CELEX: 51981PC0128
Language: en
Date: 1981-03-24
Title: Proposal for a COUNCIL DIRECTIVE amending Directive 73/405/EEC on the approximation of the laws of the Member States relating to methods of testing the biodegradability of anionic surfactants (submitted to the Council by the Commission)

ARCHIVES HISTORIQUES
DE LA COMMISSION
COLLECTION RELIEE DES
DOCUMENTS "COM"
COM (81) 128
Vol. 1981/0034
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 ---pagebreak--- COMMISSION OF THE EUROPEAN COMMUN1TIE
                                                  COM(81)128 final
                                                  Brussels , 24 March 1981
                          Proposal for a
                        COUNCIL    DIRECTIVE
 amending Directive 73 / 405 / EEC on the approximation of the laws of
 the Member States relating to methods of tesring the biodegradabi­
                   lity of anionic surfactants
          ( submitted to the Council by the Commission )
C0MC81 ) 128 final
 ---pagebreak---                                                                Λ 2-*
                          Explanatory Memorandum
I. General
   Council Directive 73 / 405 / EEC relating to methods of testing the bio-
                                            1
   degradabi I ity of anionic surfactants     , which is to be amended by this
   proposal , forms part of the general programme for the removal of
   technical barriers to trade approved by the Council on 28 May 1969 .
   There are so far two Directives on detergents ( specific washing and
   c leaning agentsT .  The Commission transmitted a proposal for a third
   Directive to the Council for approval on 18 .February 1980 .
   All these Directives have two main aims : to improve environmental
   protection and to secure free trade within the European Community .
The main purpose of the present proposal is to adapt to technical progress
the method described in the Annex ( confirmatory test procedure ) for
testing the biodegradabi lity of anionic surfactants .     The need for this
adaptation became obvious during the preparation of the proposal on
methods of testing non-ionic surfactants put before the ^Council a year ago .
The confirmatory test procedures proposed in the two Directives must
correspond as far as possible .     This test is to be carried out in the event
of objections pursuant to Article 5 ( 2 ) of Directive 73 / 404 / EEC on
detergents , and serves as a basis for the Commission 's decision .
This proposal also updates the measuring methods used in the Member States
and incorporates an additional method .
The opportunity is also taken to define the scope of the Directive more
precisely as the existing text has given rise to difficulties of inter­
pretation .                                                          .
1  0J N° L 347 , 17.12.1973 , p.    53
2  0J N° C 104 , 28 . 4.1980 , p. 112
3  0J N° L 347 , 17.12.1973 , p.    51
 ---pagebreak---                                       -2-
 II .  Comments on the Articles
      Article 1            1
      The words added to Artidle 1 are intended to define the scope of the
      Directive more precisely .   They make it clear that the Directive
      applies only to surfactants used in detergents .    Consequently the
      Directive does not apply to surfactants in products other than detei–
      gents , a point that was not clear in the existing text .
       In addition , the references to existing methods in certain Member
      States are brought up to date and the method in use in the United
      Kingdom is incorporated .   The text of the existing Articles 2 and 3
      is also made more precise in the light of experience gained .
      Article 2
      The reference method described in the Annex ( confirmatory test
      procedure ) is adapted , on the lines of the method to be used for
      non-ionic surfactants , to take account of the latest developments
      in science and technology .   In future , adaptations to technical
      progress will be made by the Committee procedure laid down in the
      proposal for a third Directive mentioned in Section I above .
      Articles 3 and 4
      These Articles are common to all Directives .
III . Consultation of the parties concerned
      The proposal for a Di recti ve' was drafted in close cooperation with
      experts from the Member States .    Account was taken of the opinions
                  y
      given by representatives of the relevant industries .
 IV . Consultation of the European Parliament and of the Economic and
      Social Committee
      Pursuant to the second paragraphe of Article 100 of the EEC Treaty ,
      the opinions of these two bodies are required .
