оп гса1
БaInр!ев аБ opposed (о synthetic samples (i.e, glucose/g1utamic acid) analYLed ав а BOD standaId.)

9. Statistics Applied to Control Charting

а ContIo! ehaIting is а теаns of'visually tIacking performance to detcrmine when а

рrосеdше i8 "оut-оf.,еопtшl" (i.e" по1 meeting data quality oЬjectives). Even better, control
сhшts indicate when а procedul'e i8 Ьеадоо оut-оf-еontш! 80 the analyst сап раиве, eliminate the
sошсе of the ртоЫет, and prevent thc оut-·оf-сопtшl situation. In BODICBOD testing, ther·c аге
two contro! charts that сan Ье used vcry effective!y 10 continuously monitor· тсаи!ш, а cont!O!
сЬат! based оп I'epeated апаlувев ofthe check standard, and а сhш! based оп the diffe!ence
between duplicate pairB Both of these eharts ате diseussed in detail in Appendix J of the
Procedural Маnuа/ (от the EnVlfvnтenta! Laboratoтy Accreditation Progfaт and wШ Ье described
only b!iefly Ье!е.

Ь. А contro! сЬат! сап Ье used (о morutor peIfoImance оп (Ье g!ucose/g1utamic ac1d standшd
Ъу analyzing results fi:om (Ье last 20 tests (от the lав! 22 tests, elim1nating the bighest, and the lowest,
and using the 20 Icmaining )

(1) ТЬе chart 1S constIUcted Ьу dIllwing а centelline оп а graph reptesenting the

average (теап) Iesult, anд аЬоуе it, а line replesenting the average рlив t,vo standard deviations, and
anothe! representing tшее standard deviations ТЬе вате is done below the теan, subtracting two
and t1uee standard deviations Веfше this cha!t 18 constructed, the lab must тзkе sше the аvешgе
уа!ие is close enough to the 198 mgIL guidance of·Standard l'vfethods. Once constructed, this chart is
uвед to monito! subsequent resu!ts fo! the GGA test АН subsequent lesults should !ie between the
outer lirnits ofthe chart (the lines at plus and minus tшее standaI·d deviations, Ie[cIIed (о ав action

Е-4
!imits). Оnlу 5% о! subsequent points should lie outside the plus and mЩus two standard deYiation
limits, I'eferтed to as war'ning limits, Vety imp01tant1y, there вЬоиЫ ье по noticeable tгепd (е g.,
upwaI'd 01' downwaI'd) in the subsequent lesults.

(2) Figшс Е-I i8 а сопtю! сhшt based оп J'eal дata rcsulting fiom lepeated
analyses of а glucose/glutamic acid standaId f01 BOD . The 'аЬ'в aveI'age fOI 20 results was
196,8 mg!L, the centra11inc оп thc сhшt, and thc standar d deviation fo1' those saше 20 results
was 82 mg!L, ТhiB lab was obviously "iп-сопtюl" at the time thc сhшt was constJucted. ТЬе
1968 avcIage 18 certainly close enough to the Standard Methods guidance of 198 mg!L, and the
standard deviation of 8,.2 mg/L indicates excellent pIecision ТЪе lаЬ kncw that jt had а problem
whelc а Iesult \vould ье fш bclow what could Ье considered ассерtзblе., This meant to them that
it was time to get ftesh seed fiom а local waste\vate! tr'eatment plant.. lЬе cycle was repeated
seveIaI times in the рзs! уеш'S Ву plotting theiI I'esults, the autho! showed the 'аЬ that, had they
been using а control cha!t, they would have undoubtedly noticed the downwatd tl'end in results
wel1 befote the unacceptably-Iow Iesult.. Ву using висЬ а chaIt and collecting fiesh seed as вооп
as the dоwnwшd trend i8 noticed, the 'аЬ Ьзs sinсе avoided оut-оf-сопtюl situations,.

Figше Е-l - Сопtюl Chatt Based оп Repeated Analysis of а ааА Standatd

16000
1234567&
1 est Number'

Е-5
 (3) FigU1e Е-2 is а control сЬшt based on fictiti'Ous but {еавопаЫе results f'O!'
ana!ysis 'Of effluent BOD duplicates. 1ЪiB c'Ontrol chart is based 'Оп а сеnпа!
Jine 'Of 0.0 mglL, the expecred difference between dup!icates in а РЮI.. Uppet
and l'Owe! warning and acti'On limits Ш'е charted 'Оп еасЬ side 'Ofthe cenna!
Нnе based оп the standard deviati'On of the difference between the pairs in 20
duplicates, ТЫ!; сЬшt il!ustJ:ates h'Ow а lab's precision ЬМ Ьееn d'Oing well,
with I'esults #1 thr'Ough #17 'О! s'Obeing within the wашing !imits. But, aftet'
that, the precision had deteIiOlated, indicating а change f'01' the WOlse., 1Ъiэ
lаЬ w'Ould Ьауе then paused to find the pr'Oblem eausing the greateI'
impreeisi'On О! e'Ould Ьауе Ьееп а new analyst, а new seed, а ma1functi'Oning
DO mete!, о! 'Опе of тanу 'Othet sоШ'сes of I'andom error).

