Fatty acids, C12-18 and C18-unsatd., 2-sulfoethyl esters, sodium salts

Biodegradation in water and sediment: simulation tests

Administrative data

Endpoint:

biodegradation in water: sewage treatment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-07-20 to 2009-11-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study has been conducted according to OECD Guideline 303A; however, no claim for GLP compliance is made for this study. However, the study was carried out in the spirit of GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 303 A (Simulation Test - Aerobic Sewage Treatment. A: Activated Sludge Units)

Deviations:

yes

Remarks:

In this continuous activated sludge test, the protocol was modified accordingly so that the fate of the test items could be assessed at environmentally realistic concentrations using radiolabelled test items along with un-labelled test item at a nominal c

GLP compliance:

no

Remarks:

No claim for GLP compliance is made for this study; however, the study was carried out in the spirit of GLP.

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): Sodium Lauryl [14C] Isethionate
- Molecular formula (if other than submission substance): Na.SO3-CH2-CH2-O-CO-(CH2)10-CH3
- Radiochemical purity (if radiolabelling): 95.2%
- Specific activity (if radiolabelling):86.3 uCi/mg
- Storage condition of test material: stored in a freezer and had been allocated the Hazard Code R


- Name of test material (as cited in study report): Sodium [1-14C]Stearyl Isethionate
- Radiochemical purity (if radiolabelling):98.4%
- Specific activity (if radiolabelling): 63.31 uCi/mg
- Storage condition of test material: stored in a deep freeze – typically -70°C and was allocated the Hazard Code R

- Name of test material (as cited in study report): Sodium [1-14C] Lauryl Isethionate
- Molecular formula (if other than submission substance): Na.SO3-CH2-CH2-O-CO-(CH2)10-CH3
- Radiochemical purity (if radiolabelling): 94.5%
- Specific activity (if radiolabelling): 78.78 uCi/mg
- Storage condition of test material: stored in a deep freeze – typically -70°C and was allocated the Hazard Code R

- Name of test material (as cited in study report): Sodium Lauroyl Isethionate
- Molecular formula (if other than submission substance): Na.SO3-CH2-CH2-O-CO-(CH2)10-CH3
- Radiochemical purity (if radiolabelling): not applicable
- Specific activity (if radiolabelling): not applicable
- Expiration date of radiochemical substance (if radiolabelling): not applicable
- Storage condition of test material: stored under ambient conditions and was allocated hazard code N

- Name of test material (as cited in study report): SLI (76) Stripped
- Molecular formula (if other than submission substance):
- Radiochemical purity (if radiolabelling): not applicable
- Specific activity (if radiolabelling): not applicable
- Expiration date of radiochemical substance (if radiolabelling): not applicable
- Storage condition of test material: no data

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not Applicable

Radiolabelling:

yes

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on source and properties of surface water:

Not applicable - liquid influent was primary settled sewage

Details on source and properties of sediment:

Not applicable for this guideline

Details on inoculum:

The mixed liquor suspended solids of the return activated sludge was determined prior to addition to the aeration vessel. The volume of sludge required was calculated so that the aeration vessel contained nominally 2.5 g/L mixed liquor suspended solids suspended. The aeration vessel was filled with sufficient return activated sludge obtained from the return activated sludge line at Broardholme waste water treatment plant. The final separator was filled with effluent (both obtained from Anglia Water Broardholme sewage works, Ditchford, Rushden, UK).

Duration of test (contact time):

ca. 72 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

ca. 2 other: uCi/L

Based on:

other: scintillation counting (radioactivity)

Initial test substance concentration 2

Initial conc.:

ca. 0.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: 3L aeration vessel
- Composition of medium: settled sewage
- Additional substrate: not applicable
- Solubilising agent (type and concentration if used): SLI (76) Stripped was used as a solubilising agent for Sodium [14C] Stearoyl Isethionate; no solubilising agent was used for any of the other test items. Sodium Lauryl Isethionate was used as a carrier but not as a solubilising.
- Solvent: Dimethyl formamide
- Test temperature: 18-25 degrees C
- pH: 7-8
- pH adjusted: no
- Aeration of dilution water: not applicable
- Suspended solids concentration: initial 2.5g/L (sludge retention time controlled at 6 days)
- Continuous darkness: no
- Any indication of the test material adsorbing to the walls of the test apparatus: no

