REGULATION ON DETERGENTS  
 
Objective   
ARTICLE 1 – (1) The objective of this Regulation is to lay down the principles and  the 
procedures  for detergents and surfactants for detergents placed on the market  to achieve the free 
movement and  ensuring a high degree of protection of the environment and human health.  
Scope  
ARTICLE 2 – (1) This Regulation includes  the following rules for the placing on the market 
of detergents and of surfactants for detergents:  
a) The biodegradability of su rfactants in detergents,  
b) Restrictions or bans on surfactants  on grounds of biodegradability,  
c) The additional labelling of detergents , including fragrance allergens,  
ç) The information that manufacturers must hold at the disposal of the Ministry  and medical 
personnel;  
d) Limitations on the content of phosphates and other phosphorus compounds in consumer 
laundry detergents and consumer automatic dishwasher detergents.  
Basis  
ARTICLE 3  – (1) This Regulation has been introduced  relying  on Law of Preparation and 
Implementation of Technical Legislation for Products numbered 4703 and dated 29/6/2001, 
Decree Law about the Organization and Assignment of Ministry of Customs and Trade 
numbered 640 and dated 3/6/2011, Regulation on Market Surveilla nce which entered into force 
by the Council of Ministers Decision numbered 2001/3529 and dated 13/11/2001 and Decision 
numbered 97/9196 and dated 15/1/1997 on Identification of the Bodies That Will Draw Up the 
Technical Legislation for Increasing the Expor ts of Turkish Products.  
Definitions  
ARTICLE 4  – (1) For the implementation of this Regulation:  
a) ‘Ministry ’ means Ministry of Customs and Trade;  
b) ‘Primary biodegradation’ means the structural change of a surfactant by micro -organisms 
resulting in the loss of its surface -active properties due to the degradation of the parent 
substance and consequential loss of the surface -active property as measured by  test methods 
listed in Annex 2 . 
c) ‘Detergent’ means , except for the cleaning of the human body, any substance or  mixture  
containing soaps and/or other surfactants intended for washing and cleaning processes  that may 
be in any form like liquid, powder, paste, bar, cake, moulded piece, shape, etc. and marketed 
for or used in household, or institutional or industrial purposes.  
Other products to be considered as detergents are:  
1) ‘Auxiliary washing mixture’, intended for soaking (pre -washing), rinsing or bleaching 
clothes, household linen, etc.;  
2) ‘Laundry fabric -softener’, intended to modify the feel of fabrics in processes which are to 
complement the washing of fabrics;  
3) ‘Cleaning mixture’, intended for domestic all purposes cleaners and/or other cleaning of 
surfaces (e.g.: materials, products, machinery, mechanical appliances, means of transport and 
associated equipment, instruments, apparatus, etc.);  
4) ‘Other cleaning and washing mixtures’, intended for any other washing and cleaning 
processes.  
ç) ‘Industrial and institutional detergent’ means a detergent for washing and cleaning outside 
the domestic  sphere, carried out by specialised personnel using specific products.  
d) ‘Manufacturer’ means the natural or legal person responsible for placing a detergent or a 
surfactant for a detergent on the market; in particular, a producer, an importer, a packager  working for his own account, or any person changing the characteristics of a detergent or of a 
surfactant for a detergent, or creating or changing the labelling thereof,  
e) ‘Mixture’ means a mixture or solution composed of two  or more substances,  
f) ‘Comm ission’ means the European Commission;  
g) ‘Substance’ means chemical elements and their compounds in the natural state or obtained 
by any production process, including any additive necessary to preserve the stability of the 
products and any impurity derivi ng from the process used, but excluding any solvent which 
may be separated without affecting the stability of the substance or changing its composition,  
ğ)‘Ultimate aerobic biodegradation’ means the level of biodegradation achieved when the 
surfactant is totally used by micro -organisms in the presence of oxygen resulting in its 
breakdown to carbon dioxide, water and mineral salts of any other elements pres ent 
(mineralisation), as measured by test methods listed in Annex 3, and new microbial cellular 
constituents (biomass),  
h) ‘Making available on the market’ means any supply for distribution, consumption or use on 
the market in the course of a commercial a ctivity, whether in return for payment or free of 
charge,  
ı) ‘Placing on the market’ means the first making  available on the market,  
i) ‘Medical personnel’ means a registered medical practitioner, or a person working under the 
direction of a registered med ical practitioner, acting to provide patient care, make a diagnosis 
or administer treatment, and who is bound by professional confidentiality,  
j) ‘Cleaning’ means the process by which an undesirable deposit is dislodged from a substrate 
or from within a su bstrate and brought into a state of solution or dispersion,  
k) ‘Consumer laundry detergent’ means a detergent for laundry placed on the market for use by 
non-professionals, including in public laundrettes,  
l) ‘Consumer automatic dishwasher detergent’ means  a detergent placed on the market for use 
in automatic dishwashers by non -professionals,  
m) ‘Washing’ means the cleaning of laundry, fabrics, dishes and other hard surfaces,  
n) ‘Surfactant’ means any organic substance and/or  mixture  used in detergents, which has 
surface -active properties and which consists of one or more hydrophilic and one or more 
hydrophobic groups of such a nature and size that it is capable of reducing the surface tension 
of water, and of forming spreading or adsorption monolayers at the water -air interface, and of 
forming emulsions and/or microemulsions and/or micelles, and of adsorption at water -solid 
interfaces.  
The placing o n the market  
ARTICLE 5  – (1) When placed on the market, detergents and surfactants for detergents shall 
conform with the conditions, characteristics and limits laid down in this Regulation and where 
relevant, with Biocidal Products Regulation  published  in the Official Gazette no. 27449 and 
dated 31/12/2009  and with any other relevant legislation.  
(2) Surfactants that are also active substances within the meaning of Biocidal Products 
Regulation and that are used as disinfectants are exempt from the provisions of Annexes 2, 3, 
4 and 8 of this Regulation provided:  
a) They are listed in List-I or List-IA of Biocidal Products Regulation , or 
b) They are constituents of biocidal products authorised under Article 15  of Biocidal Products 
Regulation.  
(3) Surfactants referred to in paragraph (2) are deemed to be disinfectants . Detergents of which 
they are ingredients are subject to the labelling provisions for disinfectants of Annex 7/A. 
(4) Manufacturers of detergents and/or of surfactants for detergents shall be established within 
Turkey.  
(5) Manufacturers shall be responsible for the conformity of detergents and/or of surfactants for 
detergents with the provisions of this Regulation and its Annexes.  Limitations based on the biodegradability of surfactant s 
ARTICLE 6  – (1) Under this Regulation, surfactants and detergents containing surfactants that 
meet the criteria for ultimate aerobic biodegradation as laid down in Annex 3 may be placed on 
the market without further limitations relating to biodegradabili ty. 
(2) If a detergent contains surfactants for which the level of ultimate aerobic biodegradation is 
lower than that stipulated in Annex 3, manufacturers of industrial or institutional detergents 
containing surfactants, and/or of surfactants for industria l or institutional detergents, may ask 
for derogation. Requests for derogation shall be made and decided in accordance with Articles 
8, 9 and 12.  
(3) The level of primary biodegradability shall be measured for all surfactants in detergents 
failing ultimate  aerobic biodegradation tests. Detergent surfactants, for which the level of 
primary biodegradability is lower than that stipulated in Annex 2, shall not be granted 
derogation.  
(4) NTA (Nitrilotriacetic acid) and its salts shall not be used in detergents.  
Limitations on the content of phosphates and of other phosphorus compounds  
ARTICLE 7  – (1) Detergents listed in Annex 6a that do not comply with the limitations on 
the content of phosphates and of other phosphorus compounds laid down in that Annex shall 
not be placed on the market from the dates set out therein.  
Granting of derogation  
ARTICLE 8  – (1) The request by a manufacturer for derogation shall be made by sending a 
written application to the Ministry, referred to in Article 11(1) providing evidence relating to 
the criteria mentioned under Article 9(1). 
(2) Applications shall include a technical file supplying all the information and justifications 
necessary for evaluating the safety aspects related to the specific use of surfac tants in detergents 
failing to comply with the biodegradability limits, as set out in Annex 3. Furthermore,  
a) In addition to the results of tests stipulated in Annex 3, the technical file shall include 
information and results of tests, as stipulated in An nexes 2 and 4. 
b) The tests laid down in Annex 4, point 4 shall be carried out on the  basis of a tiered approach. 
This tiered approach will be defined in a technical guidance document and be published in 
Ministry’s website.  This guidance document will also  specify, where appropriate, those tests 
for which the principles of good laboratory practice should be applied.  
(3) The Ministry receiving applications for derogation in accordance with paragraphs 1 and 2, 
shall examine the requests, evaluate their compli ance with the conditions for derogation. While 
evaluating application for a derogation,  
a) Ministry deems it necessary for the evaluation of the risk which may be caused by a substance 
and/or a mixture, it shall, within three months of receiving the appli cation, ask for further 
information, verification and/or confirmatory tests concerning these substances and/or mixtures 
or their transformation products, of which they have been notified or have received information 
under this Regulation.  The time period f or the evaluation of the dossier by the Ministry will 
start only after the dossier is completed with the additional information. If the requested 
information is not provided within 12 months to the Ministry, the application shall be 
considered incomplete a nd thus invalid. In such a case Article 9(2) shall not apply.  
b) If further information on metabolites is sought, stepwise testing strategies should be 
employed to ensure maximum use of in -vitro and other non -animal test methods.  
(4) On the basis of, in pa rticular, the evaluation carried, the Ministry may take its decision 
within 12 months. If necessary, before granting such derogation the Ministry shall evaluate 
further the matters indicated in paragraph 3 of this Article.  
(5) Such derogations may allow, l imit or severely restrict the placing on the market and the use 
of surfactants as ingredients in detergents, depending on the results of the complementary risk 
assessment, as defined in Annex 4. They may include a phase -out period for placing on the market  and the use of surfactants as ingredients in detergents.  The Ministry may review a 
derogation as soon as information comes to light which would justify a significant revision of 
the technical file that was included in the application for derogation. For this purpose, the 
manufacturer shall, upon request, supply to the Ministry a technical file that has been updated 
regarding the items mentioned in Annex 4, point 2. On the basis of this updated information, 
the Ministry may decide to prolong, modify or ter minate the derogation. In this case, paragraphs 
1 to 4 and 6 of this Article and Article 9 shall apply mutatis mutandis.  
(6) The Ministry shall publish the list of surfactants that have obtained derogation, with the 
corresponding conditions or limitations of use, as provided in Annex 5 and shall notify the 
Commission through the Ministry of Economy.  
Conditions for granting a derogation  
ARTICLE 9 – (1) The Ministry considers the request for a derogation on the basis of the 
following criteria:  
a) Use in low -dispersive applications, rather than in wide -dispersive applications,  
b) Use in specific industrial and/or institutional applications only;  
c) The risk to the environment or to health posed by the volume of sales and the pattern of use 
throughout the Turkey is small compared to the socio -economic benefits, including food safety 
and hygiene standards.  
(2) As long as the Ministry has not decided on a request for derogation, the placing on the 
market and use of the surfactant in question may be maintained , provided the manufacturer can 