 ---pagebreak---                                  Proposal - for a
                               COUNCIL DIRECTIVE
amending Directive 73/ 405 / EEC on the approximation of the laws of the Member
States relating to methods of testing the biodegradabi lity of anionic
surfactants                                                    /
                                                                                   \
THE COUNCIL OF THE EUROPEAN COMMUNITIES /
Having regard to the Treaty establishing the European Economic Community , and
in particular Article 100 thereof ,
Haying regard to the proposal of the Commission ,
Having regard to the Opinion of the European Parliament ,
Having regard to the Opinion of the Economic and Social Committee ,
                                        1
Whereas Council Directive 73/ 405 / EEC     must be adapted to , take account of the
latest developments in science and technology , thus necessitating :
- the updating of the references relating to the methods listed in Article 2 ;
- the addition to Article 2 of a further , method of measurement which is in
    use in the United Kingdom ;                       -    ,
- the improvement of the 'Confirmatory test procedure' " provided for in the
    event of a dispute ;
Whereas pursuant to Article 4 . of Council Directive 73/ 404 / EEC of 22 November
                                              I
1973 on the approximation of the laws of the Member States relating to
            2
detergents    suitable tolerances for the measurement of biodegradabi lity should
be determined in order to take due' account of the unreliability of text
methods which could result in rejection decisions with important economic
 consequences ; whereas a rejection decision may therefore only be taken where
the results obtained with .one of the test methods specified in Article 2 of
 Directive 73/ 405 / EEC reveal a biodegradabi'lity of - less than 80% ;
 " Whereas certain confusion having arisen as to the scope of Directive 73 / 405 / EEC,
 it is necessary to make quite clear that 'the Directive applies only to
 surfactants used in detergents ; it is also necessary to make quite clear that
 it is the level of biodegradabi I ity of the anionic surfactants contained in
 a detergent , and not the level of biodegradabi lity of the detergent itself ,
which is dealt with in Article 2 ;"
 1 0J No L 347, 17.12.1973, p . 53
 20J No L 347, 27.12.1973, p . 51
 ---pagebreak---                   f                       -2-
HAS ADOPTED THIS DIRECTIVE :        ■
                                         Article 1
Directive 73 / 405 / EEC is hereby amended as follows :
1.    The following      is      added to Article 1 :
      " used in detergents ".
2.    Articles 2 and 3      are       replaced by the following :
                                       " Article 2
Pursuant to Article 4 of Directive 73 / 404 / EEC relating to detergents ,
due account being taken of the unreliability of the test methods , Member
States shall prohibit the placing 'On the . market and use on their terri­
tory     of a detergent if the biodegradabi I ity of the anionic surfactants
contained therein is less than 80 % determined in accordance with one of
                               t
the following methods :
- the OECD method , published in the OECD 's technical report of 11 June 1976
   on a " Proposed method for the Determination of the Biodegradabi I ity of
   Surfactants used in Synthetic Detergents ";
- the method in use in France , approved by decree of 28 December 1978
   published in the " Journal Officiel de la Republique Frangaise " of
   18 January 1979 , pages 514 / 515 , and by experimental standard T 73-260
   of February 1971 published by the " Association Frangaise de Normalisation "
   ( AFNOR );
- the . method in use in the Federal Republic of Germany , laid down under
   the " Verordnung über die Abbaubarkeit anionischer und ni cht ioni scher
   grenzflächenaktiver Stoffe in Wasch- und Reinigungsmitteln " of
   30 January 1977 and incorporated in the Order amending that Order of
   18 June 1980 , as published in the Bundesgesetzblatt 1980 , Part I ,
   page 706 ;
- the method in use in the United Kingdom , called the " Porous Pot . Test "
   and described in Technical Report No 70 ( 1978 ) of the Water Research
   Centre .
 ---pagebreak---           ^                       Article 3
 Under the procedure laid down in Article 5 < 2 ) of Directive 73 / 4Q4 / EEC
                               r
 relating to detergents , the laboratory opinion on anionit surfactants
shall be based on the Reference method ( confirmatory test procedure )
described in the Annex to this Directive ."
3 . The Annex is replaced by the Annex to this Directive .
