ARCHIVESHISTORIQUES
DELACOMMISSION
COLLECTION RELIEEDES
DOCUMENTS "COM"
COM(81)128
Vol.1981/0034

Disclaimer
Conformément aurèglement (CEE,Euratom) n°354/83duConseildu1erfévrier1983
concernant l'ouverture aupublicdesarchiveshistoriques delaCommunauté économique
européenne etdelaCommunauté européenne del'énergieatomique(JOL43du15.2.1983,
p.1),telquemodifiéparlerèglement (CE,Euratom) n°1700/2003 du22septembre 2003
(JOL243du27.9.2003, p.1),cedossierestouvertaupublic.Lecaséchéant,lesdocuments
classifiésprésentsdanscedossierontétédéclassifiés conformément àl'article5dudit
règlement.
Inaccordance withCouncilRegulation (EEC,Euratom) No354/83of1February 1983
concerning theopeningtothepublicofthehistoricalarchivesoftheEuropean Economic
Community andtheEuropean AtomicEnergyCommunity (OJL43,15.2.1983, p.1),as
amended byRegulation (EC,Euratom) No1700/2003 of22September 2003(OJL243,
27.9.2003, p.1),thisfileisopentothepublic.Wherenecessary, classifieddocuments inthis
filehavebeendeclassified inconformity withArticle5oftheaforementioned regulation.
InÜbereinstimmung mitderVerordnung (EWG,Euratom) Nr.354/83desRatesvom1.
Februar 1983überdieFreigabe derhistorischen Archive derEuropäischen
Wirtschaftsgemeinschaft undderEuropäischen Atomgemeinschaft (ABI.L43vom15.2.1983,
S.1),geändertdurchdieVerordnung (EG,Euratom) Nr.1700/2003 vom22.September 2003
(ABI.L243vom27.9.2003, S.1),istdieseDateiderÖffentlichkeit zugänglich. Soweit
erforderlich, wurdendieVerschlusssachen indieserDateiinÜbereinstimmung mitArtikel5
dergenannten Verordnung freigegeben.

COMMISSION OFTHEEUROPEAN COMMUN1TIE
COM(81)128 final
Brussels,24March1981
Proposalfora
COUNCILDIRECTIVE
amendingDirective 73/405/EEContheapproximation ofthelawsof
theMemberStatesrelatingtomethodsoftesringthebiodegradabi­
lityofanionicsurfactants
(submittedtotheCouncilbytheCommission )
C0MC81)128final

Λ2-*
Explanatory Memorandum
I.General
CouncilDirective 73/405/EECrelating tomethodsoftestingthebio-
1degradabi Iityofanionicsurfactants ,whichistobeamended bythis
proposal,formspartofthegeneral programme fortheremovalof
technical barriers totradeapproved bytheCouncil on28May1969.
TherearesofartwoDirectives ondetergents (specific washing and
cleaningagentsT.TheCommission transmitted aproposal forathird
Directive totheCouncilforapproval on18.February 1980.
AlltheseDirectives havetwomainaims:toimproveenvironmental
protection andtosecurefreetradewithintheEuropean Community .
Themainpurposeofthepresentproposal istoadapttotechnical progress
themethoddescribed intheAnnex(confirmatory testprocedure )for
testingthebiodegradabi lityofanionicsurfactants .Theneedforthis
adaptation becameobviousduringthepreparation oftheproposal on
methodsoftestingnon-ionic surfactants putbeforethe^Council ayearago.
Theconfirmatory testprocedures proposed inthetwoDirectives must
correspond asfaraspossible .Thistestistobecarriedoutintheevent
ofobjections pursuant toArticle 5(2)ofDirective 73/404/EECon
detergents ,andservesasabasisfortheCommission 'sdecision .
Thisproposalalsoupdatesthemeasuring methodsusedintheMemberStates
andincorporates anadditional method.
Theopportunity isalsotakentodefinethescopeoftheDirective more
precisely astheexistingtexthasgivenrisetodifficulties ofinter­
pretation . .
10JN°L347,17.12.1973 ,p.53
20JN°C104,28.4.1980,p.112
30JN°L347,17.12.1973 ,p.51