show that the surfactant was already in use at the date of entry into force of this Regulation and 
that the request for derogation was made within two years from that date.  
(3) If the Ministry refuses to grant a derogation,  it may set a transitional period during which 
the placing on the market and use of the surfactant in question shall be phased out. This 
transitional period shall not exceed two years from the date of the Ministry’s decision.  
(4) The Ministry shall publish in Annex 6 the list of surfactants that have been identified as not 
complying with this Regulation and shall notify the Commission through the Ministry of 
Economy.  
Testing of surfactants  
ARTICLE 10 – (1) All tests referred to in Articles 5 and 6 and in Annexes 2, 3, 4 and 8 shall 
be conducted in compliance with the standards mentioned in Annex 1/1 and in accordance with 
testing requirements under Regulation on Test Methods for Determination of the Physico -
chemical, Toxicological and Ecotoxicological Properties of the Substances and Mixtures  
published in the Official Gazette no. 28848 and dated 11/12/2013.  For this purpose, it is 
sufficient to apply either the TS EN ISO/IEC standard or the principles  of good laboratory 
practice, except for those tests for which the principles of good laboratory practice have been 
made mandatory.  In cases where surfactants are used in detergents which were placed on the 
market before the entry into force of the above s tandard, existing tests that were performed 
using the best scientific knowledge available, and that were performed to a standard comparable 
to those of the standards mentioned in Annex 1 may be accepted on a case -by-case basis. The 
manufacturer or the Mini stry may submit to the Commission through the Ministry of Economy 
any case over which there is doubt or dispute.  
Determination of authorized institutions  
ARTICLE 11 – (1) Ministry is responsible for communicating and exchanging information 
relating to the management of this Regulation. Ministry shall provide necessary information to 
the Commission through the Ministry of Economy.  
(2) Ministry shall notify the Commission th rough the Ministry of Economy the list of approved 
laboratories, w ith their full name and address  that are competent and authorised to carry out the 
tests required by this Regulation.  Laboratories that demonstrate the competence according to 
the standard T S EN ISO /IEC 17025 mentioned in Annex 1/ 1 or Article 2 of Regulation on Principles of Good Laboratory Practices, Harmonisation of Test Units and Controlling Good 
Laboratory Practices  published in the Official Gazette no. 27516 and dated 9/3/2010  shall be 
deemed verified laboratory by the Ministry.  
(3) Where the Ministry has grounds for believing that an approved laboratory does not possess 
the competence referred to in paragraph 2, the name of the approved laboratory shall be 
removed from the lis t referred to in paragraph 4.  Ministry shall notify the situation to the 
Commission through the Ministry of Economy.  Article 16 shall apply, except in the case of 
laboratories claiming compliance with the requirements of good laboratory practice, for which  
the non -compliance provisions of Article 7 of Regulation on Principles of Good Laboratory 
Practices, Harmonisation of Test Units and Controlling Good Laboratory Practices  shall apply.  
(4) Lists  of approved laboratories mentioned in paragraph 2  will be published in the Ministry ’s 
website and notified to the Commission through the Ministry of Economy . 
Information to be provided by manufacturers  
ARTICLE 12 – (1) Without prejudice to the obligation of manuf acturers and importers placing  
on the market mixtures which are determined to be dangerous due to their health effects and 
physico -chemical properties within the scope of this Regulation, to provide detailed 
information on the chemical composition and hazard properties of the mixture t o the Ministry 
of Health National Toxic Center , manufacturers placing on the market the substances and/or 
mixtures covered by this Regulation shall hold following information and documents at the 
disposal of the Ministry : 
a) Information on one or more resu lts of the tests mentioned in Annex 3; 
b) For those surfactants failing to pass tests mentioned in Annex 3, and for which a request for 
derogation was made as referred to in Article 8: 
1) A technical file on results of the tests mentioned in Annex 2, 
2) A technical file on results of the tests and information mentioned in Annex 4 . 
(2) Whenever substances and/or  mixtures  covered by this Regulation are placed on the market, 
the manufacturer shall be responsible for the correct performance of the relevant tes ts 
mentioned above. He shall also have available documentation on the testing carried out to 
demonstrate compliance with this Regulation, and to show that he is allowed to benefit from 
the property rights concerning the test results, other than for those t est results already in the 
public domain.  
(3) Manufact urers placing on the market the mixtures  covered by this Regulation shall, upon 
request, make available without delay and free of charge, to any medical personnel, an 
ingredient data sheet as stipulated in Annex 7/ C. This is without prejudice to the right of a  public 
authority  to request that such a datasheet be made available to a specific public body to which 
the Ministry  has assigned the task of providing this information to medical personnel.  The 
information contained in the datasheet shall be kept confidential by the specific public body 
and by the medical personnel, and shall be used only for medical purposes.  
Control measures  
ARTICLE 13 – (1) Ministry may apply, as appropriate, all necessary con trol measures to 
detergents placed on the market which ensure the compliance of the product with the provisions 
of this Regulation.  The reference method shall be the test and analytical methods referred to in 
Annex 8. These control measures shall not oblige manufacturers to repeat tests made by 
laboratories fulfilling the conditions indicated in Article 11(2), or to pay for any repeat or 
additional test, provided the initial test has shown compliance of detergents, or s urfactants used 
as ingredients in detergents, with this Regulation.  
(2)  In cases of concern that a test carried out in accordance with the methods listed in Annex 
2, 3, 4 or 8 has produced false positive results, the Ministry  shall verify those results an d take 
the necessary measures.  Ministry shall  notify the case to the Commission through the Ministry 
of Economy.  Labelling  
ARTICLE 14  – (1) This Regulation shall apply  without prejudice to the provisions relating to 
the classification, labelling and packaging of substances and mixtures in Regulation on 
Classification, Labelling and Packaging of Substances and Mixtures  published in the Official 
Gazette no. 28848 and dated 11/12/2013.  
(2) The following information must appear in legible, visible and indelible characters on the 
packaging in which the detergents are put up for sale to the consumer:  
(a) The name and trade n ame of the product;  
(b) The name or trade name or trademark and full address and telephone number of the party 
responsible for placing the product on the market;  
(c) The address, email address, where available, and telephone number from which the 
datasheet  referred to in Article 12(3) can be obtained.  
(3) The information  mentioned in paragraph 2 must appear on all documents accompanying 
detergents transported in bulk.  
(4) The packaging of detergents shall indicate the content, in accordance with the specifications 
provided for in Annex 7/A. It shall also indicate instructions for use and special precautions, if 
required.  
(5) Additionally, the packaging of consumer laundry detergents and consumer automatic 
dishwasher detergents shall bear the information provided for in Annex 7/ B. 
(6) The information stated in the above paragraphs must appear on the label in Turkish.  
(7) Paragr aphs 1 to 5 are without prejudice to existing other legislation  according to which 
graphic representations of fruits which may lead the user into error as to the use of liquid 
products, shall not appear on the packaging in which the detergents are put up f or sale to the 
consumer.  
Free movement clause  
ARTICLE 15 – (1) Ministry  shall not prohibit, restrict or impede the making available on the 
market of detergents, and/or of surfactants for detergents, which comply with the requirements 
of this Regulation, on grounds that are dealt with in this Regulation.  
Safeguard clause  
ARTIC LE 16 – (1) Where the Ministry  has justifiable grounds for believing that a specific 
detergent, although complying with the requirements of this Regulation, constitutes a risk to 
the safety or health of humans or of animals or a risk to the environment, it  may take all 
appropriate provisional measures, commensurate with the nature of the risk, in order to ensure 
that the detergent concerned no longer presents that risk, is withdrawn from the market or 
recalled within a reasonable period or its availability is otherwise restricted.  
(2) Ministry shall immediately inform the Commission through the Ministry of Economy, 
giving the reasons for its decision.  
Other provisions  
ARTICLE 1 7 – (1) The proportion of nitric acid contained in household cleaning products 
cannot exceed 20%.  
(2) Where a liquid consumer laundry detergent  or a liquid consumer automatic dishwasher 
detergent  in dosages for single use is contained in a soluble packaging, without prejudice to the 
provisions of Regulation on Classification, Labelling and Packaging of Substances and 
Mixtures, the following additional provisions shall apply:  
a) Liquid consumer lau ndry detergents or liquid consumer  automatic dishwasher detergents  
contained in soluble packaging for single use shall be contained in an outer packaging. The 
outer packaging shall fulfil the requirements of subparagraph  (b) and the soluble packaging 
shall fulfil the requirements of subparagraph (c) . 
b) The outer packaging shall:  
1) Be opaque or obscure etc. so that it impedes the visibility of the product or individual doses;  2) Without prejudice to Article 3 4(3) of Regulation on Classification, Labelling and Packaging 
of Substances and Mixtures , bear the precautionary statement P102 “Keep out of reach of 
children” at a visible place and in a format that attracts attention;  
3) Be an easily reclosable, self -standing container;  
4) Without prejudice to the requirements of Regulation on Classification, Labelling and 
Packaging of Substances and Mixtures  Annex -2 section 3.1, be fitted with a closure that:  
(i) impedes the ability of young children to open the packaging by r equiring coordinated action 
of both hands with a strength that makes it difficult for young children to open it;  
(ii) maintains its functionality under conditions of repeated opening and closing for the entire 
life span of the outer packaging.  
c) The solub le packaging shall:  
1) Contain  an aversive agent in a concentration which is safe, and which elicits oral repulsive 
behaviour within a maximum time of 6 seconds, in case of accidental oral exposure;  
2) Retain  its liquid content for at least 30 seconds when the soluble packaging is placed in water 
at 20 °C;  
3) Resist  mechanical compressive strength of at least 300 N under standard test conditions.  
Harmonized EU legislation  
ARTICLE  18- (1) This Regulation has been prepared in accordance with the Regulation (EC) 
No 648/2004 of the European Parliament and of the Council of 31 March 2004 on detergents 
within the scope of harmonisation of European Union legislation.  
Market  surveillance  
ARTICLE 19  - (1) Ministry shall perform market surveillance of detergents placed on the 
market in accordance with this Regulation with the Law no. 4703 and Regulation on Ministry 
of Customs and Trade Market Surveillance published in the Official Gazette no. 29028 and 
dated 1 2/6/2014 .  
Penalties  
ARTICLE 20 - (1) For the actions contrary to the provisions of this Regulation, the provisions 
laid down in the Law no. 4703 and other relevant legislation shall be applied.  
Repealed legislation  
ARTICLE 21- (1) The Communique on Detergent and surfactants for detergents and 
Communiqué Pertaining to Production, Importation and Notification of Cleaning Products 
Containing Strong Acid or Base  published in the Official Gazette no. 28807 and dated 
31/10/2013 shall be repealed on the effective date of this Regulation.  References made to these 
Communiqués in other legislation shall be deemed to have been made to this Regulation.  
Entry into force  
ARTICLE 22- (1) This Regulation shall enter into force six months after the publication in the 
Official Gazette of Turkey.  
Execution  
ARTICLE 23 - (1) The provisions of this Regulation shall be enforced by the Minister of 
Customs and Trade.  
 
