Names in the IUPAC nomenclature

Other names

CAS number and CAS name (if available)

Einecs29 or Elincs30 numbers (if available)

Any of the tests described in Annex III, may be run with pre adapted inoculum in order to provide evidence of the relevance of pre-adaptation for the surfactant.

At least one of the tests referred to below shall be included:

• F112method of EU Regulation 440/2008, Annex, Part C, C.12. (biodegradation – modified SCAS test).

• F113method of EU Regulation 440/2008, Annex, Part C, C.9. (biodegradation – Zahn-Wellens test).

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 6 indicate that there is no justification for refusing a derogation.

The following tests referred below shall be included:

• F114method of EU Regulation 440/2008, Annex, Part C, C.10. (biodegradation – activated sludge simulation tests).

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.

• 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.).

Fish: the test recommended is that in F101EU Regulation 440/2008, Annex, Part C, C.1. (acute toxicity for fish)

Daphnia: the test recommended is that in F102EU Regulation 440/2008, Annex, Part C, C.2. (daphnia SP. acute immobilisation test)

Algae: the test recommended is that in F103EU Regulation 440/2008, Annex, Part C, C.3. (algal inhibition test)

Bacteria: the test recommended is that in F104EU Regulation 440/2008, Annex, Part C, C.11. (biodegradation – activated sludge respiration inhibition)

Biotic: the test recommended is that in F115EU Regulation 440/2008, Annex, Part C, C.5. (degradation – biochemical oxygen demand)

Abiotic: the test recommended is that in F105EU Regulation 440/2008, Annex, Part C, C.7. (degradation – abiotic degradation: hydrolysis as a function of PH). 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.

Dissolve 24 g sodium bicarbonate, NaHCO₃ AR, and 27 g anhydrous sodium carbonate (Na₂CO₃) AR in deionised water and dilute to 1 000 ml.

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 nm.

Dissolve 0,35 g methylene blue AR in 500 ml deionised water and mix with 6,5 ml H₂SO₄ (d = 1,84 g/ml). Dilute to 1 000 ml with deionised water. 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 nm.

Chloroform (trichloromethane) AR freshly distilled

Dodecyl benzene sulphonic acid methyl ester

Ethanolic potassium hydroxide solution, KOH 0,1 M

Ethanol pure, C₂H₅OH

sulphuric acid, H₂SO₄ 0,5 M

Phenolphthalein solution

Dissolve 1 g phenolphthalein in 50 ml ethanol and add 50 ml deionised water while stirring continuously. Filter off any precipitate obtained.

Methanolic hydrochloric acid: 250 ml hydrochloric acid AR and 750 ml methanol

Separating funnel, 250 ml

Graduated flask, 50 ml

Graduated flask, 500 ml

Graduated flask, 1 000 ml

Round-bottomed flask with ground glass stopper and reflux condenser, 250 ml; boiling granules

pH meter

Photometer for measurements at 650 nm, with 1 to 5 cm cells

Qualitative grade filter paper

Pure ethyl acetate, freshly distilled.

Sodium bicarbonate, NaHCO₃ AR.

Dilute hydrochloric acid [20 ml concentrated acid (HCl) diluted to 1 000 ml with water]

Methanol AR, freshly distilled, stored in a glass bottle.

Bromocresol purple, 0,1 g in 100 ml methanol.

Glacial acetic acid 99-100 % (lower concentrations are unsuitable).

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).

Dilute ammonia solution: 40 ml ammonia solution AR (d = 0,910 g/ml) diluted to 1 000 ml with water.

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 acetic acid (3.2.7). Mix thoroughly, cool and transfer to a 1 000 ml volumetric flask. Make up to the mark with water.

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.

Sodium chloride AR.

Gas-stripping apparatus (see Figure 5).

The diameter of the sintered disc must be the same as the internal diameter of the cylinder.

Separating funnel, 250 ml.

Magnetic stirrer with magnet 25-30 mm.

Gooch crucible, diameter of the perforated base = 25 mm, Type G4.

Circular glass-fibre filter papers, 27 mm diameter with fibre diameter 0,3-1,5 m.

Two filter flasks with adapters and rubber collars, 500 and 250 ml respectively.

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.

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, dissolve 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 repeated 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 acetate 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 dryness using a water-bath (fume cupboard). Direct a gentle stream of air over the surface of the solution to accelerate the evaporation.

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 returned to the precipitating beaker, and the remaining precipitate will then be dissolved.

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.

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 ml/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 electrode (by polishing with emery paper).

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.

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 buffer (3.2.10). If the amount used is a ml, the factor f is:

f=10a

and all the results of the titration are multiplied by this factor.

The treatment of anionic 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.

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.

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.

Deionised water

Ethanol, 95 % (v/v) C₂H₅OH (permissible denaturant: methyl ethyl ketone or methanol)

• 50 parts by volume isopropanol, CH₃CHOH.CH₃, and

• 50 parts by volume water (4.3.1)

Solution of carbon dioxide in ethanol (approximately 0,1 % CO₂): using a delivery tube with a built-in sinter, pass carbon dioxide, CO₂, through the ethanol (4.3.2) for ten minutes. Use fresh solutions only

Ammonium bicarbonate solution (60/40 v/v): 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 (4.3.1)

Cation exchanger (KAT), strongly acidic, resistant to alcohol (50-100 mesh)

Anion exchanger (AAT), macro-porous, Merck Lewatit MP 7080 (70-150 mesh) or equivalent

Hydrochloric acid, 10 % HCl (w/w)

2 000 ml round-bottomed flask with ground glass stopper and reflux condenser

90 mm diameter suction filter (heatable) for filter papers

2 000 ml filter flask

Exchange columns with heating jacket and tap: inner tube 60 mm in diameter and 450 mm in height (see Figure 4)

Water-bath

Vacuum drying oven

Thermostat

Rotary evaporator

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.

Add 250 g of the synthetic detergent to 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 approximately 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 ethanolic filtrate to dryness, preferably by means of a rotary evaporator. Repeat the operation if a greater quantity of extract is required. Dissolve the residue in 5 000 ml isopropanol/water mixture.

Preparation of ion-exchange columns

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 combination 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 fatty 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

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

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.

Ion-exchange procedure

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

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 extraction is not necessary; however, at least 80 % of the non-ionic surfactants should be extracted. Usually, 90 % or more is obtained.

Principle

Ethanol, C₂H₅OH 95 % (v/v) (permissible denaturant: methyl-ethyl ketone or methanol)

Isopropanol/water mixture (50/50 v/v):

• 50 parts by volume water (5.3.1)

• Ammonium bicarbonate solution (60/40 v/v):

• Cation exchanger (KAT), strongly acidic, resistant to alcohol (50-100 mesh)

Anion exchanger (AAT), macro-porous, Merck Lewatit MP 7080 (70-150 mesh) or equivalent

Hydrochloric acid, 10 % HCl w/w

2 000 ml round-bottomed flask with ground glass stopper and reflux condenser

90 mm diameter suction Filter (heatable) for filter papers

2 000 ml filter flask

Exchange columns with heating jacket and tap: inner tube 60 mm in diameter and 450 mm in height (see Figure 4)

Water-bath

Vacuum drying oven

Thermostat

Rotary evaporator

Preparation of extract and separation of non-ionic active agents

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 extractions rather than one large extraction.

Isolation of alcohol-soluble constituents

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 liquid 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 powders 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.

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 collect 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 used as a standard solution of non-ionic surfactants for the degradation test. The solution should be kept at a temperature below 5 ° C.

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.