                                  Article 2
Member States shall bring into force the provisions necessary in order to
comply with this Directive not later than.1 June 1982 and shall forthwith
inform the Commission thereof .
                                  Article 3
 This Directive is addressed to the Member States .
 ---pagebreak---                              'ANNEX
   DETERMINATION OF THE BIODEGRADABILITY OF
              ANIONIC SURFACE ACTIVE AGENTS
            ,     feEFFgFNCir . METHDJ>      V  ■
           { CONFIRMATORY TEST PROCEDURE/
                     ' ' CHAPTER 1
Definition                 T               *
Anionic surface active agents in the sense of this directive
are those surface active agents , which , after passage through
cationic and anionic ion exchangers are . separated by fractional
elution and determined as methylene blue " active substance ( MBASl
according to the analytical procedure described in Chapter 3 .
Equipment needed for measurement                     ^
The method of measurement employs the small activated sludge
plant shown in Figure 1 , and in greater detail in Figure 2 .
The equipment consists of a storage vessel A for synthetic
sewage , dosing pump B , aeration vessel C , settling vessel D ,
air-lift pump E to recycle the activated sludge , and vessel
F for collecting the treated effluent .
Vessels A and F must be of glass or suitable plastic and hold
at least 24 litres .          Pump B must provide a constant flow of
synthetic sewage to the aeration vessel ; this vessel , during
normal operation , contains 3 litres of mixed liquor .         A sintered
aeration Cube G is suspended in the vessel C at the apex of
the cone .      The quantity of air blown through the aerator should
be monitored by means of a flowmeter H .
Synthetic s ewage                                  ' .
A synthetic sewage is employed for the' test .         Dissolve in each
litre of tap water :
                   160 nig peptone
                   110 mg meal extract
                    30 nig urea (CO (Nil ) 2 )
 ---pagebreak---                                        τ* 2 **
                                 7 rag sodium chloride (NaCl )
     *                           4 mg calcium chloride , (CaCl ^ , 2Ho0i
                                 2 mg magnesium sulphate (MgSO^y^O).                  ^
                                28 mg of mondhydrogeri dipotassium ortho phosphate
                                  '              •    -        -.     CK2HPO4 >
                   and          20 - 2 mg MBAS
               The MBAS is extracted from the product to be tested by the method
            • given in Chapter 2 . , The synthetic sewage is freshly prepared daily .
 i  •,                                                                             •
   1.4 .       Preparation of samples
   1.4.1 .     Uncompounded surfactants may be examined in the original state .
               The MBAS content must be determined in order to prepare the
               synthetic sewage (1.3 ).
   1.4.2 .     Formulated products are analysed for MBAS and soap content .
             . They must be subjected to an alcoholic extraction and
• •
               separation of MBAS (see Chapter 2 ).         *
               The MBAS content of the extract must be known in order to prepare
               the synthetic sewage .
   1.5 -       Operation' of equipment
               Initially fill aeration vessel C and settling vessel D with
               synthetic sewage .     The height of the vessel D should be so fixed
               that " the volume contained in the aeration vessel C is 3 litres .
               Inoculation is made by introducing 3 ml of a secondary effluent of
               good quality , freshly collected from a treatment plant dealing with a
               predominantly domestic sewage . The effluent must be kept under
               aerobic conditions in the period between sampling and application .
               Then set the aerator , air lift E and dosing device B in operation .
               The synthetic sewage must pass through aeration vessel C at the
               rate of one litre per hour ;, this gives a mean retention time             N
               of 3 hours .
               The rate of aeration should be regulated so that the contents of
               vessel C are kept constantly in suspension while the dissolved
               oxygen content is at least 2 mg/ 1 .     Foaming must be prevented by
               appropriate means . . Antifoaming agents which inhibit the activated
         - . sludge or contain MBAS must not be used .         The air-lift pump, E
               must be set so that the activated sludge from the settling vessel
               is continually and regularly recycled to aeration vessel C.              .