 
 
 
 
 ANNEX 1  
Standards of accreditation, good laboratory practice and animal protection concerning 
the laboratories that are competent and authorised to provide the necessary service for 
checking compliance of detergents with the requirements of this Regulation and its 
Annexes  
 
1.   Standards applicable at the level of the laborato ries:  
TS EN ISO/IEC 17025, General requirements for the competence of testing and calibration 
laboratories;  
 
Provisions of the Regulation on Principles of Good Laboratory Practices, Harmonisation of 
Test Units and Controlling Good Laboratory Practices  published in the Official Gazette ; 
 
Provisions of the Regulation on the Welfare and Protection of Animals Used for Experimental 
and Other Scientific Purposes dated 13/12/2011 and numbered 28141 . 
 
2.   Standards applicable at the level of the accreditation  bodies and good laboratory 
practice monitoring authorities:  
TS EN ISO/IEC 17011 , Conformity assessment - General requirements for accreditation bodies 
accrediting conformity assessment bodies ; 
 
Provisions of the Regulation on Principles of Good Laboratory Practices, Harmonisation of 
Test Units and Controlling Good Laboratory Practices  published in the Official Gazette . 
 
 
 
 
 
 
 
 
 
 
 
  
ANNEX 2  
Primary Biodegradability Test Methods for Surfactants in Detergents  
Primary biodegradability is measured by the determination in biodegraded liquors of the 
remaining level of parent surfactants. This Annex begins with a list of the test -methods common 
to all classes of surfactants, and then lists  under headings A to D the analytical test procedures 
specific to each class of surfactant.  
The pass criterion for primary biodegradability shall be a level of at least 80 %, as measured 
according to the test methods below.  
The reference method for the laboratory testing of surfactants in this Regulation is based on the 
Confirmatory test procedure in the OECD method, described in Annex 8/1. Changes to the 
Confirmatory test procedure are permissible provided that they comply w ith TS EN ISO 11733.  
Test methods  
(1) The OECD method published in the OECD's technical report of 11 June 1976 on the 
‘Proposed Method for the Determination of the Biodegradability of Surfactants in Synthetic 
Detergents’.  
(2) The method in use in France, approved by the ‘arrêté du 24 décembre 1987’ published in 
the Journal officiel de la République française of 30 December 1987, p. 15385, and by the 
standard NF 73 -260 of June 1981, published by the Association française de normalisation 
(AFNOR).  
(3) The me thod in use in Germany, established by the ‘Verordnung über die Abbaubarkeit 
anionischer und nichtionischer grenzflächenaktiver Stoffe in Wasch - und Reinigungsmitteln’ 
of 30 January 1977, published in the Bundesgesetzblatt of 1977, Part I, p. 244, as set o ut in the 
Regulation amending that Regulation of 4 June 1986, published in the Bundesgesetzblatt of 
1986, Part I, p. 851.  
(4) The method in use in the United Kingdom called the ‘Porous Pot Test’ and described in 
Technical Report No 70 (1978) of the Water R esearch Centre.  
(5) The ‘Confirmatory test procedure’ in the OECD method, described in Annex 8/1 (including 
possible changes in operating conditions as proposed in EN ISO 11733). This is also the 
reference method used for the settlement of litigation.  
A.   Analytical Methods for Anionic Surfactants  
The determination of anionic surfactants in the tests shall be done by the Methylene Blue Active 
Substance (MBAS) analysis according to the criteria established in Annex 8/2. For those 
anionic surfactants not reacting to the abovementioned MBAS method, or if it seems more 
appropriate for reasons of efficiency or precision, appropriate specific instrumental analyses 
such as high performance liquid chromatography (HPLC) or gas  chromatography (GC) are to 
be applied. Samples of the pure surfactant of interest shall be provided by the manufacturer to 
the Ministry upon request.  
 
B.   Analytical Methods for Non -Ionic Surfactants  
The determination of non -ionic surfactants in the tests shall be done by the Bismuth Active 
Substance (BiAS) method, according to the analytical procedure established in Annex 8/3. 
For those non -ionic surfactants not reacting to the abovementioned BiAS met hod, or if it seems 
more appropriate for reasons of efficiency or precision, appropriate specific instrumental analyses such as HPLC or GC are to be applied. Samples of the pure surfactant of interest shall 
be provided by the manufacturer to the Ministry  upon request.  
 
C.   Analytical Methods for Cationic Surfactants  
The determination of cationic surfactants in the tests shall be done by the Disulfine Blue Active 
Substance (DBAS) analysis according to the following DBAS procedures  in use in the Federal 
Republic of Germany (1989 - DIN 38 409 — Ausgabe: 1989 -07) 
For those cationic surfactants not reacting to the abovementioned test method, or if it seems 
more appropriate for reasons of efficiency or precision (this must be justified),  appropriate 
specific instrumental analyses such as HPLC or GC are to be applied. Samples of the pure 
surfactant of interest shall be provided by the manufacturer to the Ministry upon request.  
 
D.   Analytical Methods for Amphoteric Surfactants  
The determi nation of amphoteric surfactants in the tests shall be done by analysis following the 
procedures listed below:  
1. If cationics absent:  The method in use in the Federal Republic of Germany, (1989) DIN 38 
409-Teil 20.  
2. Otherwise:  Orange II method (Boiteux,  1984).  
For those amphoteric surfactants not reacting to the abovementioned tests, or if it seems more 
appropriate for reasons of efficiency or precision (this must be justified), appropriate specific 
instrumental analyses such as HPLC or GC are to be appl ied. Samples of the pure surfactant of 
interest shall be provided by the manufacturer to the Ministry upon request.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 ANNEX  3 
Ultimate Biodegradability (Mineralisation) Test Methods for Surfactants in Detergents  
 
A. The reference method for laboratory testing of surfactant ultimate biodegradability in this 
Regulation is based on the TS EN ISO standard 14593 (CO 2 headspace test).  
Surfactants in detergents shall be considered as biodegradable if the level of biodegrada bility 
(mineralisation) measured according to one of the following tests  (These tests are identified as 
the most suitable for surfactants) is at least 60 % within 28 days:  
1.  TS EN ISO Standard 14593 — Water quality — Evaluation of ultimate aerobic 
biodeg radability of organic compounds in aqueous medium — Method by analysis of inorganic 
carbon in sealed vessels (CO 2 headspace test). Pre -adaptation is not to be used. The 10 -day 
window principle is not applied (reference method).  
2. Regulation on Test Methods for Determination of the Physico -chemical, Toxicological and 
Ecotoxicological Properties of the Substances and Mixtures method, C.4-C (carbon dioxide 
(CO 2) Eeolution modified Sturm test): pre -adaptation is not to be used. The 10 -day window 
principle is not applied.  
3. Regulation on Test Methods for Determination of the Physico -chemical, Toxicological and 
Ecotoxicological Properties of the Substances and Mixtures method, C.4 -E (closed Bottle): pre -
adaptation is not to be used. The 10-day window principle is not applied.  
4. Regulation on Test Methods for Determination of the Physico -chemical, Toxicological and 
Ecotoxicological Properties of the Substances and Mixtures method, C.4 -D (manometric 
respirometry): pre -adaptation is not to be used. The 10 -day window principle is not applied.  
5. Regulation on Test Methods for Determination of the Physico -chemical, Toxicological and 
Ecotoxicological Properties of the Substances and Mixtures method, C.4 -F (MITI: Ministry of 
International Trade and Industry, Japan): pre -adaptation is not to be used. The 10 -day window 
principle is not applied.  
6. TS ISO 10708:1997 — Water quality — Evaluation in an aqueous medium of the ultimate 
aerobic biodegradability of organic compounds — Determination of bioc hemical oxygen 
demand in a two -phase closed bottle test. Pre -adaptation is not to be used. The 10 -day window 
principle is not applied.  
 
B. Depending on the physical characteristics of the surfactant, one of the methods listed below 
may be used if appropriately justified  ( The DOC methods could give results on the removal 
and not on the ultimate biodegradation. The m anometric respirometry and the MITI and two -
phase BOD methods would not be appropriate in some cases because the high initial test 
concentration could be inhibitory.  ). It should be noted that the pass criterion of at least 70 % 
of these methods is to be c onsidered as equivalent to the pass criterion of at least 60 % referred 
to in methods listed in point A. The adequacy of the choice of the methods listed below shall 
be decided on a case -by-case confirmation, in accordance with Article 8 of this Regulation . 
1. Regulation on Test Methods for Determination of the Physico -chemical, Toxicological and 
Ecotoxicological Properties of the Substances and Mixtures method, C.4-A (dissolved organic 
carbon DOC die -away): pre -adaptation is not to be used. The 10 -day wind ow principle is not 
applied. The pass criteria for biodegradability measured according to the test shall be at least 
70 % within 28 days.  
2. Regulation on Test Methods for Determination of the Physico -chemical, Toxicological and 
Ecotoxicological Properties  of the Substances and Mixtures  method,  C.4-B (modified OECD 
screening -DOC die -away): pre -adaptation is not to be used. The 10 -day window principle is 
not applied. The pass criteria for biodegradability measured according to the test shall be at 
least 70 %  within 28 days.  ANNEX  4 
Complementary Risk Assessment for Surfactants in Detergents  
 
For those surfactants for which an environmental risk assessment is available in the context of 
Regulation on the Inventory and Control of Chemicals, published in the Official Gazette dated 
26.12.2008 and numbered 27092,  other related legislation and Technical Guidance Documents, 
this risk assessment shall be considered together with the complementa ry risk assessment run 
in the scope of this Regulation.  
 