-2-
II.Comments ontheArticles
Article 1 1
ThewordsaddedtoArtidle 1areintended todefinethescopeofthe
Directive moreprecisely .TheymakeitclearthattheDirective
appliesonlytosurfactants usedindetergents .Consequently the
Directive doesnotapplytosurfactants inproducts otherthandetei–
gents,apointthatwasnotclearintheexisting text.
Inaddition,thereferences toexisting methodsincertain Member
Statesarebrought uptodateandthemethodinuseintheUnited
Kingdomisincorporated .Thetextoftheexisting Articles 2and3
isalsomademoreprecise inthelightofexperience gained.
Article 2
Thereference methoddescribed intheAnnex(confirmatory test
procedure )isadapted,onthelinesofthemethodtobeusedfor
non-ionic surfactants ,totakeaccountofthelatestdevelopments
inscience andtechnology .Infuture,adaptations totechnical
progress willbemadebytheCommittee procedure laiddowninthe
proposal forathirdDirective mentioned inSection Iabove.
Articles 3and4
TheseArticles arecommontoallDirectives .
III.Consultation oftheparties concerned
Theproposal foraDirective'wasdraftedinclosecooperation with
experts fromtheMemberStates.Account wastakenoftheopinions
y
givenbyrepresentatives oftherelevant industries .
IV.Consultation oftheEuropean Parliament andoftheEconomic and
Social Committee
Pursuant tothesecondparagraphe ofArticle 100oftheEECTreaty,
theopinions ofthesetwobodiesarerequired .

Proposal -fora
COUNCILDIRECTIVE
amendingDirective 73/405/EEContheapproximation ofthelawsoftheMember
Statesrelatingtomethodsoftestingthebiodegradabilityofanionic
surfactants /
\
THECOUNCILOFTHEEUROPEAN COMMUNITIES/
HavingregardtotheTreatyestablishing theEuropeanEconomicCommunity,and
inparticularArticle100thereof,
HayingregardtotheproposaloftheCommission,
HavingregardtotheOpinionoftheEuropeanParliament,
HavingregardtotheOpinionoftheEconomicandSocialCommittee,
1WhereasCouncilDirective 73/405/EECmustbeadaptedto,takeaccountofthe
latestdevelopments inscienceandtechnology,thusnecessitating :
-theupdatingofthereferencesrelatingtothemethodslistedinArticle2;
-theadditiontoArticle2ofafurther,methodofmeasurement whichisin
useintheUnitedKingdom; -,
-theimprovement ofthe'Confirmatory testprocedure' "providedforinthe
eventofadispute;
WhereaspursuanttoArticle4.ofCouncilDirective 73/404/EECof22November
I
1973ontheapproximation ofthelawsoftheMemberStatesrelatingto
2detergents suitabletolerancesforthemeasurement ofbiodegradabilityshould
bedetermined inordertotakedue'accountoftheunreliability oftext
methodswhichcouldresultinrejectiondecisionswithimportanteconomic
consequences ;whereasarejectiondecision maythereforeonlybetakenwhere
theresultsobtainedwith.oneofthetestmethodsspecifiedinArticle2of
Directive73/405/EECrevealabiodegradabi'lity of-lessthan80%;
"WhereascertainconfusionhavingarisenastothescopeofDirective 73/405/EEC,
itisnecessarytomakequiteclearthat'theDirectiveappliesonlyto
surfactants usedindetergents;itisalsonecessarytomakequiteclearthat
itisthelevelofbiodegradabi Iityoftheanionicsurfactants containedin
adetergent,andnotthelevelofbiodegradabilityofthedetergentitself,
whichisdealtwithinArticle2;"
10JNoL347,17.12.1973,p.53
20JNoL347,27.12.1973,p.51

f -2-
HASADOPTEDTHISDIRECTIVE :■
Article 1
Directive 73/405/EECisherebyamendedasfollows:
1.Thefollowing isaddedtoArticle 1:
"usedindetergents".
2.Articles 2and3arereplacedbythefollowing :
"Article 2
PursuanttoArticle4ofDirective 73/404/EECrelatingtodetergents,
dueaccountbeingtakenoftheunreliability ofthetestmethods,Member
Statesshallprohibittheplacing'Onthe.marketanduseontheirterri­
toryofadetergentifthebiodegradabi Iityoftheanionicsurfactants
containedthereinislessthan80%determined inaccordance withoneof
tthefollowing methods:
-theOECDmethod,publishedintheOECD'stechnicalreportof11June1976
ona"ProposedmethodfortheDetermination oftheBiodegradabi Iityof
Surfactants usedinSyntheticDetergents";
-themethodinuseinFrance,approvedbydecreeof28December1978
publishedinthe"JournalOfficieldelaRepublique Frangaise"of
18January1979,pages514/515,andbyexperimental standard T73-260
ofFebruary1971published bythe"Association Frangaise deNormalisation "
(AFNOR);
-the.methodinuseintheFederalRepublicofGermany,laiddownunder
the"Verordnung überdieAbbaubarkeit anionischer undnichtionischer
grenzflächenaktiver StoffeinWasch-undReinigungsmitteln "of
30January1977andincorporated intheOrderamendingthatOrderof
18June1980,aspublishedintheBundesgesetzblatt 1980,PartI,
page706;
-themethodinuseintheUnitedKingdom,calledthe"PorousPot.Test"
anddescribedinTechnicalReportNo70(1978)oftheWaterResearch
Centre.