               Sludge which has accumulated around the top of the aeration
               vessel C , in the base of the settling vessel D , or in the circula-
          -    tion circuit must be returned to the circulation at least once .
               each day by brushing or some other appropriate means .         When sludge
 ---pagebreak---                            ^ 3  r\
fails to settle , its density may be increased by addition of
2 ml portions of a ^ per cent solution of ferric chloride ,
repeated as necessary .
The effluent from separator D is accumulated in vessel F for
24 hours , following which a sample is taken after thorough
mixing .     Vessel F must then be carefully cleaned .
Checking measuring equipment
The MBAS content ( in mg /1 ) of the synthetic sewage is determined
immediately before use ,
The M£AS content ( in mg / 1 ) of the effluent collected over 24 hours
in vessel F should be determined analytically by the same method ,
immediately after collection ; otherwise the samples must be
preserved , preferably by freezing .     The concentrations must be
determined to the nearest 0.1 mg / 1 MBAS .
As a check on the efficiency of the process the chemical oxygen
demand ( COD)^ or the dissolved organic carbon (DOC ) of the
filtered effluent accumulated in vessel F , is measured at least
twice "weekly , as well as that of the filtered synthetic sewage
in vessel A.
The reduction in COD or DOC should level off when a roughly
regular daily MBAS biodegradation is obtained i.e. at the end of
the running-in period shown in Figure 3 .
The loss on ignition of the dried suspended solids in the activated
sludge in the aeration tank should be determined twice a week
 (in g/ 1 ).   If it is more than 2.5 g/1 , the excess activated
sludge must be discarded .
The test is performed at room temperature ;        this should be steady
and should be kept between 291 and 298K (18 - 25°C) .
Càlculation of biodégradation »
The percentage biodegradation of MBAS must be calculated every
day on the basis of the MBAS content in mg / 1 of the synthetic sewage
and the corresponding effluent accumulated in vessel F.
The biodegradation figures thus obtained should be presented
graphically as in Figure 3 .
The biodegradation of the MBAS should be ^ calculated as the arithmetic
mean of the figures obtained over the 21 days which follow the
running-in period ,       during which biodegradation has been regular and
 the operation of the plant trouble-free .     In any case the duration
of the running-in period should not exceed six weeks .
                                                                         ./•
                      >
                                                • ·Τ·Γ«ΤΤ·Γ'    – –Ι-
 ---pagebreak--- The daily biodegradation values are calculated to the nearest of
0.1 per cent but the final result is given to the nearest whole
number .              -
In some cases it may be permissible to reduce the frequency of
sampling but at least 14 results collected over the 21 days which
follow the running-in period should be used in calculating the
average .                                                   .
 ---pagebreak---    *
                                       • CHAPTER 2
                        • PRELIMINARY TREATMENT OF PRODUCTS
                                    • TO BE TESTED
                      »
                                                                        •     *
2.1 .   ' ' Preliminary notes
2.1.1 .     Treatment of samples '
            The treatment of anionic surface active agents and formulated
            detergents prior to the determination of biodegra dability in the
            confirmatory test is :-
 ι   .
        ' PRODUCTS                           " TREATMENT
            Anionic surfactants                None
            Formulated detergents              alcoholic extraction
                                               followed by ion exchange
                                               separation arnd fractional
                                               elution from the anion exchanger
            The purpose of the alcoholic extraction is to eliminate the insoluble
            and inorganic ingredients of the commercial product which in some
            circumstances might upset the biodegrada bility test .
2.1.2 .     Ion exchange procédure
               «                                        *
                              I
            Isolation and separation of anionic surface active agents from
            soap , nonionics' and cationics are required for correct biodegraaability
            tests .
                                               0
                                                 *
            This is        achieved by an ion exchange technique using a macro­
           porous anion exchange resin and suitable elu-ants for fractional
            elution .    Thus soap , anionic and nonionic surf actants . may be
            isolated in one procedure .
2.1.3 .     Analytical control
           After homogenising , the content of anionic surfactants in the
            synthetic detergent is determined according to the MBAS analytical
            procedure .    The soap content is determined by a suitable analytical
           method .     This analysis of the products is necessary to calculate
            the quantities required co prepare        fractions, for the biodegrada bility
            test .