The complementary risk assessment run in the scope of this Regulation, in case it is likely that 
recalcitrant metabolites are produced, shall be considered in the context of assessments made 
on the b asis of Regulation on the Inventory and Control of Chemicals . This is to be assessed 
case by case and in particular on the basis of the results of the tests referred to in part 3.  
 
The study shall cover the aquatic environmental compartment. Additional inf ormation relating 
to specific risk assessment concerns might be required by the Ministry on a case by case basis. 
Additional information might include other environmental compartments such as sewage 
sludge and soil. A tiered approach shall be adopted concerning the information required for the 
technical file referred to in Articles 8 and 12. The file shall contain at least the information 
described under points 1, 2 and 3 below.  
 
However, to minimise testing, and especially to avoid unnecessary animal testing, the additional 
studies listed under point 4.2.2 should be requested only where such i nformation is necessary 
and proportionate. In case of dispute concerning the extent of additional information required , 
Ministry may take a d ecision  about it.   
 
The guidelines included in this annex for the decisions on derogation may be adapted as 
appropr iate on the basis of the accumulated experience  in view of scientific and technological 
developments . 
 
1.   Identity of the surfactant (in accordance with the provisions laid down Regulation on Test 
Methods for Determination of the Physico -chemical, Toxicological and Ecotoxicological 
Properties of the Substances and Mixtures)  
1.1. Name  
1.1.1. Names in the IUPAC nomenclature  
1.1.2.  Other names  
1.1.3.  CAS number and CAS name (if available)  
1.1.4.  Einecs ( European Inventory of Existing Commercial Substances.  ) or Elincs ( European 
List of Notified Chemical Substances. ) numbers (if available)  
1.2. Molecular and structural formula  
1.3. Composition of the surfactant  
2.   Information on the surfactant  
2.1. Quantities of the surfactant used in detergents  
2.2. The information on use patterns given in this section shall be sufficient to allow an 
approximate but realistic estimate of function and environmental exposure to the surfactant as 
associated with its use in detergents. It shall include the following : 
- importance of the application ( societal value),  
- use conditions (release scenario),  
- use volume,  
- availability and suitability of alternatives (performance and economic considerations),  - assessment of relevant environmental information.  
3. Information on the potential recalcitrant metabolites  
Toxicity information on test liquors shall be provided.  If no data are available on residue 
identity, the information referred to in point 4.2.1 may be requested, depending on the potential 
risk, the importance and the quantity of the surfactant used in detergents. In conflicting cases 
concerning this informati on, Ministry may take a decision about it.  
4. Additional studies  
4.1. Biodegradability tests  
4.1.1. Pre -adapted inoculum  
Any of the tests described in Annex 3, may be run with pre adapted inoculum in order to provide 
evidence of the relevance of pre -adapta tion for the surfactant.  
4.1.2. Inherent Biodegradability Tests  
At least one of the tests referred to below shall be included:  
- Regulation on Test Methods for Determination of the Physico -chemical, Toxicological and 
Ecotoxicological Properties of the Subs tances and Mixtures method, C.12 (Modified SCAS 
test),  
- Regulation on Test Methods for Determination of the Physico -chemical, Toxicological and 
Ecotoxicological Properties of the Substances and Mixtures method, C.9 (Zahn -Wellens).  
Failure to pass the inherent biodegradability test would indicate potential for persistency which 
may be considered, in general terms, as sufficient to prohibit the placing on the market of such 
a surfactant except in cases where the criteria set out in Article 9 indicate tha t there is no 
justification for refusing a derogation.  
4.1.3. Activated Sludge Simulation Biodegradability Tests  
The following tests referred below shall be included:  
- Regulation on Test Methods for Determination of the Physico -chemical, Toxicological and  
Ecotoxicological Properties of the Substances and Mixtures method,  C.10 (including possible 
changes in operating conditions as proposed in TS EN ISO 11733).  
Failure to pass the activated sludge simulation biodegradability test would indicate potential for  
the release of the metabolites by sewage treatment, which may be considered, in general terms, 
as evidence of need for a more complete risk assessment.  
4.2. Toxicity testing of biodegradation test liquors  
Toxicity information on test liquors is to be prov ided on:  
4.2.1. Chemical and physical information, such as:  
— identity of the metabolite (and analytical means by which it was obtained);  
— key physical chemical properties (water solubility, Octanol: Water partition coefficient (Log 
Po/w, etc.).  
4.2.2. Ef fects on organisms. Tests to be conducted in compliance with the principles of good 
laboratory practice.  
Fish: The test recommended is that in C.1 of Regulation on Test Methods for Determination of 
the Physico -chemical, Toxicological and Ecotoxicological P roperties of the Substances and 
Mixtures  
Daphnia: The test recommended is that in C.2 of Regulation on Test Methods for Determination 
of the Physico -chemical, Toxicological and Ecotoxicological Properties of the Substances and 
Mixtures  
Algae: The test re commended is that in C.3 of Regulation on Test Methods for Determination 
of the Physico -chemical, Toxicological and Ecotoxicological Properties of the Substances and 
Mixtures  
Bacteria: The test recommended is that in C.11 of Regulation on Test Methods for 
Determination of the Physico -chemical, Toxicological and Ecotoxicological Properties of the 
Substances and Mixtures  4.2.3. Degradation  
Biotic: The test recommended is that in C.5 of Regulation on Test Methods for Determination 
of the Physico -chemical, Toxicological and Ecotoxicological Properties of the Substances and 
Mixtures.   
Abiotic:  The test recommended is that in C.7 of Regulation on Test Methods for Determination 
of the Physico -chemical, Toxicological and Ecotoxicological Properties of the Substa nces and 
Mixtures.  The information to be provided will consider as well the potential of metabolites for 
bio-concentration and their partitioning to the sediment phase.  
Moreover, if some metabolites are suspected for endocrine disrupting activity, it is 
recommended to determine if these have potential to result in adverse affects as soon as 
validated testing schemes to assess such adverse effects are available.  
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 ANNEX 5  
List of Surfactants That Have Obtained a Derogation  
 
By way of derogation granted in a ccordance with Articles 6, 8 and 9, the following detergent 
surfactants, which pass the tests stipulated in Annex 2 , but  which fail t he tests stipulated in 
Annex 3 , may be placed on the market and used subject to the limitations ment ioned below.  
 
Name in the IUPAC 
nomenclature  EC 
Number  CAS 
number  Limitations  
Alcohols, Guerbet, C16 -20, 
ethoxylated, n -butyl ether (7 -
8EO)  None 
(polymer)  147993 -
59-7 May be used for the following 
industrial applications until 27 June 
2019:  
— bottle washing,  
— cleaning -in-place,  
— metal cleaning,  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 ANNEX 6  
List of Banned or Restricted Detergent Surfactants  
 
The following detergent surfactants have been identified as not complying with the provisions 
of this Regulation:  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Name in the IUPAC nomenclature  EC Number  CAS number  Limitations  
        ANNEX 6 a 
Limitations on the Content of Phosphates and  of Other Phosphorus Compounds  
 
Detergent  Limitations  Date as of which the 
limitation applies  
1.  Consumer 
laundry detergents  Shall not be placed on the market if the total content 
of phosphorus is equal to or greater than 0,5 grams in 
the recommended quantity of the detergent to be used 
in the main cycle of the washing process for a 
standard washing machine load as de fined in section 
B of Annex 7 for water of hard water hardness  
— for ‘normally soiled’ fabrics in the case of 
heavy -duty detergents,  
— for ‘lightly soiled’ fabrics in the case of 
detergents for delicate fabrics,  1 year after the date of 
publication of the 
Regulation  
2. Consumer 
automatic 
dishwasher 
detergents  Shall not be placed on the market if the total content 
of phosphorus is equal to or greater than 0,3 grams in 
the standard dosage as d efined in section B of Annex 
7 1 year after the date of 
publication of the 
Regulation  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 ANNEX 7  
Labelling and Ingredient Data Sheet  
 
A.   Labelling of contents  
The following provisions on labelling shall apply to the packaging of detergents sold to the 
general public.  
 
The following weight  percentage ranges:  
— less than 5 %,  
— 5 % or over but less than 15 %,  
— 15 % or over but less than 30 %,  
— 30 % and more,  
shall be used to indicate the content of the constituents listed below where they are added in a 
concentration above 0,2 % by weight:  
— phosphates,  
— phosphonates,  
— anionic surfactants,  
— cationic surfactants,  
— amphoteric surfactants,  
— non-ionic surfactants,  
— oxygen -based bleaching agents,  
— chlorine -based bleaching agents,  
— EDTA and salts thereof,  
— phenols and halogenated phenols,  
— paradichlorobenzene,  
— aromatic hydrocarbons,  
— aliphatic hydrocarbons,  
— halogenated hydrocarbons,  
— soap,  
— zeolites,  
— polycarboxylates.  
 
The following classes of constituent, if added, shall be listed irrespective of their concentration:  
— enzymes,  
— disinfectants,  
— optical brighteners,  
— perfumes.  
 
If added, preservation agents shall be listed, irrespective of their concentration, using where 
possible the common nomenclature established under Cosmetic Products  Regulation published 
in the Official Gazette no. 25823  and dated 23/5/2005 . 
If adde d at concentrations exceeding 0, 01 % by weight, the allergenic fragrances that appear on 
the list of substances in Annex III of Cosmetic Products Regulation , shall be listed using the 
nomenclature of that Regulation , as shall any other allergenic fragrances that are subsequently 
added to Annex III of Cosmetic Products Regulation by adaptation of that Annex to technical 
progress.  
The website address, from whic h the list of ingredients mentioned in section D of Annex 7 can 
be obtained, shall be given on the packaging.  For detergents intended to be used in the industrial and institutional sector, and not made 
available to members of the general public, the abovem entioned requirements do not have to 
be fulfilled if the equivalent information is provided by means of technical data sheets, safety 
data sheets, or i n a similar appropriate manner.  
 