^ Article 3
Undertheprocedure laiddowninArticle 5<2)ofDirective 73/4Q4/EEC
rrelating todetergents ,thelaboratory opinion onanionitsurfactants
shallbebasedontheReference method (confirmatory testprocedure )
described intheAnnextothisDirective ."
3.TheAnnexisreplaced bytheAnnextothisDirective .
Article 2
MemberStatesshallbringintoforcetheprovisions necessary inorderto
complywiththisDirective notlaterthan.1June1982andshallforthwith
informtheCommission thereof.
Article 3
ThisDirective isaddressed totheMemberStates.

'ANNEX
DETERMINATION OFTHE BIODEGRADABILITY OF
ANIONIC SURFACE ACTIVE AGENTS
, feEFFgFNCir .METHDJ > V ■
{CONFIRMATORY TEST PROCEDURE /
''CHAPTER 1
Definition T *
Anionic surface active agents inthesense ofthis directive
are those surface active agents ,which ,after passage through
cationic andanionic ionexchangers are .separated byfractional
elution anddetermined asmethylene blue "active substance (MBASl
according totheanalytical procedure described inChapter 3.
Equipment needed formeasurement ^
Themethod ofmeasurement employs thesmall activated sludge
plant shown inFigure 1,and ingreater detail inFigure 2.
Theequipment consists ofastorage vessel Aforsynthetic
sewage ,dosing pump B,aeration vessel C,settling vessel D,
air-lift pump Etorecycle theactivated sludge ,andvessel
Fforcollecting thetreated effluent .
Vessels AandFmust beofglass orsuitable plastic andhold
atleast 24litres .Pump Bmust provide aconstant flow of
synthetic sewage totheaeration vessel ;this vessel ,during
normal operation ,contains 3litres ofmixed liquor .Asintered
aeration Cube Gissuspended inthevessel Cattheapex of
thecone .Thequantity ofairblown through the aerator should
bemonitored bymeans ofaflowmeter H.
Synthetic sewage '.
Asynthetic sewage isemployed for the'test .Dissolve ineach
litre oftapwater :
160 nigpeptone
110mgmeal extract
30nigurea (CO(Nil)2)

τ*2**
7ragsodium chloride (NaCl )
* 4mgcalcium chloride ,(CaCl ^,2Ho0i
2mgmagnesium sulphate (MgSO^y^O ). ^
28mgofmondhydrogeri dipotassium ortho phosphate
' • - -. CK2HPO4>
and 20-2mgMBAS
TheMBAS isextracted from theproduct tobetested bythemethod
•given inChapter 2.,Thesynthetic sewage isfreshly prepared daily .
i•, •
1.4 . Preparation ofsamples
1.4.1 .Uncompounded surfactants maybeexamined intheoriginal state .
TheMBAS content must bedetermined inorder toprepare the
synthetic sewage (1.3).
1.4.2 .Formulated products areanalysed forMBAS andsoap content .
.They must besubjected toanalcoholic extraction and
separation ofMBAS (seeChapter 2). *
••
TheMBAS content oftheextract must beknown inorder toprepare
thesynthetic sewage .
1.5 - Operation' ofequipment
Initially fill aeration vessel Candsettling vessel Dwith
synthetic sewage .Theheight ofthevessel Dshould besofixed
that "the volume contained inthe aeration vessel Cis3litres .
Inoculation ismade byintroducing 3mlofasecondary effluent of
good quality ,freshly collected from atreatment plant dealing with a
predominantly domestic sewage .Theeffluent must bekept under
aerobic conditions intheperiod between sampling andapplication .
Then set theaerator ,airlift Eanddosing device Binoperation .
Thesynthetic sewage must pass through aeration vessel Catthe
rate ofonelitre perhour ;,this gives amean retention time N
of3hours .
Therate ofaeration should beregulated sothat thecontents of
vessel Carekept constantly insuspension while thedissolved
oxygen content isatleast 2mg/1.Foaming must beprevented by
appropriate means . .Antifoaming agents which inhibit theactivated
-.sludge orcontain MBAS must not beused .Theair-lift pump ,E
must besetsothat theactivated sludge from thesettling vessel
iscontinually andregularly recycled toaeration vessel C. .
Sludge which hasaccumulated around thetopoftheaeration
vessel C,inthebase ofthesettling vessel D,orinthecircula -
-tion circuit must bereturned tothecirculation atleast once .
each daybybrushing orsome other appropriate means .When sludge