FigШ'е Е-2 - С'ОпtюJ СЬшt Based оп Ana1ysis 'Of Duplicate Samples

AnaJysis of' Duplicate Samples

1500

10 ОО

500

j 000

-500

-1000

"iS-QО

Jest Number

с, An MS Ехсеl 8pI'eadsheet file was used t'O gеnешte еасЬ of' the preceeding СOnП'О!
eharts. C'Opies 'Of the Ехее! files ше аvШ!аЫе to accredited !аЬБ Ьу c'Ontactirig the Dершt!nent 'Of
Ecol'Ogy Quality Аssшanсе Section,

Е-б
 Appendix }<'

Troubleshooting the BODlCBOD Test

ТЬе results of quality contro! (QC) tests analyzed with each BOD/CBOD batch, combined \vith the resu!ts for tЬе эеед control and
environrnental samples, сап Ье иэед to teH (Ье analyst that а рroЫет eXlsts. ТЬе results сan шэо givc hints оп what mlg!lt Ье ca\lsing the
probIem, апd what to до about Н. Absence of any шdiсаtoгs of а probIem is reassunng evidence that thc analytical process is "in control."
This appendix addresses воте of the соmшоп ртоЫетэ ехрспспсед In BOD/CBOD lаЬэ and possible саиэеэ, and suggests асtiопs to
eliminate the рroЫетэ. ТЬе fоllоwшg i8 огgашzеd ш sections according to the эатрlе to which the results apply. Possible саиэеэ ате listed
in отдет oflikelihood with the most рroЬаЫе first.

Sаmш Indicator PossibIe Сапэе Possible Solution Reference

Blank Usua!ly exceeds 0.2 mg!L Source water 18 u11suitabJe IпспЬаtе several Ыaпks USi11g Pg 26, рата 9Ь( 5)
alternate water(э); choose best Pg 13, para 7а(З)(Ь)

Sometimes exceeds 0.2 DiJ utюп water 1В supersaturated Aerate дау before test, storc ш Pg 25, рата 9Ь(2)
mg!L (i.e., DO depletlO11 wllen poured i11to BOD bottle шсuЬаtог, add nUtrientslbuffers Pg 13, para 7а(З)(Ь)
ехсеоов 0.2 mglL) day of test, swirl to щiх, siрhоп Pg 1О, рата 6f
i11to bottom ofBOD bottles

Labware is сопtаmшаtcd More aggressive сlеащпg of Pg 25, para 9b(l)

labware шсludiпg acid гшsе Pg 13, para 7а(3)(Ь)
if IIccessary

Sometimes negatlve (i.e., Tempeтature of bottle c011tcnts Ксср воитсе water, dilutiоп Pg 25, para 9Ь(З)
final DO 18 greater tha11 18 sometJmes >20 ± 1ос w!lel1 water ш шсuЬаtог Pg 13, рша 7а(3)(Ь)
i111t!al ОО) illittal ОО 18 read *

РhоtоsупthеSlS is occuring ctunng Cleall BOD bottles, incubate Pg 26, р,шt 9Ь(З)
incubation шdагk Pg 13, para 7а(З)(Ь)
Sашрlе Indicator I.ikely Сапsе Possiblc Sоlпtiоп Refercncc

Blank Sometimes negative, DO !Б nо! Ьешg шеаsuгеd Check entire DO measurement Pg 26, para 9Ь(б)
(Collt'd) sometlmes posit!ve correct1y procedure Pg 13, рша 7а(З)(Ь)

АtшоэрЬелс pressure not Use barometer and ТаЫе 4 when Pg 25, para 9Ь(4)
Ьешg accounted for when calibrating DO meter Pg 13, para 7а(3)(Ь)
calibrating DO meter

Any combination of the аЬоуе Ве more cOnS1stent at domg the Pg 25, раш 9Ь
possibIe саиэеэ entire procedure correct1y Pg 13, para 7а(3)(Ь)

Glucosc! Standard dеviаtюn of Analysis not Ьешg dOl1e the эате, Look for sources ofvariabi1ity, Pg 26, para 9с
Glutamic last 20 results lS less tban tllne after Нте. Could Ье variable еlimшаtс tflem Pg 27, рша 10b
Acid Ьи! approaclllng 30.5 mg/L веед, temperature от ргеssпrе
рroЫет, faulty DO readil1gs,
1l1attel1tjon of al1alyst(s), or
аl1У of СОШltlсss other th1l1gs