B. Labelling of dosage information  
As prescribed in Article 14 (5), the following provisions on labelling shall apply to the 
packaging of detergents sold to the general public.  
Consumer laundry detergents  
The packaging of detergents sold to the general public intended to be used as laundry detergents 
shall bear the following information:  
- the recommended quantities and/or dosage instructions expressed in millilitres or grams 
appropriate to a standard washing machine load, for soft, medium and hard water hardness 
levels and making provision for one or two cycle washing process es, 
- for heavy -duty detergents, the number of standard washing machine loads of ‘normally 
soiled’ fabrics, and, for detergents for delicate fabrics, the number of standard washing machine 
loads of ‘lightly soiled’ fabrics, that can be washed with the cont ents of the package using water 
of medium hardness, corresponding to 2,5 millimoles CaCO 3/l, 
- the capacity of any measuring cup, if provided, shall be indicated in millilitres or grams, and 
markings shall be provided to indicate the dose of detergent appr opriate for a standard washing 
machine load for soft, medium and hard water hardness levels,  
The standard washing machine loads are 4,5 kg dry fabric for heavy -duty detergents and 2,5 kg 
dry fabric for light -duty detergents. A detergent shall be considered  to be a heavy -duty detergent 
unless the claims of the manufacturer predominantly promotes fabric care, i.e. low temperature 
wash, delicate fibres and colours.  
Consumer automatic dishwasher detergents  
The packaging of detergents sold to the general public intended to be used as automatic 
dishwasher detergents shall bear the following information:  
- the standard dosage expressed in grams or ml or number of tablets for the main washing 
cycle for normally soiled tableware in a fully loaded 12 place settings dishwasher, making 
provisions, where relevant, for soft, medium, and hard water hardness.  
 
C.   Ingredient data sheet  
The following provisions shall apply to the listing of ingredients on the data sheet referred to 
in Article 12(3). 
 
The data sheet shall list the name of the detergent and that of the manufacturer.  
 
All ingredients shall be listed; in order of decreasing abundance by weight, and the list shall be 
sub-divided into the following weight percentage ranges:  
 
— 10 % or more,  
— 1 % or over, but le ss than 10 %,  
— 0,1 % or over, but less than 1 %,  
— less than 0,1 %.  Impurities shall not be considered to be ingredients.  
‘Ingredient’ means any chemical substance, of synthetic or natural origin, intentionally included 
in the composition of a detergent. For the purpose of this Annex, a perfume, an essential oil, or 
a colouring agent shall be considered to be a single ingredient and none of the substances that 
they contain shall be listed . But, allergenic fragrance substances that appear on the list of 
substances in Annex III of Cosmetic Products Regulation, if the total concentration of the 
allergenic fragrance substance in the detergent exceeds the limit mentioned in section A.  
The common chemi cal name or IUPAC  (International Union of Pure and Applied Chemistry ) 
name and, where available, the INCI  (International Nomenclature Cosmetic Ingredient ) name, 
the CAS number, and the European Pharmacopoeia name, shall be given for each ingredient.  
D.   Publication of the list of ingredients  
Manufacturers shall make available on a website the ingredient data sheet mentioned above 
except for the following:  
— information on weight percentage ranges is not required  
— CAS numbers are not required  
— the ingredient names shall be given in INCI nomenclature, or where this is not available, the 
European Pharmacopoeia name, shall be given. If neither name is available, the common 
chemical name or IUPAC name shall be used instead. For a perfume the word ‘parfu m’ shall 
be used and for a colouring agent, the word ‘colorant’. A perfume, an essential oil, or a 
colouring agent shall be considered to be a single ingredient and none of the substances th at 
they contain shall be listed. But, allergenic fragrance substan ces that appear on the list of 
substances in Annex III of Cosmetic Products Regulation,  if the total concentration of the 
allergenic fragrance substance in the detergent exceeds the limit mentioned in section A.  
Access to the website shall not be subject to any restriction or condition and the content of the 
website shall be kept up to date. The website shall include a link to the Commission Pharmacos 
website or to any other suitable website that provides a table of correspondence between INCI 
names, Europ ean Pharmacopoeia names, and CAS numbers.  
This obligation shall not apply to industrial or institutional detergents, or to surfactants for 
industrial or institutional detergents, for which a technical data sheet or safety data sheet is 
available.  
 
 
 
 
 
 
 
 
 
 ANNEX 8  
Test Methods and Analytical Methods  
The following test and analytical methods apply to control procedures for detergents on the 
market carried out by Ministry : 
 
1.   Reference method (confirmatory test)  
 
1.1.   Definition  
This method describes a laboratory model of the activated sludge + secondary settler which is 
designed to simulate municipal sewage treatment. Improved state -of-the-art operating 
conditions can be applied to this test method as described in TS EN ISO 11733 . 
 
1.2.   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 sewage 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 twenty -four litres. Pump 
B must provide a constant flow o f synthetic sewage to the aeration vessel; this vessel, during 
normal operation, contains three 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 shall be 
monitored by means of a flow meter H.  
 
1.3.   Synthetic sewage  
A synthetic sewage is employed for the test. Dissolve in each litre of tap water:  
- 160 mg peptone;  
- 110 mg meat extract;  
- 30 mg urea, CO(NH 2)2; 
- 7 mg sodium chloride, NaCl;  
- 4 mg calcium chloride, CaCl 2.2H 2O; 
- 2 mg magnesium sulphate, MgSO 4.7H 2O; 
- 28 mg of di -potassium hydrogen phosphate, K 2HPO 4; 
- and 10 ± 1 mg of the surfactant.  
 
The synthetic sewage is freshly prepared daily.  
 
1.4.   Preparation of samples  
Uncompounded surfactants are examined in the  original state. Active content of surfactant 
samples must be determined in order to prepare the synthetic sewage (1.3).  
 
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 three 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 G, air -lift E and dosing device B in operation. The synthetic sewage must pass 
through the aeration vessel C at a rate of one litre per  hour; this gives a mean retention time of 
three hours.   
The rate of aeration should be so regulated that the contents of vessel C are kept constantly in 
suspension and the dissolved oxygen content is at least 2 mg/l. Foaming must be prevented by 
appropria te means. Anti -foaming agents that inhibit the activated sludge or contain surfactants 
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. Sludg e which has accumulated 
around the top of the aeration vessel C, in the base of the settling vessel D, or in the circulation 
circuit must be returned to the circulation at least once each day by brushing or some other 
appropriate means. When the sludge fai ls to settle, its settleability may be increased by the 
addition of 2 ml portions of a 5 % solution of ferric chloride, repeated as necessary.  
 
The effluent from the settling vessel D is accumulated in vessel F for twenty -four hours, 
following which a samp le is taken after thorough mixing. Vessel F must then be carefully 
cleaned.  
 
1.6.   Checking measuring equipment  
The surfactant content (in mg/l) of the synthetic sewage is determined immediately before use.  
 
The surfactant content (in mg/l) of the effluent collected over twenty -four 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/l surfactant . 
 
As a check on the efficiency of the process, the chemical oxygen demand (COD) or the 
dissolved organic carbon (DOC) of the glass fibre filtered effluent accumulated in vessel F and 
of the filtered synthetic sewage in vessel A is measured at least twice per week.  
 
The reduction in COD or DOC should level off when a roughly regular daily surfactant 
degradation is obtained at the end of the running -in period shown in Figure 3.  
 
The content of dry matter in the activated sludge contai ned in the aeration vessel should be 
determined twice a week in g/l. If it is more than 2,5 g/l, the excess activated sludge must be 
discarded.  
 
The degradation test is performed at room temperature; this should be steady and kept between 
19-24 ° C.  
 
1.7.   Calculation of biodegradability  
The percentage degradation of surfactant must be calculated every day on the basis of the 
surfactant content in mg/l of the synthetic sewage and of the corresponding effluent 
accumulated in vessel F.  
 
The degradability values thus obtained should be presented graphically as in Figure 3.  
 
The degradability of the surfactant should be calculated as the arithmetic mean of the values 
obtained over the twenty -one days that follow the running -in and acclimatisation period, dur ing 
which degradation has been regular and the operation of the plant trouble -free. In any event the 
duration of the running -in period should not exceed six weeks.  
The daily degradation values are calculated to the nearest 0,1 % but the final result is giv en to 
the nearest whole number.   
In some cases it may be permissible to reduce the frequency of sampling but at least fourteen 
results collected over the twenty -one days which follow the running -in period should be used 
in calculating the average.  
 
2.   Determination of anionic surfactants in biodegradability tests  
 
2.1.   Principle  
The method is based on the fact that the cationic dye methylene blue forms blue salts with 
anionic surfactants (MBAS), which can be extracted with chloroform. To eliminate 
interference, the extraction is first effected from alkaline solution and the extract is then shaken 
with acidic methylene blue solution. The absorbency of the separated organic phase is measured 
photometrically at the wavelength of maximum absorption of 6 50 nm.  
 
2.2.   Reagents and equipment  
2.2.1.  Buffer solution pH 10  
Dissolve 24 g sodium bicarbonate, NaHCO 3 AR, and 27 g anhydrous sodium carbonate 
(Na 2CO 3) AR in deionised water and dilute to 1  000 ml.  
 
2.2.2.  Neutral methylene blue solution  
Dissolve 0,35  g methylene blue AR in deionised water and dilute to 1  000 ml. Prepare the 
solution at least twenty -four hours before use. The absorbency of the blank chloroform phase, 
measured against chloroform must not exceed 0,015 per 1 cm of layer thickness at 650 n m. 
 
2.2.3.  Acidic methylene blue solution  
Dissolve 0,35 g methylene blue AR in 500 ml deionised water and mix with 6,5 ml H 2SO 4 (d = 
1,84 g/ml). Dilute to 1  000 ml with deionised water. Prepare the solution at least twenty -four 
hours before use. The absorb ency of the blank chloroform phase, measured against chloroform 
must not exceed 0,015 per 1 cm of layer thickness at 650 nm.  
 
2.2.4. Chloroform (trichloromethane) AR freshly distilled  
 
2.2.5. Dodecyl benzene sulphonic acid methyl ester  
 
2.2.6. Ethanolic potassium hydroxide solution, KOH 0,1 M  
 
2.2.7. Ethanol pure, C 2H5OH 
 
2.2.8. Sulphuric acid, H2SO4 0,5 M  
 
2.2.9. Phenolphthalein solution  
Dissolve 1 g phenolphthalein in 50 ml ethanol and add 50 ml deionised water while stirring 
continuously. Filter off any precipitate obtained.  
 