^3r\
failstosettle,itsdensity maybeincreased byaddition of
2mlportions ofa^percentsolution offerricchloride ,
repeated asnecessary .
Theeffluent fromseparator Disaccumulated invesselFfor
24hours,following whichasampleistakenafterthorough
mixing.VesselFmustthenbecarefully cleaned.
Checking measuring equipment
TheMBAScontent (inmg/1)ofthesynthetic sewageisdetermined
immediately beforeuse,
TheM£AScontent (inmg/1)oftheeffluent collected over24hours
invesselFshouldbedetermined analytically bythesamemethod,
immediately aftercollection ;otherwise thesamplesmustbe
preserved ,preferably byfreezing .Theconcentrations mustbe
determined tothenearest0.1mg/1MBAS.
Asacheckontheefficiency oftheprocessthechemical oxygen
demand(COD)^orthedissolved organiccarbon(DOC)ofthefilteredeffluent accumulated invesselF,ismeasured atleast
twice"weekly,aswellasthatofthefilteredsynthetic sewage
invesselA.
Thereduction inCODorDOCshouldleveloffwhenaroughly
regulardailyMBASbiodegradation isobtainedi.e.attheendof
therunning-in periodshowninFigure3.
Thelossonignition ofthedriedsuspended solidsintheactivated
sludgeintheaeration tankshouldbedetermined twiceaweek
(ing/1).Ifitismorethan2.5g/1,theexcessactivated
sludgemustbediscarded .
Thetestisperformed atroomtemperature ;thisshouldbesteady
andshouldbekeptbetween291and298K(18-25°C).
Càlculation ofbiodégradation »
Thepercentage biodegradation ofMBASmustbecalculated every
dayonthebasisoftheMBAScontentinmg/1ofthesynthetic sewage
andthecorresponding effluent accumulated invesselF.
Thebiodegradation figuresthusobtained shouldbepresented
graphically asinFigure3.
Thebiodegradation oftheMBASshouldbe^calculated asthearithmetic
meanofthefiguresobtained overthe21dayswhichfollowthe
running-in period,duringwhichbiodegradation hasbeenregularand
theoperation oftheplanttrouble-free .Inanycasetheduration
oftherunning-in periodshouldnotexceedsixweeks.
./•
>
•·Τ·Γ«ΤΤ·Γ' ––Ι-

Thedailybiodegradation valuesarecalculated tothenearestof
0.1percentbutthefinalresultisgiventothenearestwhole
number. -
Insomecasesitmaybepermissible toreducethefrequency of
samplingbutatleast14resultscollected overthe21dayswhich
followtherunning-in periodshouldbeusedincalculating the
average . .

•CHAPTER 2
•PRELIMINARY TREATMENT OFPRODUCTS
•TOBETESTED
»
• *
2.1.''Preliminary notes
2.1.1.Treatment ofsamples '
Thetreatment ofanionicsurfaceactiveagentsandformulated
detergents priortothedetermination ofbiodegradabilityinthe
confirmatory testis:-
ι.
'PRODUCTS "TREATMENT
Anionicsurfactants None
Formulated detergents alcoholic extraction
followed byionexchange
separation arndfractional
elutionfromtheanionexchanger
Thepurposeofthealcoholic extraction istoeliminate theinsoluble
andinorganic ingredients ofthecommercial productwhichinsome
circumstances mightupsetthebiodegrada bilitytest.
2.1.2.Ionexchange procédure
« *
IIsolation andseparation ofanionicsurfaceactiveagentsfrom
soap,nonionics' andcationics arerequiredforcorrectbiodegraaability
tests.
0
*
Thisisachieved byanionexchange technique usingamacro­
porousanionexchange resinandsuitableelu-ants forfractional
elution.Thussoap,anionicandnonionic surfactants .maybe
isolated inoneprocedure .
2.1.3.Analytical control
Afterhomogenising ,thecontentofanionicsurfactants inthe
synthetic detergent isdetermined according totheMBASanalytical
procedure .Thesoapcontentisdetermined byasuitableanalytical
method.Thisanalysis oftheproducts isnecessary tocalculate
thequantities required coprepare fractions ,forthebiodegrada bility
test.*
2.1.