 ---pagebreak---             Quantitative extraction is not necessary ; however at least 802
            of the anionic surfactants should be extracted .         Usually , 90% -
            or more is obtained .
 •- • • •                                                              .
 2.2 .      Principle                                                                   -
            From an homogeneous sample (powders , dried pastes , and dried liquids )
             an ethanol extract is obtained which contains the surfactants ,
             soap and other alco'nol'-soluble constituents of the synthetic
             detergent sample .                * .
            'The ethanol extract is evaporated and dissolved in an isopropanol /
            water mixture , and the solution obtained is passed through a
      "    .'strongly acidic cation exchange /macro-porous anion exchange
             combination heated to 323 K ( 50 c ) .    tThis temperature is necessary
             to prevent precipitation of fatty acids in acidic media .
            The nonionic surfactants remain in the effluent .
     ' ,     The soap fatty acids are separated by elution with ethanol containing
             CO2'     The anionic surfactants are then obtained as ammonium salts .
             by elution with an aqueous isopropanolic solution of ammonium
             bicarbonate .     These ammonium salts are used for the degradation
             test .               ••                      '    '
             Cationics , which might upset the bi'odegradability test and the' analytical
             procedure , are eliminated by the cation exchanger placed on top of the
             anion exchanger .                      ...                               -
2.3 ..       Chemicals and equipment
                • "
2.3.1 .      Deionised water
2.3.2 . Ethanol , 95% (v/v) C,H OH
              /     •    • < 4       v J •
             (permissible as denaturant :    methyl ethyl ketone or methanol )
                                           •              *-
2.3.3 .      Isopropanol /water mixture (50 /50 ):
             50 parts by volume isopropanol (CH^CHOH.CH^) and
             50 parts by volume water (2.3.1 .).
                       *                                     A
2.3.4 . Solution of carbon dioxide in ethanol (approx . 0.1% (X^) :
             using a delivery tube with a built-in frit , pass carbon dioxide (C0^)
          . through t-hc ethanol (2.3.2 ) for 10 minutes .       Use fresh solutions
             only .
 ---pagebreak---                                          7
2.3.5 ,   Ammonium bicarbonate solution (60 /40):
          0.3 mol NH^HCOg in 1000 ml isopropanol /water mixture of 60 parts
          by volume isopropanol and 40 parts by volume water (2.3.1 .)
2.3.6 .   Cation exchanger (KAT ) , strongly acidic , resistant to alcohol
          (50 - 100 mesh ).
                                                                S
2.3.7 .   Anion exchanger (AAT ) , macro-porous , Merck Lewatit MP 7080
          ( 70 - 150 mesh ) or equivalent . \
2.3.8 .   Hydrochloric acid , 10% HC1 (w /w )
2.3.9 .   2000 ml round-bottomed* flask with ground glass stopper and reflux
          condenser               •
2.3.10 . 90 mm dia . suction filter (heatable ) for paper filters                       '
2.3.11 . 2000 ml filter flask
2.3.12 . Exchange columns with heating jacket and cock :
          Inner tube 3D mm in diameter and 200 mm in height (Fig . 4 ).
2,3.13 . Water-bath
             •*                                      «
2.3.14 . Vacuum drying oven
2.3.15 . Thermostat                                                                         .
2.3.16 . Rotary evaporator
2.4 .     Extraction . and Separation of Anionic surface active agents
2.4.1 .   Preparation of extract                 .          '
          The quantity of surface active agents necessary for the biodegradntion
          test is about 50 g MBAS .
          Uorriis.! ly the quantity of product to be extracted will not . xc -. cd -10C0 g,
          but it may be necessary to extract further quantities of Kar.-.nlc .
        . For praotxcal reasons 5000 g will in most circumstances be t'r.e unoer limit .
                                                                                          ./.
 ---pagebreak---        v        In preparing extracts " for biodegradability test ,
            1   experience has shown that there are advantages in using a number
                of small extractions rather than one large extraction .                  -
                The exchanger quantities specified are designed for a working capacity
                of 600-700 mmoles of surfactants and soap .