2.2.10. Methanolic hydrochloric acid: 250 ml hydrochloric acid AR and 750 ml methanol  
 
2.2.11. Separating funnel, 250 ml  
 
2.2.12. Graduated flask, 50 ml  
 2.2.13. Graduated flask, 500 ml  
 
2.2.14. Graduated flask, 1  000 ml  
 
2.2.15.  Round -bottomed flask with ground glass stopper and reflux condenser, 250 ml; boiling 
granules  
 
2.2.16. pH meter  
 
2.2.17. Photometer for measurements at 650 nm, with 1 to 5 cm cells  
 
2.2.18. Qualitative grade filter paper  
 
2.3. Procedure   
The samples for analysis must not be taken through a layer of foam.  
 
After thorough cleaning with water, the equipment used for the analysis must be thoroughly 
rinsed with methanolic hydrochloric acid (2.2.10) 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.  
 
Place a measured volume of the sample, neutralised if necessary, into a 250 ml separating funnel 
(2.2.11). The volume of sample  should contain between 20 and 150 g 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 (2.2.1), 5 ml of neutral 
methylene b lue solution (2.2.2) and 15 ml of chloroform (2.2.4). Shake the mixture uniformly 
and not too vigorously for one minute. After phase separation, run the chloroform layer into a 
second separating funnel, containing 110 ml of deionised water and 5 ml of acid ic methylene 
blue solution (2.2.3). Shake the mixture for one minute. Pass the chloroform layer through a 
cotton -wool filter previously cleaned and wetted with chloroform into a graduated flask 
(2.2.12).  
 
Extract the alkaline and acid solutions three times, using 10 ml of chloroform for the second 
and third extractions. Filter the combined chloroform extracts through the same cotton wool 
filter and dilute to the mark in the 50 ml flask (2.2.12) with chloroform used for rewashing the 
cotton wool. Measur e the absorbency of the chloroform solution with a photometer at 650 nm 
in 1 to 5 cm cells against chloroform. Run a blank determination through the whole procedure.  
 
2.4.   Calibration curve  
Prepare a calibration solution from the standard substance dodec ylbenzene sulphonic acid 
methyl ester (tetrapropylene type mol. wt. 340) after saponification into the potassium salt. The 
MBAS is calculated as sodium dodecyl benzene sulphonate (mol. wt. 348).  
 
From a weighing pipette, weigh 400 to 450 mg of dodecyl -benzene -sulphonic -acid-methyl -
ester (2.2.5) to the nearest 0,1 mg in a round -bottomed flask and add 50 ml of ethanolic 
potassium hydroxide solution (2.2.6) and some boiling granules. After mounting th e reflux 
condenser, boil for one 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 phenolphthalein until it beco mes colourless. Transfer this solution 
to a 1  000 ml graduated flask (2.2.14), 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 (2.2.13), dilute to the mark with deionised water and mix.  
 
This standard solution contains:  
 
where E is the sample weight in mg.  
 
To establish the calibration curve, withdraw 1, 2, 4, 6, 8 ml portions of the standard solution 
and dilute each to 100 ml with deionis ed water. Then proceed as stated under item 2.3 including 
a blank determination.  
 
2.5.   Calculation of results  
The amount of anionic surfactant (MBAS) in the sample is read from the calibration curve (2.4). 
The MBAS content of the sample is given by:  
 
where: V = ml volume of the sample used.  
Express the results as sodium dodecylbenzene sulphonate (MW 348).  
 
2.6.   Expression of results  
Express the results as MBAS mg/l to the nearest 0,1.  
 
3.   Determination of non -ionic surfactants in biodegradation test liquors  
 
3.1.   Principle  
Surface active agents are concentrated and isolated by gas stripping. In the sample used, the 
quantity of non -ionic surfactant should be in the range 250 -800 g.  
 
The stripped surfactant is dissolved in ethyl acetate.  
 
After phase separation and evaporation of the solvent, the non -ionic surfactant is precipitated 
in aqueous solution with modified Dragendorff reagent (KBiI 4 + BaCl 2 + glacial acetic acid).  
 
The precipitate is filtered, washed with glacial acetic acid and dissolved in ammonium tartrate 
solution. The bismuth in the solution is titrated potentiometrically with 
pyrrolidinedithiocarbamate solution at pH 4 -5 using a bright platinum indicator electrode and a 
calomel or silver/silver chloride reference electrode. The method is  applicable to non -ionic 
surfactants containing 6 -30 alkylene oxide groups.  
 
The titration result is multiplied by the empirical factor of 54 for conversion to the reference 
substance nonylphenol condensed with 10 mols ethylene oxide (NP 10).  
 
3.2.   Reage nts and Equipment  
Reagents are to be made up in deionised water.  
3.2.1. Pure ethyl acetate, freshly distilled.  
 
3.2.2. Sodium bicarbonate, NaHCO3 AR.  
 
3.2.3. Dilute hydrochloric acid [20 ml concentrated acid (HCl) diluted to 1 000 ml with water]  
 
3.2.4. Methanol AR, freshly distilled, stored in a glass bottle.  
 
3.2.5. Bromocresol purple, 0,1 g in 100 ml methanol.  
 
3.2.6. Precipitating agent: the precipitating agent is a mixture of two volumes of solution A and 
one volume of solution B. The mixture is stored in a brown bottle and can be used for up to one 
week after mixing.  
 
 3.2.6.1. Solution A  
 Dissolve 1,7 g bismuth nitrate, BiONO3.H2O AR, in 20 ml glacial acetic acid, and make 
up to 100 ml with water. Then dissolve 65 g potassium iodide AR in 200 ml  water. Mix these 
two solutions in a 1 000 ml measuring flask, add 200 ml glacial acetic acid (3.2.7) and make up 
to 1 000 ml with water.  
 
 3.2.6.2. Solution B  
 Dissolve 290 g barium chloride, BaCl2.2H2O AR, in 1 000 ml of water.  
  
3.2.7. Glacial acetic ac id 99 -100 % (lower concentrations are unsuitable).  
 
3.2.8. Ammonium tartrate solution: mix 12,4 g tartaric acid AR and 12,4 ml of ammonia 
solution AR (d = 0,910 g/ml) and make up to 1 000 ml with water (or use the equivalent amount 
of ammonium tartrate AR) . 
 
3.2.9. Dilute ammonia solution: 40 ml ammonia solution AR (d = 0,910 g/ml) diluted to 1 000 
ml with water.  
 
3.2.10. Standard acetate buffer: dissolve 40 g solid sodium hydroxide AR, in 500 ml water in a 
beaker and allow to cool. Add 120 ml glacial aceti c acid (3.2.7). Mix thoroughly, cool and 
transfer to a 1 000 ml volumetric flask. Make up to the mark with water.  
 
3.2.11. Pyrrolidinedithiocarbamate solution (known as ‘carbate solution’): dissolve 103 mg 
sodium pyrrolidinedithiocarbamate, C5H8NNaS2.2H2O,  in about 500 ml water, add 10 ml of 
n-amyl alcohol AR and 0,5 g NaHCO3 AR, and make up to 1 000 ml with water.  
 
3.2.12. Copper sulphate solution (for standardisation of 3.2.11).  
 
STOCK SOLUTION  
Mix 1,249 g copper sulphate, CuSO4.5H2O AR, with 50 ml 0,5 M sulphuric acid and make 
up to 1 000 ml with water.  
 
STANDARD SOLUTION  
Mix 50 ml stock solution with 10 ml 0,5 M H2SO4 and make up to 1 000 ml with water.  
 
3.2.13. Sodium chloride AR.  
3.2.14. Gas -stripping apparatus (see Figure 5).  The diameter of the sintered disc must be the same as the internal diameter of the cylinder.  
 
3.2.15. Separating funnel, 250 ml.  
 
3.2.16. Magnetic stirrer with magnet 25 -30 mm.  
 
3.2.17. Gooch crucible, diameter of the perforated base = 25 mm, Type G4.  
 
3.2.18. Circular glass -fibre filter papers, 27 mm diameter with fibre diameter 0,3 -1,5 m.  
 
3.2.19. Two filter flasks with adapters and rubber collars, 500 and 250 ml respectively.  
 
3.2.20. Recording potentiometer fitted with a bright platinum indicator electrode and a calomel 
or silver/silver chloride reference electrode with a 250 mV range, with automatic burette of 20 -
25 ml capacity, or alternative manual equipment.  
 
3.3.   Method   
3.3.1.    Concentration and separation of the surfactant  
Filter the aqueous sample through a qualitative filter paper. Discard the first 100 ml of the 
filtrate.  
 
Into the stripping apparatus, previously rinsed with ethyl acetate, place a measured quantity of 
the sample, such that it contains between 250 -800 g non -ionic surfactant.  
 
To improve the separation add 100 g sodium chloride and 5 g sodium bicarbonate.  
 
If the volume of the sample exceeds 500 ml, add these salts to the stripping apparatus in solid 
form, and dissolve by passing nitrogen or air through.  
 
If a smaller -sized sample is used, dis solve the salts in 400 ml water and then add to the stripping 
apparatus.  
 
Add water to bring the level to the upper stopcock.  
 
Cautiously add 100 ml ethyl acetate on top of the water.  
 
Fill the wash -bottle in the gas -line (nitrogen or air) two -thirds full with ethyl acetate.  
 
Pass a gas stream of 30 -60 l/h through the apparatus; the use of a flowmeter is recommended. 
The rate of aeration must be increased gradually at the beginning. The gas rate must be so 
adjusted that the phases remain noticeably separate  to minimise the mixing of the phases and 
the solution of the ethyl acetate in the water. Stop the gas flow after five minutes.  
 
If there is a reduction of more than 20 % in the volume of the organic phase through solution 
in water, the sublation must be r epeated paying special attention to the rate of gas flow.  
 
Run off the organic phase into a separating funnel. Return any water in the separating funnel 
from the aqueous phase — it should only be a few ml — to the stripping apparatus. Filter the 
ethyl acet ate phase through a dry qualitative filter paper into a 250 ml beaker.  
 Put a further 100 ml ethyl acetate into the stripping apparatus and again pass nitrogen or air 
through for five minutes. Draw off the organic phase into the separating funnel used for the first 
separation, reject the aqueous phase and run the organic phase through the same filter as the 
first ethyl acetate portion. Rinse both the separating funnel and the filter with about 20 ml ethyl 
acetate.  
 