Quantitative extraction isnotnecessary ;however atleast 802
oftheanionic surfactants should beextracted .Usually ,90% -
ormore isobtained .
•-• •• .
2.2 .Principle -
From anhomogeneous sample (powders ,dried pastes ,anddried liquids )
anethanol extract isobtained which contains thesurfactants ,
soap andother alco'nol'-soluble constituents ofthesynthetic
detergent sample . * .
'The ethanol extract isevaporated anddissolved inanisopropanol /
water mixture ,and thesolution obtained ispassed through a
" .'strongly acidic cation exchange /macro-porous anion exchange
combination heated to323K(50c).tThis temperature isnecessary
toprevent precipitation offatty acids inacidic media .
The nonionic surfactants remain inthe effluent .
',Thesoap fatty acids areseparated byelution with ethanol containing
CO2' Theanionic surfactants arethenobtained asammonium salts .
byelution with anaqueous isopropanolic solution ofammonium
bicarbonate .These ammonium salts are used for thedegradation
test . •• ' '
Cationics ,which might upset thebi'odegradability test and the'analytical
procedure ,areeliminated bythecation exchanger placed ontopofthe
anion exchanger . ... -
2.3.. Chemicals andequipment
•"
2.3.1 .Deionised water
2.3.2 .Ethanol ,95% (v/v)C,HOH
/ • •<4 vJ •(permissible asdenaturant :methyl ethyl ketone ormethanol )
• *-
2.3.3 .Isopropanol /water mixture (50/50):
50parts byvolume isopropanol (CH^CHOH.CH ^)and50parts byvolume water (2.3.1 .).
* A
2.3.4 .Solution ofcarbon dioxide inethanol (approx .0.1% (X^):
using adelivery tubewith abuilt-in frit ,pass carbon dioxide (C0^)
.through t-hcethanol (2.3.2 )for 10minutes .Usefresh solutions
only .

7
2.3.5,Ammonium bicarbonate solution (60/40):
0.3molNH^HCOgin1000mlisopropanol /watermixtureof60partsbyvolumeisopropanol and40partsbyvolumewater(2.3.1.)
2.3.6.Cationexchanger (KAT),strongly acidic,resistant toalcohol
(50-100mesh).
S
2.3.7.Anionexchanger (AAT),macro-porous ,MerckLewatit MP7080
(70-150mesh)orequivalent .\
2.3.8.Hydrochloric acid,10%HC1(w/w)
2.3.9.2000mlround-bottomed *flaskwithgroundglassstopper andreflux
condenser •
2.3.10.90mmdia.suctionfilter(heatable )forpaperfilters '
2.3.11.2000mlfilterflask
2.3.12.Exchange columnswithheatingjacketandcock:
Innertube3Dmmindiameter and200mminheight(Fig.4).
2,3.13.Water-bath
•* «2.3.14.Vacuumdryingoven
2.3.15.Thermostat .
2.3.16.Rotaryevaporator
2.4.Extraction .andSeparation ofAnionicsurfaceactiveagents
2.4.1.Preparation ofextract . '
Thequantity ofsurfaceactiveagentsnecessary forthebiodegradntion
testisabout50gMBAS.
Uorriis.!lythequantityofproducttobeextractedwillnot.xc-.cd-10C0g,butitmaybenecessary toextractfurtherquantities ofKar.-.nlc.
.Forpraotxcal reasons5000gwillinmostcircumstances bet'r.eunoerlimit.
./.