Standard dеviаtюn of Маnу Бошсеэ оfШl1dоm error; Request asslstal1Ce fюm Ecology Pg 26, para 9с
la8t 20 re8uJts 18 BOD procedure 18 ш big trouble ( e.g., techrrical аSSlstапсе special- Pg 27, para 10Ь
> 30.5 mg/L * i8t8 or Quality Аssuгапсе SесtЮI1

(Note: Before worrying аЬои! the averagc rcsult (эее пехt two entnes) for several апаlуsеs of the
g!ucose/glutamlc acid solut10n, tlle analyst must Ье виге that the analY81S 18 Ьеiпg done consistently
(i.e., that {Ье standard dеv!аtюn 18 acceptable, well below 30.5 mg/L).

А verage result for last Seed 18 100 8trong Тгу altemate seed Pg 26, para 9с
20 results 18 » 198.5*

А verage resuJt for /ast Seed 18 too weak TI'y altemate seed (seed with mапу Pg 26, para 9с
20 геsпJts 18« 198.5* bacteIJa of thc right typc 18 пееded)

.. > 18 "grea!er tl1aIl;" »18 "тисЬ grcater tbап;" < 18 "Iess (Ьап;" «18 "тисЬ leS8 thап"

F-2
Sample Indicator
Duplicate RPOs (вее Арр Е) f'or
Effluent duplicate pa!rs ехсеед
Samples 50% (for samplcs ш tllC
range of5-20 mglL)
Seed 00 depleHon i8 <0.6 mgIL
Contl"ol per шL of seed ш bottle
Bottle(s)
DO dерlеtюп i8 >1.0 mgll,
per mL of Беед ш bottle
00 depletions sometlmes
«0.6, sometimes »1.0
mgll., per mL of Беед ш bottle
Environ- No dilutlOuS 'еауе а! least
mental 1.0 шgIL 00 after incuba-
Samples Но!!
No (lilutюпs deplete а!
least 2.0 шglL 00
Significant BOD шсrеаsе
[or more-dilute Ьоttlев
Likely СаПБе Possible Solution Reference
Random errors with1n the batch; Тту to eliminate possible sources Pg 27, рата 9d
coиlд Ье lmproperly mixed seed, of random error within а batch Pg 26, рата IOb
fau1ty шеаsuremепt of seed, cont-
ашшаtеd BOD bottles, faulty DO
рroЬе, and countless other things
that сIшпgе from ЬоШе to bottle
Seed 18 100 weak Try altemate seed (seed with тапу Pg! 4, para 7с(2)
bacteria of thc right (уре 18 needed)
Seed i8 too strong Tryaltemate seed Pg 14, рауа 7с(2)
Seed 18 too vanable Need 10 find а lllore consistent Pg 14, para 7с(2)
8eed (for exampJe, raw шfltLепt
18 often too vanable)
Samples rlOt dilute enough Add еуеп more-dilute samples Pg 24, para 9а
to batcb (soте labs do preliml- Pg 9, ТаЫе 3
пату TSS or COD to еstlшаtе Pg 1З, para 7Ь
ВОО апа pтoper dilution)
Sашрlеs too di1ute Run essentially 100% sample (c.g., Pg 24, para 9а
285 mL samp]e, 10 mL bufferl Pg 9, ТаЫе 3
nutrient solution, 5 rnL seed)
Matnx 18 iпtеrfеппg Witl1 Report BOD Oflllost dil\1te bottle Pg 24, Note 11
the test and ТаЫе 7
F-З
 Influent ВОD (and TSS)
suddenly higher thal1
поrшзJ, or always hlghel'
when done Ьу опе апаIуst
than when done Ьу others
Sample 18 not being ТhоroиgЫу ШlХ sample Ьу Pg. 4, para За
thoroughIy lшхеd before shаkшg, ro\Jing, stшiпg,
taklllg a!iquots (sub-samples) blending (at low speed)

NOTE: Кnowledge сan Ье gail1cd Ьу studying thlS doeument; wisdom еап Ье gашеd only Ьу observation. When trying to
discover what 18 causing а problcm Wlth thc BOD51CBODs test, (Ье wise thing to do is change опе likely cause at а
time ... rnaуЬе two ... and observe what impact the change has оп the шdicаtor being mOl1itorcd. Кеер good records, апd if
sOlnethil1g \vorks, иве Jt!