Evaporate the ethyl acetate extract to dry ness using a water -bath (fume cupboard). Direct a 
gentle stream of air over the surface of the solution to accelerate the evaporation.  
 
3.3.2.    Precipitation and filtration  
Dissolve the dry residue from 3.3.1 in 5 ml methanol, add 40 ml water and 0,5 ml dilute HCl 
(3.2.3) and stir the mixture with a magnetic stirrer.  
 
To this solution add 30 ml of precipitating agent (3.2.6) from a measuring cylinder. The 
precipitate forms after repeated stirring. After stirring for ten minutes leave the mixture to stand 
for at least five minutes.  
 
Filter the mixture through a Gooch crucible, the base of which is covered with a glass -fibre 
filter paper. First wash the filter under suction with about 2 ml glacial acetic acid. Then 
thoroughly wash the beaker, magnet, and crucible with glacial acetic acid, of which about 40 -
50 ml is necessary. It is not necessary to quantitatively transfer the precipitate adhering to the 
sides of the beaker, to the filter, because the solution of the precipitate for the titration is returne d 
to the precipitating beaker, and the remaining precipitate will then be dissolved.  
 
3.3.3.    Dissolution of the precipitate  
Dissolve the precipitate in the filter crucible by the addition of hot ammonium tartrate solution 
(about 80 ° C) (3.2.8) in three portions of 10 ml each. Allow each portion to stand in the crucible 
for some minutes before being sucked through the filter into the flask.  
 
Put the contents of the filter flask into the beaker used for the precipitation. Rinse the sides of 
the beaker with  a further 20 ml of tartrate solution to dissolve the rest of the precipitate.  
 
Carefully wash the crucible, adapter and filter flask with 150 -200 ml water, and return the 
rinsing water to the beaker used for the precipitation.  
 
3.3.4.    The titration  
Stir the solution using a magnetic stirrer (3.2.16), add a few drops of bromocresol purple (3.2.5) 
and add the dilute ammonia solution (3.2.9) until the colour turns violet (the solution is initially 
weakly acid from the residue of acetic acid used for rinsing ). 
 
Then add 10 ml standard acetate buffer (3.2.10), immerse the electrodes in the solution, and 
titrate potentiometrically with standard ‘carbate solution’ (3.2.11), the burette tip being 
immersed in the solution.  
 
The titration rate should not exceed 2 m l/min.  
 
The endpoint is the intersection of the tangents to the two branches of the potential curve.  
 
It will be observed occasionally that the inflection in the potential curve becomes flattened; this 
can be eliminated by carefully cleaning the platinum e lectrode (by polishing with emery paper).   
3.3.5.    Blank determinations  
At the same time run a blank determination through the whole procedure with 5 ml methanol 
and 40 ml water, according to the instructions in 3.3.2. The blank titration should be below 1 
ml, otherwise the purity of the reagents (3.2.3, 3.2.7, 3.2.8, 3.2.9, 3.2.10) is suspect, especially 
their content of heavy metals, and they must be replaced. The blank must be taken into account 
in the calculation of the results.  
 
3.3.6.    Control of th e factor of the ‘carbate solution’  
Determine the factor for the carbate solution on the day of use. To do this, titrate 10 ml of the 
copper sulphate solution (3.2.12) with ‘carbate solution’ after the addition of 100 ml water and 
10 ml standard acetate buf fer (3.2.10). If the amount used is a ml, the factor f is:  
 
and all the results of the titration are multiplied by this factor.  
 
3.4.   Calculation of results  
Every non -ionic surfactant has its own factor, depending on its composition, particularly on the  
length of the alkene oxide chain. The concentration of non -ionic surfactant is expressed in 
relation to a standard substance — a nonyl phenol with ten ethylene oxide units (NP 10) — for 
which the conversion factor is 0,054.  
 
Using this factor the amount o f surfactant present in the sample is found expressed as mg of NP 
10 equivalent, as follows:  
 
(b — c) xfx 0,054 = mg non -ionic surfactant as NP 10  
where:  
b = volume of ‘carbate solution’ used by the sample (ml),  
c = volume of ‘carbate solution’ used by the  blank (ml),  
f = factor of the ‘carbate solution’.  
 
3.5.   Expression of results  
Express the results in mg/l as NP 10 to the nearest 0,1.  
 
4.   Preliminary treatment of anionic surfactants to be tested  
 
4.1.   Preliminary notes  
 
4.1.1.    Treatment of samples  
The treatment of anionic surface -active agents and formulated detergents prior to the 
determination of primary biodegradability in the confirmatory test is:  
 
Products  Treatment  
Anionic 
surfactants  None  
Formulated 
detergents  Alcoholic extraction followed by separation of the 
anionic surfactants by ion exchange  
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 biodegradability test.  
 
4.1.2.    Ion-exchange procedure  
Isolation and separation of anionic surface active agents from soap, non -ionic and cationic 
surfactants are required for correct biodegradability tests.  
This is achieved by an ion -exchange technique using a macro -porous exchange resin and 
suitable eluants for fractional elution. Thus soap, anionic and non -ionic surfactants may be 
isolated in one procedure.  
 
4.1.3.    Analytical control  
After homogenising, the concentration 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 for preparing 
fractions for the biodegradability test . 
 
Quantitative extraction is not necessary; however, at least 80 % of the anionic surfactants should 
be extracted. Usually, 90 % or more is obtained.  
 
4.2.   Principle  
From a homogeneous sample (powders, dried pastes and dried liquids) an ethanol extract is 
obtained which contains the surfactants, soap and other alcohol -soluble constituents of the 
synthetic detergent sample.  
 
The ethanol extract is evaporated to dryness, 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 50 ° C. This temperature is necessary to prevent the 
precipitation of any fatty acids which may be present in acidic media.  
 
Any non -ionic surfactants remain in the effluen t. 
 
Soap fatty acids are separated by extraction with ethanol containing CO 2. 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 degr adation test.  
 
Cationic surfactants that might upset the biodegradability test and the analytical procedure are 
eliminated by the cation exchanger placed above the anion exchanger.  
 
4.3.   Chemicals and equipment  
 
4.3.1.  Deionised water  
 
4.3.2.  Ethanol, 95  % (v/v) C 2H5OH (permissible denaturant: methyl ethyl ketone or methanol)  
 
4.3.3.  Isopropanol/water mixture (50/50 v/v):  
— 50 parts by volume isopropanol, CH 3CHOH.CH 3, and  
— 50 parts by volume water (4.3.1)  
 4.3.4.  Solution of carbon dioxide in ethanol (approximately 0,1 % CO 2): using a delivery tube 
with a built -in sinter, pass carbon dioxide, CO 2, through the ethanol (4.3.2) for ten minutes. Use 
fresh solutions only  
 
4.3.5.  Ammonium bicarbonate solution (60/40 v/v): 0,3 mol NH 4HCO 3 in 1 000 ml of an 
isopropanol/water mixture consisting of 60 parts by volume isopropanol and 40 parts by volume 
water (4.3.1)  
 
4.3.6.  Cation exchanger (KAT), strongly acidic, resistant to alcohol (50 -100 mesh)  
 
4.3.7.  Anion exchanger (AAT), macro -porous, Merck Lewatit MP 7080  (70-150 mesh) or 
equivalent  
 
4.3.8.  Hydrochloric acid, 10 % HCl (w/w)  
 
4.3.9.  2 000 ml round -bottomed flask with ground glass stopper and reflux condenser  
 
4.3.10.  90 mm diameter suction filter (heatable) for filter papers  
 
4.3.11.  2 000 ml filter flask  
 
4.3.12.  Exchange columns with heating jacket and tap: inner tube 60 mm in diameter and 450 
mm in height (see Figure 4)  
 
4.3.13.  Water -bath 
 
4.3.14.  Vacuum drying oven  
 
4.3.15.  Thermostat  
 
4.3.16.  Rotary evaporator  
 
4.4.   Preparation of extract and separation of anionic active agents  
 
4.4.1.    Preparation of extract  
The quantity of surfactants necessary for the biodegradation test is about 50 g MBAS.  
 
Normally, the quantity of product to be extracted will not exceed 1  000 g, but it may be 
necessary to extract further quantities of sample. For practical reasons, the quantity of product 
used should in most cases be limited to 5  000 g in preparing extracts for the biodegradation test.  
 
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.  
 
4.4.2.    Isolation of alcohol -soluble constituents  
Add 250 g of the synthetic detergent t o be analysed to 1  250 ml ethanol, heat the mixture to 
boiling point and reflux for one hour with stirring. Pass the hot alcoholic solution through a 
coarse -pored suction filter heated to 50 ° C and filter rapidly. Wash the flask and suction filter 
with ap proximately 200 ml hot ethanol. Collect the filtrate and filter washings in a filter flask.   
In the case of pastes or liquid products to be analysed, make sure that not more than 55 g anionic 
surfactants and 35 g soap are contained in the sample. Evaporate  this weighed sample to 
dryness. Dissolve the residue in 2  000 ml ethanol and proceed as described above. In the case 
of powders of low apparent density (< 300 g/l) it is recommended to increase the ethanol ratio 
in the relation 20:1. Evaporate the ethanol ic 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 5 000 ml isopropanol/water mixture.  
 
4.4.3.   Preparation of ion -exchange columns  
 
CATION -EXCHANGE COLUMN  
Place 600 ml cation -exchange resin (4.3.6) in a 3  000 ml beaker and cover by adding 2  000 ml 
hydrochloric acid (4.3.8). Allow to stand for at least two hours, with occasional stirring.  
 
Decant the acid and transfer the resin into the  column (4.3.12) by means of deionised water. 
The column should contain a glass -wool plug.  
 
Wash the column with deionised water at a rate of 10 -30 ml/min until the eluate is free of 
chloride.  
 
Displace the water with 2  000 ml isopropanol/water mixture (4. 3.3) at a rate of 10 -30 ml/min. 
The exchange column is now ready for operation.  
 
ANION -EXCHANGE COLUMN  
Place 600 ml anion -exchange resin (4.3.7) in a 3  000 ml beaker and cover by adding 2  000 ml 
deionised water.  
 
Allow the resin to swell for at least two h ours.  
 
Transfer the resin into the column by means of deionised water. The column should contain a 
glass -wool plug.  
 
Wash the column with 0,3 M ammonium bicarbonate solution (4.3.5) until free of chloride. 
This requires about 5  000 ml solution. Wash again with 2  000 ml deionised water. Displace the 
water with 2  000 ml isopropanol/water mixture (4.3.3) at a rate of 10 -30 ml/min. The exchange 
column is now in the OH -form and ready for operation.  
 