vInpreparing extracts "forbiodegradability test,
1experience hasshownthatthereareadvantages inusinganumber
ofsmallextractions ratherthanonelargeextraction . -
Theexchanger quantities specified aredesignedforaworkingcapacity
of600-700mmolesofsurfactants andsoap.
» •
2.4.2.Isolation ofalcohol-soluble Constituents *
Extract250galiquots ofsamplebyaddingto1250mlportions of-
ethanol,heatthemixturetoboilingpointandreflux .for1hour
with
r- .rassthehotalcoholic solution "throughacoarse-pored suction ""filter ,heatedto323K(50°C)andfilterrapidly.Washtheflaskand/
suctionfilterwithapprox.200mlhotethanol.Collectthefiltrate
' andfilterwashingsinafilterflask.
Inthecaseofpastesorliquidproducts ,makesurethatnotmore
than55ganionicsurfactant 'and35gsoaparecontained inthe
sample.Evaporate thisweighedsampletodryness.Dissolve
theresiduein2000mlethanolandproceedasdescribed above.
Evaporate theethanolic filtrate todryness,preferably bymeans
ofarotaryevaporator .Repeattheoperation ifagreaterquantity...ofextractisrequired .Dissolve theresiduein5000mlisopropanol /
watermixture .
\•
*2.4.3.Preparation ofIonexchange columns
Cationexchange column
Place600mlcationexchangeresin(2.3.6.)ina3000mlbeakerand
coverbyadding2000mlhydrochloric acid(2.3.8.).Allowtostand
foratleast2hoursstirringoccasionally .Decanttheacidand
transfer theresinintothecolumn(2.3.12)bymeansofdeionised
water.Thecolumnshouldcontainaglasswoolplug.Washthecolumn
.'withdeionised waterattherateof10.-30ml/minuntiltheeluate
isfreeofchloride .Displacethewaterwith2000mlisopropanol /
watermixture (2.3.3.)atarateof10–30ml/min.Theexchangecolumn
isnowreadyforuse. • • .
'Anionexchange column
Place600mlanionexchangeresin(2.3.7.)^.nabeakerandcoverby
adding2000mldeionised water.Allowtheexchanger toswellforat
least2hours.Transfer theresinintothecolumnbymeansof
deionised water.Thecolumnshouldcontainaglasswoolplug
asexchanger supporting layer.

-9-
Washthecolumnwith0.3Mammonium bicarbonate solution (2..?..-5.)^-
untilfreeofchloride .Thisrequires about5000mlsolution .
Washagainwith2000mldeionised waeer.Displace thewaeerwith
2000mlisopropanol /watermixture (2.3.3.)attherateof10-30ml/min.
Theexchange columnisnowintheOH-forraandreadyforuse.
2.4.4.Ionexcnange procédure
Connecttheexchange columns ,soth,T,tthecationexchange columnis
.placedontopoftheanionexchangecolumn.Usingathermostat heattheexchange columnsto323K(50C).Heat5000mlofthesolution
obtained in2.4.2.to333K(60C)andpassthesolution throughthe
exchanger combination attherateof20ml/min.Washthecolumns
with1000mlhotisopropanol /watermixture (2.3.3.).
I•
Toobtaintheanionicsynthetic surfactants (MBAS),disconnect the
'KATcolumn.Using5000mlethanol/CO,,solution (323K;50°C)
(2.3.4.),elutethesoapfattyacidsoutoftheAATcolumn.Reject
theeluate .
V
ThenelutetheMBASoutoftheAATcolumnwith5000mlammonium
bicarbonate solution (2.3.5.).Evaporate theeluatetodryness
onasteambathorinarotaryevaporator .Theresiduecontains the
MBAS(asammoniumsalt)andpossiblynon-surfactant anionics whichhave
nodetrimental effectonthebiodegradation test.Adddeionised water
totheresidueuntiladefinite volumeisobtained anddetermine the
MBAScontentinanaliquotasinChapter 3.Thesolution isused
asastandard solution oftheanionicsynthetic detergents forthe
biodegradation test.Thesolution shouldbekeptatatemperature
below278K(5°C).
2.4.5.Regeneration ofionexchange resins
Thecationexchanger isrejectedafteruse. .
Theanionexchange resinisregenerated bypassingabout5000-6000 ml
ofammonium bicarbonate solution (2.3.5.)downthecolumnataflow
rateofapproximately 10ml/minuntiltheeluateisfreefrom
anionics (methylene blue-test ).Thenpass2000mlisopropanol /
watermixture (2.3.3.)downtheanionexchanger towash.
Theanionexchanger isagainreadyforuse.
■•