                                       »                                  •
      2.4.2 . Isolation of alcohol-soluble Constituents                    *
                Extract 250 g aliquots of sample by adding to 1250 ml portions of -
                ethanol , heat the mixture to boiling point and reflux .for 1 hour
                with
    r -         .rass the hot alcoholic solution " through a coarse-pored suction ""filter,
                heated to 323 K (50°C) and filter rapidly .     Wash the flask and/
                suction filter with approx . 200 ml hot ethanol .     Collect the filtrate
  '             and filter washings in a filter flask .
                In the case of pastes or liquid products , make sure that not more
                than 55 g anionic surfactant 'and 35 g soap are contained in the
                sample .   Evaporate this weighed sample to dryness .     Dissolve
                the residue in 2000 ml ethanol and proceed as described above .
                Evaporate the ethanolic filtrate to dryness , preferably by means
                of a rotary evaporator .     Repeat the operation if a greater quantity
    ...         of extract is required .     Dissolve the residue in 5000 ml isopropanol /
                water mixture .
\ •
    * 2.4.3 . Preparation of Ion exchange columns
                Cation exchange column
                Place 600 ml cation exchange resin (2.3.6 .) in a 3000 ml beaker and
                cover by adding 2000 ml hydrochloric acid (2.3.8 .).      Allow to stand
                for at least 2 hours stirring occasionally . Decant the acid and
                 transfer the resin into the column ( 2.3.12 ) by means of deionised
                water .   The column should contain a glass wool plug .      Wash the column
  .       '     with deionised water at the rate of 10 .- 30 ml /min until the eluate
                 is free of chloride .    Displace the water with 2000 ml isopropanol /
                water mixture ( 2.3.3 .) at a rate of 10 – 30 ml/min . The exchange column
                is now ready for use .     •                 •                              .
              ' Anion exchange column
                Place 600 ml anion exchange resin (2.3.7 .) ^.n a beaker and cover by
                adding 2000 ml deionised water .     Allow the exchanger to swell for at
                least 2 hours .    Transfer the resin into the column by means of
                deionised water .    The column should contain a glass wool plug
                 as exchanger supporting layer .
 ---pagebreak---                                       - 9 -
         Wash the column with 0.3 M ammonium bicarbonate solution ( 2 . .?..-5 .)^   -
         until free of chloride .     This requires about 5000 ml solution .
         Wash again with 2000 ml deionised waeer .        Displace the waeer with
         2000 ml isopropanol /water mixture ( 2.3.3 .) at the rate of 10-30 ml /min .
         The exchange column is now in the OH-forra and ready for use .
2.4.4 . Ion excnange procédure
         Connect the exchange columns, so th,T,t the cation exchange column is
       . placed on top of the anion exchange column .        Using a thermostat heat
         the exchange columns to 323 K ( 50 C ) .      Heat 5000 ml of the solution
         obtained in 2.4.2 . to 333 K (60 C ) and pass the solution through the
         exchanger combination at the rate of 20 ml /min .       Wash the columns
         with 1000 ml hot isopropanol /water mixture (2.3.3 .).
  I •
         To obtain the anionic synthetic surfactants (MBAS ), disconnect the
        'KAT column .    Using 5000 ml ethanol /CO,, solution (323 K ; 50°C)
         (2.3.4 .), elute the soap fatty acids out of the AAT column .        Reject
         the eluate .
                        V
         Then elute the MBAS out of the AAT column with 5000 ml ammonium
         bicarbonate solution (2.3.5 .).      Evaporate the eluate to dryness
         on a steam bath or in a rotary evaporator .        The residue contains the
         MBAS (as ammonium salt)and possibly non-surfactant anionics which have
         no detrimental effect on the biodegradation test .        Add deionised water
         to the residue until a definite volume is obtained and determine the
         MBAS content in an aliquot as in Chapter 3 .        The solution is used
         as a standard solution of the anionic synthetic detergents for the
         biodegradation test .     The solution should be kept at a temperature
         below 278 K (5°C) .