Ion-exchange procedure  
4.4.4.  Connect the exchange columns so that the cation -exchange column is placed on top of 
the anion -exchange column.  
 
Heat the exchange columns to 50 ° C using thermostatic control.  
 
Heat 5 000 ml of the solution obtained in item 4.4.2 to 60 ° C and pass the solution through the 
exchanger comb ination at a rate of 20 ml/min. Wash the columns with 1 000 ml hot 
isopropanol/water mixture (4.3.3).  
 To obtain the anionic surface active agents (MBAS), disconnect the KAT column. Using 5 000 
ml ethanol/CO2 solution at 50 ° C (4.3.4), elute the soap fatt y acids out of the KAT column. 
Reject the eluate.  
 
Then elute the MBAS out of the AAT column with 5 000 ml ammonium bicarbonate solution 
(4.3.5). Evaporate the eluate to dryness using a steam bath or in a rotary evaporator.  
 
The residue contains the MBAS (as ammonium salt) and possible non -surfactant anionics that 
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. 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 5 ° C.  
 
Regeneration of ion exchange resins  
 
4.4.5.  The cation exchanger is rejected after use.  
 
Passing an additional quantity of ammonium bicarbonate solution (4.3.5) down the column at 
a flow rate of approximately 10 ml/min until the eluate is free from anionic surfactants 
(methylene blue test) regenerates the anion -exchange resin.  
 
Then pass 2 000 ml isopropanol/water mixture (4.3.3) down the anion exchanger to wash. The 
anion exchanger is again ready for operation.  
 
Preliminary treatment of non -ionic surfactants to be tested  
 
5.   Preliminary notes  
 
5.1.   Treatment of samples  
 
5.1.1. The treatment of non -ionic surface -active agents and formulated detergents prior to the 
determination of primary biodegradability in the confirmatory test is:  
 
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 biodegradability test.  
 
 
Products  Treatment  
Non-ionic 
surfactants  None  
Formulated 
detergents  Alcoholic extraction followed by separation of the non -
ionic surfactants by ion exchange  
 
Ion-exchange procedure  
5.1.2.  Isolation and separation of non -ionic surface active agents from soap, anionic and 
cationic surfactants are required for correct biodegradability tests.  This is achieved by an ion exchange technique using a macro -porous exchange resin and 
suitable eluants for fractional elution. Thus soap, anionic and non -ionic surfactants may be 
isolated in one procedure.  
Analytical control  
5.1.3.  After homogenising, the concentration of anionic and non -ionic surfactants in the 
detergent is determined according to the MBAS and BiAS analytical procedure. The soap 
content is determined by a suitable analytical method.  
This analysis of the product is necessary to calculate the quantities required preparing fractions 
for the biodegradability tests.  
Quantitative extracti on is not necessary; however, at least 80 % of the non -ionic surfactants 
should be extracted. Usually, 90 % or more is obtained.  
Principle  
5.2. From a homogeneous sample (powders, dried paste and dried liquids) an ethanol 
extract is obtained which contains  the surfactants, soap and other alcohol -soluble 
constituents of the detergent sample.  
The ethanol extract is evaporated to dryness, dissolved in an isopropanol/water mixture and the 
solution obtained is passed through a strongly acidic cation exchange/mac ro-porous anion 
exchange combination heated to 50 ° C. This temperature is necessary to prevent the 
precipitation of any fatty acids which may be present in acidic media. The non -ionic surfactants 
are obtained from the effluent by evaporation.  
 
Cationic su rfactants, which might upset the degradation test and the analytical procedure, are 
eliminated by the cation exchanger placed above the anion exchanger.  
 
Chemicals and equipment  
 
5.3. Deionised water  
 
5.3.1. Ethanol, C2H5OH 95 % (v/v) (permissible denaturant: methyl -ethyl ketone or methanol)  
 
5.3.2. Isopropanol/water mixture (50/50 v/v):  
 
5.3.3. 50 parts by volume isopropanol, CH3CHOH.CH3, and  
— 50 parts by volume water (5.3.1)  
— Ammonium bicarbonate solution (60/40 v/v):  
 
5.3.4. 0,3 mol NH4HCO3 in 1 000 ml of an isopropanol/water mixture consisting of 60 parts 
by volume isopropanol and 40 parts by volume water (5.3.1)  
 
Cation exchanger (KAT), strongly acidic, resistant to alcohol (50 -100 mesh)  
 
5.3.5. Anion exchanger (AAT), macro -porous, Merck Lewat it MP 7080 (70 -150 mesh) or 
equivalent  
 
5.3.6. Hydrochloric acid, 10 % HCl w/w  
 
5.3.7. 2 000 ml round -bottomed flask with ground glass stopper and reflux condenser   
 
  
5.3.8. 90 mm diameter suction Filter (heatable) for filter papers  
 
5.3.9. 2 000 ml filter f lask 
 
5.3.10. Exchange columns with heating jacket and tap: inner tube 60 mm in diameter and 450 
mm in height (see Figure 4)  
 
5.3.11. Water -bath 
 
5.3.12. Vacuum drying oven  
 
5.3.13. Thermostat  
 
5.3.14. Rotary evaporator  
 
5.3.15. Preparation of extract and separation of non -ionic active agents  
 
5.4. Preparation of extract  
 
5.4.1.  The quantity of surfactant necessary for the degradation test is about 25 g BiAS.  
 
In preparing extracts for the degradation tests, the quantity of product to be used should be 
limited to a maximum of 2 000 g. Therefore it may be necessary to carry out the operation two 
or more times in order to obtain sufficient quantity for the degradation tests.  
 
Experience has shown that there are advantages in using a number of small extract ions rather 
than one large extraction.  
 
Isolation of alcohol -soluble constituents.  
 
5.4.2. Add 250 g of the synthetic detergent to be analysed to 1 250 ml ethanol and heat the 
mixture to boiling point and reflux for one hour with stirring. Pass the hot alcoholic solution 
through a coarse -pored suction filter heated to 50 ° C and filter rapidly. Wash the flask and 
suction filter with approximately 200 ml hot ethanol. Collect the filtrate and filter washings in 
a filter flask.  
 
In the case of pastes or liq uid products to be analysed, make sure that not more than 25 g anionic 
surfactants and 35 g soap are contained in the sample. Evaporate this weighed sample to 
dryness. Dissolve the residue in 500 ml ethanol and proceed as described above.  
 
In the case of p owders of low apparent density (< 300 g/l) it is recommended to increase the 
ethanol ratio in the relation 20:1.  
 
Evaporate the ethanolic filtrate to complete dryness, preferably by means of rotary evaporator. 
Repeat the operation if a greater quantity of extract is required. Dissolve the residue in 5 000 
ml isopropanol/water mixture.  
 
5.4.3. Preparation of ion -exchange columns  
CATION -EXCHANGE COLUMN   Place 600 ml cation -exchange resin (5.3.5) in a 3 000 ml beaker and cover by adding 2 000 ml 
hydrochloric ac id (5.3.7). Allow to stand for at least two hours, with occasional stirring.  
 
Decant the acid and transfer the resin into the column (5.3.11) by means of deionised water. 
The column should contain a glass -wool plug. Wash the column with deionised water at a rate 
of 10 -30 ml/min until the eluate is free of chloride.  
 
Displace the water with 2 000 ml isopropanol/water mixture (5.3.3) at a rate of 10 -30 ml/min. 
The exchange column is now ready for operation.  
 
ANION -EXCHANGE COLUMN  
Place 600 ml anion -exchange r esin (5.3.6) in a beaker and cover by adding 2 000 ml deionised 
water. Allow the resin to swell for at least two hours. Transfer the resin into the column by 
means of deionised water. The column should contain a glass -wool plug.  
 
Wash the column with 0,3 M  ammonium bicarbonate solution (5.3.4) until free of chloride. 
This requires about 5 000 ml solution. Wash again with 2 000 ml deionised water.  
 
Displace the water with 2 000 ml isopropanol/water mixture (5.3.3) at a rate of 10 -30 ml/min. 
The exchange colu mn is now in the OH form and ready for operation.  
 
5.4.4. Ion -exchange procedure  
Connect the exchange columns so that the cation -exchange column is placed on top of the 
anion -exchange column. Heat the exchange columns to 50 ° C using thermostatic control. Heat 
5 000 ml of the solution obtained in item 5.4.2 to 60 ° C and pass the solution through the 
exchanger combination at a rate of 20 ml/min. Wash the columns with 1 000 ml hot 
isopropanol/water mixture (5.3.3).  
 
To obtain the non -ionic surfactants collec t the filtrate and filter washings and evaporate to 
dryness, preferably by means of a rotary evaporator. The residue contains the BiAS. Add 
deionised water until a defined volume is obtained and determine the BiAS content in an 
aliquot. The solution is use d as a standard solution of non -ionic surfactants for the degradation 
test. The solution should be kept at a temperature below 5 ° C.  
 
5.4.5. Regeneration of ion exchange resins  
The cation exchanger is rejected after use.  
 
Passing about 5 000 - 6 000 ml of  ammonium bicarbonate solution (5.3.4) down the column at 
a flow rate of approximately 10 ml/min until the eluate is free from anionic surfactants 
(methylene blue test) regenerates the anion -exchange resin. Then pass 2 000 ml 
isopropanol/water mixture (5.3 .3) down the anion exchanger to wash. The anion exchanger is 
again ready for operation.  
 
  
 
 
 
   
 
Figure 1:  
Activated sludge plant: overviews  
 
 
 
  
  
 
 
 
 
 
 
 
A Storage vessel   
B Dosing device  
C Aeration chamber (three litres 
capacity)  
D Settling vessel  
E Air -lift pump  
F Collector  
G Sintered aerator  
H Air -flow meter  
I Air   
 
Figure 2  
Activated sludge plant: detail  
 (dimensions in millimetres)   
 
 
A Liquid level  
B Hard PVC  
C Glass or waterproof plastic (hard PVC)  
 
 
 
 
 
 
Figure 3  
Calculation of biodegradability — Confirmatory test  
 
 
 
 
A Running -in period  
B Period used for calculation (twenty -one days)  
C Readily biodegradable surfactant  
D Surfactant not readily biodegradable  
E Biodegradation ( %)  
F Time (days)  
 
 
Figure 4  
Heated exchange column  
(dimensions in millimetres)  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 5  
Gas stripping apparatus  
(dimensions in millimetres)