CHAPTER 3
DETERMINATION OFANIONIC SURFACE ACTIVE AGENTS .
■INBIODEGRADABILI TYTEST
Principle
\ . ' . . .-
ThemethodisBasedonthefactthatthecationic dyemethylene
blueformsbluesaltswithanionicsurfactants :whichcanbe
extracted withchloroform .Toeliminate interferences ,the
extraction isfirsteffected fromalkaline solution andthe
extractisthenshakenwithacidicmethylene bluesolution .The
absorbance oftheseparated organicphaseismeasured photometrically
atthewavelength ofmaximumabsorption of650nm.
Reagents andequipment
I ■ •
Buffersolution pH10: .'
Dissolve24gsodiumbicarbonate (NaHCO^)A.R;and27ganhydroussodiumcarbonate (Na~C0_)A.R.indeionised wateranddiluteto
1000ml. - '
Neutralmethylene bluesolution :
Dissolve 0.35£methylene blue(B.P.grade)indeionised waterand
diluteto1000ml.Preparethesolutionatleast24hoursbefore
use.'Theabsorbence oftheblankchloroform phase,measured
againstchloroform ,mustnotexceed0.015per1cmoflayerthickness
at650nm.
Acidicmethylene bluesolution:, ' \
Dissolve 0.35gmethylene blue(B.P.grade)in500mldeionised
waterandmixwith6.5mlI^SO^.(d=1.84).Dilutewithdeionisedwater1000ml.Preparethesolutionatleast24hoursbeforeuse.The
absorbence oftheblankchloroform phase,measured againstchloroform ,
mustnotexceed0.015per1cmoflayerthickness at650nm.
*
Trichloromethane (Chloroform )CHC1,orDichloromethane(Methylene
dichloride )CH^C^,freshlydistilled. '
Dodecylbenzenesulphonic acidmethylester
s
*. ' * •Ethanolic potassium hydroxide -solution ,KOH0.1M

-11-
Λ
3.2.7.^Etlianolpure,C^H^OH
3.2.8.Sulphuric acid, 0.5M
3.2.9.Phenolphthalein solution :
Dissolve 1gphenolphthalein in50mlethanol andadd50ml
deionised waterwhilestirring continuously .Filteroffany
precipitate obtained .
3.2.10.Methanolic hydrochloric acid:250mlhydrochloric acid
■.conc.A.R.and750ml.methanol .
3.2.11.Separating funnel,250ml
3.2.12.Volumetric flask,50ml
3.2.13.Volumetric flask,'500ml -
3.2.14.Volumetric flask,1000ml'
3.2.15.Weighingpipette
3.2.16.Round^bottomed flaskwithgroundglassstopperandreflux
condenser ,250ml;boilinggranules
3.2.17.pH-meter ,
3.2.18.Photometer formeasurements at650nm,with1to5-cmcells
« •
3.2.19.Qualitative filterpaper
3.3.Procédure
Thesamplesforanalysis mustnotbetakenthroughalayerof
foam.
Afterthoroughcleaning withwnter,theequipment usedforthe
analysis mustbethoroughly rinsedwithmethanolichycirqchloric
acid3.2.10andthenwithdeionised waterbeforeusing.
Filtertheactivated sludgeplantinfluent andeffluent tobeexamined
immediately onsampling .Discardthefirst100mlofthefiltrates .

-12-
Place ameasured volume ofthesample ,neutralised ifnecessary ,
.into e250mlseparating funnel (3.2,11 ,), Thevolume ofsample
should contain between 20and150^ugofMBAS .At .thelower MBAS
content ,upto100mlofsample may beused .When using less
than 100 ml,dilute to100mlwith deionised water .Add tothe
sample 10mlofbuffer solution (3.2.1 .), 5mlofneutral /ord><.Uoroine}jtanc _
methylene blue solution (3.2.2 .)and 15mlofchloroform *(3.2.4.).,
Shake themixture uniformly andnot toovigorously for 1minute .
After phase separation ,run thechloroform layer into asecond
separating funnel containing 110mlofdeionised water and 5ml
ofacidic methylene blue solution (3,2.3 .). Shake themixture
for 1min .Pass thechloroform layer through acotton-wool • ..
filter wetted with chloroform into agraduated flask (3.2.12 .).
ι
Extract thealkaline andacid solutions three times ,using 10ml
ofchloroform for the second and third extractions . Filter tJie
combined "chloroform extracts through thesame cotton wool filter
and dilute /tothemark inthe50ml-flask (3.2.12 )with chloro-r
form used forrewashing thecotton3 ro°l»Measure theabsorhance
ofthechloroform solution with aphotometet at650nmin1-to
5-cm cells against chloroform .Runa'blank determination
through thewhole procedure .
.3,4 . Calibration curve
Prepare acalibration solution from thestandard substance dodecyl
benzene sulphonic acid methyl ester (tetrapropylene type Wo/.v/f340 )
after saponification into thepotassium salt ,TheMBAS is
calculated assodium dodecyl benzene sulphonate (w.Wt348 ).
From aweighing pipette ,weigh 400 to450mgof .dodecyl benzene
sulphonic acid methyl ester (3.2.5 ,)tothenearest 0.1mgina
round-bottomed flask andadd50mlofethanolic potassium hydroxide
solution andsome boiling granules .After mounting thereflux
condenser ,boil for 1hour .After cooling ,wash thecondenser
and .ground glass Joint with about 30mlofethanol ,and add these
washings tothecontents oftheflask .Titrate thesolution with
sulphuric acid against phenolph'thalein until itbecomes colourless .
Transfer this solution toa1000 mlgraduated flask (3.2.14.),dilute
tothemark with deionised water and mix . •
Part ofthis surfactant stock solution isthen further diluted .
Withdraw 25ml,transfer -toa500mlgraduated flask (3.2.13 .).
dilute tothemark with deionised water and mix . .
Εχ1.023This standard solution contains ■9 * mgMBAS perml,whereM • .» ■ • . • favvUUEisthesample weight inmg.