Р-4
 Appendix О

BOD~CBOD5 Checksheet

Ref: SM (16'" ed) 507; SM (18'\ 19'\ 20th ed) 52]0 В; ЕРА 405 J

Comments

1. Is approved metbod followed?

Method ____ ~_ _ _ _ _ _ __

2. 18 incubatot adequate (i .е., clean, excludes light, -- -- --------

circulates air/wateI)?

3. Лrе ватрlев stoted (iГпеееSSaIУ) at 40 С?

-- -- --------
4, 18 ватрlе вощее and type (i.e., g:rab OI
eomposite) гесot·dеd?

5. Ате samples analyzed within:

48 hошs?

6hошs?

6 If DO руоЬе is used, is it ealibrated:

_ _ against air'?

_ _ against Winklet titJation?

_ _ against 02-saturated water?

before eaeh day's lIве? -- -- --------

7.. IfDO probe is used, is it propeI'ly maintained 80 --

there are по ыlыывs undeI the membrane? -~ - - - -",-,--

the mеmЬшпе is not a!lowed to dIY out? -- -- ---------

therc is по gгowth under the membraIIe? - - - - ---------------

Y:\sllare\save\bod\bod chk дос 0-1

 8 ЛIе РlOреl' BOD bottles used
(е g., 75-,125-,250-,01 300-mL)?

Sea!ed dUI]ng иве?

9. Docs incubatol maintain temperatUIe of 20 ± 1о С?

10. I5 ineubator thermomctcr ccrtificd to ± 1о С?

11 . 18 buffe! added to dilution water.

on1y оп day ofuse foт BOD?

оп day of иве от earlier f01 CBOD? -----_.-

12 I5 buffc! 5torcd in rcfiigcrator?

13 15 SOUIce water.

_ _ Deionized

Disti1led Wnat type ofstil! _ _ _ _ _ __

PUIchased Brand Namc _ _ _ _ _ _ __

14. I5 dilution water' р! otected fiom atmospheric

contamination?

15, ЛIе di!ution water b!anks ana!yzed?

16 . I5 the Ыаnk dep!etion

usuall у zero? --- - - ---_ ...... _._-_._-

sometime5 01 mgIL?

always 1евв than О 2 mgIL?

17. Ате BOD bott!es and g1asswar'e cleaned with

non-phosphate detergent and (ав песеввату)
acid rinsed?

18. ЛIе ватр!ев neutralized to рН 6.5 7,.5?

Y;\shaIe\save\bod\bod chkdoc 0-2

 19. If doing CBOD, i8 nitrification inhibitor added to .

dilution wate!?

_ _ sample?

20 Атс !Cagcnts fOl dilution water propcrIy prcpared - -

Fепiс chloride (О 25 g/L)? ----------

Magnesium sulfate (22.5 g/L)?

Calcium chlO!ide (27.5 g!L)? - - - - - ---------------

Phosphate Buffer (рН = 7.2)?

Sodium su1fite (1.575 g!L), pr'cpared daily?

.. .от ше they purchased pre-packaged _ __ VendoI: ________________

21 Are bott1es brought to 20 ± 1о С before

reading initial DO? -- -- ---------------

22.. А samples thol'Oughly mixed Ьеfше

taking a1iquots (sub-samples)?

23.. 18 reference solution О50 mg each of glucosc

& g1utamic acid diluted w/distШеd wateI to 1 L)
run \vith еасЬ batch of sarnples? ----------
24, Do repeated results fO! the аЮА solution average ...

in the vicinity of 198 mg/L fOl BOD? А verage: ______ mg/L

in thc vicinity of 184 mg/L [О! СВО D? Average: _____ mg/L

25. I5 the standard deviation of Iepeated

arra1yses ofthe аЮА solution .

тисЬ less than 30.5 mg/L [о! BOD? ----------

much less tharr 28..3 mg/L f01 CBOD?

26 If present, i5 chlorine removed with sodium

sulfite arrd ш'с sarnples pl'Operly seeded?

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27 What is the SOUIce of seed .

fina! eff1uent fiom WWTP?

_ _ primary eff1uent fiom \\'WТP?

_ _ ArtificiaI seed (e.g., PoIybac)? Which опе?

__ Fтozen sewage?

Other? What? _ _ _ _ _ _ _ __

28 Атс рторе! di1ution techniqttes uscd?

29. Do di1ution Ьаус depletions of at least 2 mg!L?

and rctention of at Icast 1 mgiL?

зо Ate БаmрlСБ incttbated for 5 days ± 2 hOUIS?

31. А!е calculations completed properIy?

32 Лrе records рюреI1у attthenticated

(ie, signedlinitiaHed Ьу anaIyst and опе other)?

33. Атс duplicates щп regnJarly (not а requirement)?

34. Is а сопtюl сЬан available and ttsed? --- --- - - - - - - -

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