TEST SYSTEM
- Method used to create aerobic conditions: Carbon dioxide free air
- Method used to create anaerobic conditions: not applicable
- Test performed in closed vessels due to significant volatility of test substance: used close vessels but not due to volatility of test substance but to obtain a mass balance CO2
- Test performed in open system: no
- Details of trap for CO2: sodium hydroxide 1M

SAMPLING
- Sampling frequency: daily
- Sampling method: traps/effluent/waste sludge changed daily (aliquots taken for counting)
- Sterility check if applicable: not applicable
- Sample storage before analysis: scintillation counting conducted immediately. Specific sample analysis preserved with 40% methanol in a refridgerated container

CONTROL AND BLANK SYSTEM
- Inoculum blank: see below
- Abiotic sterile control: see below
- Toxicity control: see below
- Other: Carbon mass balance control

STATISTICAL METHODS: mean, 95% confidence limits (Student's T-test)
- Results calculated over a 32 day calculation period (days 40-72)

Reference substance

Reference substance:

other: Dimethyl formamide added at same application rate as test vessels so that equal amount of carbon was added to control vessel.

Results and discussion

Test performance:

Throughout the study, conditions were maintained in the test system that best represent a waste water sewage treatment plant but at a laboratory scale i.e. OECD303A Guideline (Husmann units). The test was carried out with the hydraulic and sludge retention times controlled at 6 hours and 6 days respectively. After the acclimation period (<6 weeks), the test was run for 32 days with samples collected daily and analysed for radioactivity remaining in effluent, sludge and traps. Concentrations of the test items obtained by scintillation counting in the sludge and effluent were determined daily between 40 and 72 days after a period of 40 days acclimatisation. All concentrations (sludge, effluent and mineralisation) were corrected for mass balance. In addition, HPLC analysis was conducted on samples collected at the conclusion of the calculation period (Day 72) to ascertain the concentration of parent material remaining in effluent and sludge and the percentage primary degradation that had occurred.

Mean total recovery

Compartment:

other: water / sediment, material (mass) balance

Remarks on result:

other: see "% Recovery" column in Table 1 below

% Degradation

% Degradation 1

% Degr.:

99.77

Parameter:

radiochem. meas.

Sampling time:

1 d

Remarks on result:

other: Range, every day from day 40 - day 72. Sodium Lauryl [14C] Isethionate

% Degradation 2

% Degr.:

99.61

Parameter:

radiochem. meas.

Sampling time:

1 d

Remarks on result:

other: Range, every day from day 40 - day 72. %parent in effluent is 0.11%. % parent in sludge is 2.74%. Sodium [14C] Stearyl Isethionate

% Degradation 3

% Degr.:

99.99

Parameter:

radiochem. meas.

Sampling time:

1 d

Remarks on result:

other: Range, every day from day 40 - day 72. Sodium [14C] Lauryl Isethionate. For all results see Table 1 below.

Transformation products:

not measured

Details on transformation products:

Not applicable

Evaporation of parent compound:

not measured

Volatile metabolites:

not measured

Residues:

not measured

Details on results:

The mixed liquor temperatures for all test and control plants ranged between 22 and 23 degrees C, which are within OECD guideline range of 18 - 25 degrees C.

The pH of the test and control units were, with few exceptions, within the accepted range for the growth of bacteria (i.e. between pH 6 to 8).

The dissolved oxygen [DO] levels remained above the critical concentration of 2 mg/L.

The hydraulic retention time [HRT] was calculated from the influent flow rates as determined by the mass of effluent collected per 24 hours. The HRT data remained largely within 6 ± 1 hours.

The sludge return flow rates for each plant remained largely within 8.5 ± 1 mL/min.

The sludge retention time [SRT] was calculated from the volume of sludge wasted per day. The SRT data remained largely within 6 ± 1 days.

Mixed liquor suspended solids: Initially concentrations were variable and often lower than anticipated (<2.5 g/L) during the acclimatisation period. However, throughout the calculation period the solids levels had increased to a normal level (~2.5 g/L) and appeared to be stable. Throughout the calculation period similar concentrations were observed between the test and control plants.

Throughout the calculation period similar organic carbon removal levels/concentrations were observed between the test and control plants.