2.4.5 . Regeneration of ion exchange resins
         The cation exchanger is rejected after use .                            .
         The anion exchange resin is regenerated by passing about 5000-6000 ml
         of ammonium bicarbonate solution ( 2.3.5 .) down the column      at a flow
         rate of approximately 10 ml /min until the eluate is free from
         anionics (methylene blue-test ).       Then pass 2000 ml isopropanol /
         water mixture (2.3.3 .) down the anion exchanger to wash .
         The anion exchanger is again ready for use .
                               ■ •
 ---pagebreak---                              CHAPTER 3
      DETERMINATION OF ANIONIC SURFACE ACTIVE AGENTS .
              ■   IN BIODEGRADABILI TY TEST
Principle
       \                      .    '          .         .                        . -
The method is Based on the fact that the cationic dye methylene
blue forms blue salts with anionic surfactants: which can be
extracted with chloroform .             To eliminate interferences , the
extraction is first effected from alkaline solution and the
extract is then shaken with acidic methylene blue solution .                The
absorbance of the separated organic phase is measured photometrically
at the wavelength of maximum absorption of 650 nm .
Reagents and equipment
                    I                ■      •
Buffer solution pH 10 :                             . '
Dissolve 24 g sodium bicarbonate (NaHCO^) A.R ; and 27 g anhydrous
sodium carbonate (Na~C0 _) A.R. in deionised water and dilute to
1000 ml .                                         -              '
Neutral methylene blue solution :
Dissolve 0.35 £ methylene blue (B.P. grade ) in deionised water and
dilute to 1000 ml .         Prepare the solution at least 24 hours before
use .    ' The absorbence of the blank chloroform           phase , measured
against chloroform , must not exceed 0.015 per 1 cm of layer thickness
at 650 nm .
Acidic methylene blue solution :,                                  '                 \
Dissolve 0.35 g methylene blue (B.P. grade ) in 500 ml deionised
water and mix with 6.5 ml I^SO^. (d = 1.84 ). Dilute with deionised water
1000 ml . Prepare the solution at least 24 hours before use .                The
absorbence of the blank chloroform phase , measured against chloroform ,
must not exceed 0.015 per 1 cm of layer thickness at 650 nm .
                *
Trichloromethane ( Chloroform) CHC1 , or Dichlorom ethane (Methylene
dichloride) CH^C^, freshly distilled.                          '
Dodecyl benzene sulphonic acid methyl ester
                                                      s
                        * .      '    *                   •
Ethanolic potassium hydroxide - solution , KOH 0.1 M
 ---pagebreak---                                    - 11 -
 Λ
3.2.7 .^ Etlianol pure , C^H^OH
3.2.8 .  Sulphuric acid ,         0.5 M
3.2.9 .  Phenolphthalein solution :
         Dissolve 1 g phenolphthalein in 50 ml ethanol and add 50 ml
         deionised water while stirring continuously .     Filter off any
         precipitate obtained .
3.2.10 . Methanolic hydrochloric acid :      250 ml hydrochloric acid
■  .     conc . A.R. and 750 ml . methanol .
3.2.11 . Separating funnel , 250 ml
3.2.12 . Volumetric flask , 50 ml
3.2.13 . Volumetric flask,' 500 ml                   -
3.2.14 . Volumetric flask , 1000 ml '
3.2.15 . Weighing pipette
3.2.16 . Round^bottomed flask with ground glass stopper and reflux
         condenser , 250 ml ; boiling granules
3.2.17 . pH-meter                        ,
3.2.18 . Photometer for measurements at 650 nm , with 1 to 5-cm cells
                                «                  •
3.2.19 . Qualitative filter paper
3.3 .    Procédure
         The samples for analysis must not be taken through a layer of
         foam .
         After thorough cleaning with wnter , the equipment used for the
         analysis must be thoroughly rinsed with methanol ic hycirqchloric
          acid 3.2.10 and then with deionised water before using .