-13-
Toestablish thecalibration curve,withdraw 1,2,4,6,'8ml
eachofthestandard solution anddiluteeachto100mlwith
deionised water.Thenproceedasstatedunderitem3.3
iincluding ablankdetermination .
3,5.'Calculation ofresults
Theamountofanionicsurfactant .inthesampleasMBASisread
fromthecalibration curve(3.4.).TheMBAScontentofthesample
is-givenby: ;
•..mgMBAS ..'.1000
– "~j=mgMBAS/1
V
whereV-mlvolumeofthesampleused.
Expresstheresultsassodiumdodecylbenzenesulphonate
(MW348).
3.6.'Expression ofresults .
ExpresstheresultsasmgMBAS/1tothenearest0.1.
Air
Rjucei t
i-ilflJ IJ\
ψ^\\••..•Λ"·:». \–, »\
,4,t4
ÏM&J,–1?1–.
Η IIV®ο°β°°ο/II
*^ Ε""||ν^ί ^
1I^Ï•VI
Air
A.Storagevessel
B.Dosingdevice
C.Aerationchamber (threelitrescapacity)
D.SettlingvesselE.Air-liftpumpF.Collector
G.Aerator
H.Air-flow meter

6
1500 ,200 i900, ?Fy„\--4 ►((»•■) \ L.J.AQHardPVC
I ^(|3 rI.14 T RiWTT„o-.-Ο ;ιγ^,-τμ-4-^ι I* &
i^im n .i i8o-–<
Liquiclevel \ IIg| . T- "
-:c=*=r-k• ir200,3..•■
iiih Ki,-n iru/v.7W3 m ■*ιυ ■ ιι- ·λ>/^ -£g! on I IVK7^ HIV> Iji45%r ijiRï :rri^L. •ii-h*- er11fttDi .• 1
\I j-–^pH–*- IIIH p Sii!i!(Material:Glassorwaterproofplastic(hardPVC)^-o-n. . i2si© t I 'fo •il I - r? il
?3; j !®*!!40r1 | § |,|
jJ°l^50 ' –-JÛJL;
^WÎW HiirdPVC.^n0 ^.' •
fN 0V/ r£>>>."X ' v–tt~ " 11(Dimensions inmillimétrés )Li250 . ii
jrJ ,
-11 °11–Q
200 .
Figure2

iiodt
V'/o
100
90
80
70
60
50
40
30
20
10
0gradation
Ia. mn ,irn 1Rcadilvdegradablesurfactant
Running-in period/[ Periodusedforthecalculation (21days)^ f>|<| 11■■ g;»|
I AAX\!-/\/!/i/V\I .+***% t\fi4 ® Surfactantnotreadily/\ -f\/\/V/\ dc§radab,e\ι7ν/*-/ι\/\ΜΑ V/ .ιν'. 1–1–1–I–I–I–I–I–I–IV?IW*lilI|_JL^flL_1JILIIJIII1!_JIIi1LiI–I111–I
5 10 15 20 25 30 35 40 45Time(days)
Figure3
Calculation ofbiodegradability -Conf^ma,tory ces£

->Ab–
/ I
• ii°h~ .■i/~r'>n
m
/ 1 - »
i .. 1
I'V '!
,oko. ?
.. i
O IIII (Dimensions inmillimetres )o . I ,
i j
:""Çffi}
:\
§ |06
'~+_ ''
010
Figure4
Heatedexchangecoluiaa