Throughout the calculation period similar ammonia removal levels/concentrations were observed between the test and control plants.

Variable levels of solids concentration and ammonia removal efficiency observed during the acclimatisation phase of this study were probably attributable to the addition of dimethylformamide to the test system. Dimethylformamide is known to be biodegradable, however it also known to inhibit biological activity. These data suggests that the test system acclimatised to the solvent and it did not inhibit biological activity.

Throughout the test all of the above parameters were consistent with properly operated sewage treatment systems.

Results with reference substance:

not applicable

Any other information on results incl. tables

Table 1: Distribution of test items in Continuous Activated Sludge system

Test Item	% parent^#			% Recovery
	degraded	in effluent	in sludge
Sodium Lauryl [14C] Isethionate	99.77 (90.7)	0.02 (2.4)	0.21 (6.9)	94.2
Sodium [1-14C] Lauryl Isethionate	99.99 (94.5)	0.01 (1.8)	0.00 (3.7)	87.0
Sodium [1-14C] Stearyl Isethionate	99.61 (94.5)	0.11 (2.2)	0.28 (3.3)	78.0

Figures in parentheses relate to total 14C (parent, metabolites or biomass) remaining

^#All values corrected for recovery

Applicant's summary and conclusion

Validity criteria

Validity criteria fulfilled:

yes

Conclusions:

Degradation of parent material was extensive with 99.61 to 99.99% removed in the CAS systems. The concentrations of parent in the effluent ranged from 0.01 to 0.11% and the concentration of the parent in the sludge ranged from 0.21 to 0.28% of applied test item. The observed distribution of the sodium [1-14C] lauryl isethionate and the sodium [1-14C] stearyl isethionate was similar suggesting that all chain lengths would behave in a similar manner in a CAS system.

Executive summary:

The purpose of this study was to assess the environmental distribution (percent degradation and concentrations in sludge and effluent) of radio-labelled Defi test items in a continuous activated sludge (CAS) system. It was not practical to assess the fate of a commercial sample of DEFI, as it is a mixture of different chain lengths of alkyl isethionates. Therefore, the items tested (labelled in the alkyl chain) in this study were selected to represent the shortest (lauryl isethionate)and longest (stearyl isethionate)significant chain length present in the commercial mixture. In addition, the lauryl isethionate(labelled in the isethionate group) was included in the study. 

 

The CAS systems were operated in accordance with EU L133/106 and OECD Guideline 303A. The test was carried out at the recommended temperature range (18-25 °C) with the hydraulic and sludge retention times controlled at 6 hours and 6 days respectively. The study deviated from the OECD 303A guideline in that the test items used were radio-labelled. As a consequence the apparatuswas modified to accommodate capture of¹⁴CO_2. No other modifications were made.

 

Concentrations of the test items obtained by scintillation counting in the sludge and effluent were determined daily between 40 and 72 days after a period of 40 days acclimatisation. All concentrations (sludge, effluent and mineralisation) were corrected for mass balance (see below table). In addition, HPLC analysis was conducted on samples collected at the conclusion of the calculation period (Day 72) to ascertain the concentration of parent material remaining in effluent and sludge and the percentage primary degradation that had occurred.

 

Degradation of parent material was extensive with 99.61 to 99.99% removed in the CAS systems. The concentrations of parent in the effluent ranged from 0.01 to 0.11% and the concentration of the parent in the sludge ranged from 0.21 to 0.28% of applied test item. The observed distribution of the sodium [1-14C] lauryl isethionate and the sodium [1-14C] stearyl isethionate was similar suggesting that all chain lengths would behave in a similar manner in a CAS system.

Distribution of test items in Continuous Activated Sludge system

Test Item	% parent^#			% Recovery
	degraded	in effluent	in sludge
Sodium Lauryl [14C] Isethionate	99.77 (90.7)	0.02 (2.4)	0.21 (6.9)	94.2
Sodium [1-14C] Lauryl Isethionate	99.99 (94.5)	0.01 (1.8)	0.00 (3.7)	87.0
Sodium [1-14C] Stearyl Isethionate	99.61 (94.5)	0.11 (2.2)	0.28 (3.3)	78.0

Figures in parentheses relate to total 14C (parent, metabolites or biomass) remaining

^#All values corrected for recovery