         Filter the activated sludge plant influent and effluent to be examined
         immediately on sampling .    Discard the first 100 ml of the filtrates .
 ---pagebreak---                                   - 12 -
          Place a measured volume of the sample , neutralised if necessary ,
        . into e 250 ml separating funnel ( 3.2,11 ,), The volume of sample
          should contain between 20 and 150 ^ug of MBAS . At .the lower MBAS
          content , up to 100 ml of sample may be used .      When using less
          than 100 ml , dilute to 100 ml with deionised water .       Add to the
          sample 10 ml of buffer solution ( 3.2.1 .), 5 ml of neutral / or d><.Uoroine}jtanc_
          methylene blue solution ( 3.2.2 .) and 15 ml of chloroform *( 3 . 2 . 4 .) .,
          Shake the mixture uniformly and not too vigorously for 1 minute .
          After phase separation , run the chloroform layer into a second
          separating funnel containing 110 ml of deionised water and 5 ml
          of acidic methylene blue solution ( 3,2.3 .).      Shake the mixture
          for 1 min .    Pass the chloroform layer through a cotton-wool • . .
          filter wetted with chloroform into a graduated flask (3.2.12 .).
                                      ι
          Extract the alkaline and acid solutions three times , using 10 ml
          of chloroform for the second and third extractions .        Filter tJie
          combined "chloroform extracts through the same cotton wool filter
          and dilute / to the mark in the 50 ml-flask ( 3.2.12) with chloro-r
          form used for rewashing the cotton3ro °l »      Measure the absorhance
          of the chloroform solution with a photometet at 650 nm in 1- to
          5-cm cells against chloroform .    Run a'blank determination
          through the whole procedure .
. 3,4 .   Calibration curve
          Prepare a calibration solution from the standard substance dodecyl
          benzene sulphonic acid methyl ester (tetrapropylene type Wo/.v/f 340)
          after saponification into the potassium salt ,       The MBAS is
          calculated as sodium dodecyl benzene sulphonate (w.Wt348 ) .
          From a weighing pipette , weigh 400 to 450 mg of .dodecyl benzene
          sulphonic acid methyl ester ( 3.2.5 ,) to the nearest 0.1 mg in a
          round-bottomed flask and add 50 ml of ethanolic potassium hydroxide
           solution and some boiling granules .      After mounting the reflux
          condenser , boil for 1 hour .   After cooling , wash the condenser
          and . ground glass Joint with about 30 ml of ethanol , and add these
          washings to the contents of the flask . Titrate the solution with
          sulphuric acid against phenolph'thalein until it becomes colourless .
          Transfer this solution to a 1000 ml graduated flask (3 . 2 . 14 . ) , dilute
          to the mark with deionised water and mix .                     •
          Part of this surfactant stock solution is then further diluted .
          Withdraw 25 ml , transfer- to a 500 ml graduated flask ( 3.2.13 .).
          dilute to the mark with deionised water and mix .         .
                                            Ε χ 1.023
          This standard solution contains ■ 9
          M  •   . »     ■    • .   •
                                                   *
                                             fa vvUU
                                                          mg MBAS per ml , where
          E is the sample weight in mg .
 ---pagebreak---                                      - 13 -
         To establish the calibration curve , withdraw 1 , 2 , 4 , 6,' 8 ml
         each of the standard solution and dilute each to 100 ml with
         deionised water .          Then proceed as stated under item 3.3
 i       including a blank determination .
3,5 . ' Calculation of results
         The amount of anionic surfactant .in the sample as MBAS is read
         from the calibration curve (3.4 .).                     The MBAS content of the sample
         is- given by :                                  ;
                                •. .mg MBAS . . ' . 1000
                                    –                         "~j=       mg MBAS / 1
                                               V
         where V - ml volume of the sample used .
         Express the results as sodium dodecyl benzene sulphonate
         (MW 348).
3.6 .  ' Expression of results                                                    .
         Express the results as mg MBAS /1 to the nearest 0.1 .
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 ---pagebreak---                                                                                                                    6
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 ---pagebreak--- iiodt   gradation
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