(xylenes and 4-ethylbenzene)sulfonic acids

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

other: Read across from another member of the category

Adequacy of study:

supporting study

Study period:

September 18 - October 16, 1992

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): aeration pond of local wastewater treatment plant- Laboratory culture: - Method of cultivation: - Storage conditions: aerated in carboy in the laboratory- Storage length: approximately 30 days- Preparation of inoculum for exposure: the sludge was maintained by the daily addition of synthetic sewage food; pH adjusted with dilute HCl to maintain a range of 6.5 - 8.0; dissolved oxygen maintained at least 2.0 mg/L; - Pretreatment: preacclimation phase was 9 days in duration- Concentration of sludge: 500 mL from each of two SCAS units combined- Initial cell/biomass concentration: - Water filtered: yes- Type and size of filter used, if any: ABC reagent water (equivalent to ASTM Type I) was used in the test. The water was purififed by a LABCONCO Water Pro Water Purifier PS.

Duration of test (contact time):

>= 28 d

Initial conc.:

>= 10 - <= 20 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS- Composition of medium: inorganic nutrient medium- Additional substrate: no- Solubilising agent (type and concentration if used): no data- Test temperature: 22+/- 3 degrees C- pH: 7- pH adjusted: no - Aeration of dilution water: stir bar- Suspended solids concentration: used supernatant- Continuous darkness: yes- Other: acclimation in SCAS unitsTEST SYSTEM- Culturing apparatus: sodium acetate carboy- Number of culture flasks/concentration: one- Method used to create aerobic conditions: aerate with CO2 free air- Test performed in closed vessels- KOH traps used - Other: Shimadzu Model 5050 Total Carbon Analyzer used for CO2 analysis (combustion at 680 degrees C and nondispersive infrared detectionSAMPLING- Sampling frequency: days 2,4,6,8,10,15,20,25 and 28- Sampling method: CO2 produced in the test systems was trapped in KOH solutions in the gas-washing bottles- Sterility check if applicable: no data- Sample storage before analysis: 7 mL aliquats in glass scintillation vials without headspace; teflon-lined cap; room temperature- Other: standard plate count to enumerate the microbial populations was also performedCONTROL AND BLANK SYSTEM- Inoculum blank: yes- Abiotic sterile control: yes - Toxicity control: no data- Other: reference material - sodium acetateSTATISTICAL METHODS: no data

Reference substance:

acetic acid, sodium salt

Preliminary study:

DOC analysis of the supernatant samples from the SCAS units indicated the inoculum had adapted to the test material within the 15-day period by the 81% degradation exhibited

Test performance:

49 mg CO2 evolved in the control system which is in the acceptable range per the guideline. Plate count analysis indicated the inoculum was viable and active.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

>= 103 - <= 109

Sampling time:

28 d

Details on results:

The 60% of CO2 threshold was achieved within a 6 day test period.

Results with reference substance:

The day 0 total carbon analysis value was 15.03 mg C/L. The 60% threshold value was attainged in the reference system (sodium acetate) by Day 6 verifying the microbial inoculum was viable and active. 93% of the theoretical CO2 was attained at the end of the 28 days

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Readily biodegradable

Executive summary:

Sodium cumenesulfonate was evaluated for biodegradability in the modified sturm test by exposing the test chemical to a mixed microbial inoculum in a defined inorganic salts medium for 28 days. The microbial inoculum was supplied by performing a semi-continuos activated sludge (SCAS) acclimation to the study.

This entailed an activated sludge inoculum that was fed with an artificial sewage feed and maintained within a reaction unit in the dark at 22 +/- 3°C.

For the Sturm portion of the test, four systems were prepared. A control system containing the microbial inoculum without test or reference chemical was established to determine the endogenous microbial respiration. Sodium cumenesulfonate was added at concentrations of 10 and 20 mg carbon/liter (mg C/L).

A reference system was established to determine viability of the microbial inoculum. The reference material (sodium acetate) was added at concentrations of 10 and 20 mg C/L. The systems were analyzd with a total orgaic carbon (TOC) analyzer for CO2 evolution on days 2,4,6,8,10,15,20,25, and 28 of the test.

Additional samples were withdrawn for TOC analyses of each of the carboys on day 0 and in day 27 for dissolved organic carbon analysis.

The percent biodegradability (as %THCO2) was calculated as a function of the CO2 evolution in the test systems as compared to the control system (control subtracted). Of the sodium acetate applied dose, 93.21% of the theoretical CO2 evolution was attained at the end the 28-day study. This verified that the microbial inoculum was viable and active.  Of the sodium cumenesulfonate applied dose, 103.21% of THCO2 (at 10 mg C/L) and 109.43% of THCO2 (at 20 mg C/L) were attained in the 28-day test period.

A threshold value of 60% of the theoretical CO2 must be reached within the 28-day test period to conclude ready biodegradability. As this value was attained at both levels, it was concluded from the results of this test that under the test conditions, sodium cumenesulfonate was biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

other: Read across from another member of the category

Adequacy of study:

supporting study

Study period:

7 April to May 5 1995

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

GLP compliance:

not specified

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): secondary treatment stage of the Severn Trent Water plc sewage treatment plant in Belper, Derbyshire, treating predominantly domestic sewage.- Laboratory culture: inoculated culture medium- Method of cultivation: direct dispersion in culture medium- Storage conditions: room temperature- Storage length: - Preparation of inoculum for exposure: - Pretreatment: - Concentration of sludge: - Initial cell/biomass concentration: - Water filtered: yes/no- Type and size of filter used, if any:

Duration of test (contact time):

>= 28 d

Initial conc.:

>= 7 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS- Composition of medium: standard culture medium- Additional substrate: sodium benzoate as reference substance- Solubilising agent (type and concentration if used):- Test temperature: - pH: - pH adjusted: yes/no - CEC (meq/100 g):- Aeration of dilution water: - Suspended solids concentration: - Continuous darkness: yes/no - Other: TEST SYSTEM- Culturing apparatus: - Number of culture flasks/concentration: - Method used to create aerobic conditions: - Method used to create anaerobic conditions: - Measuring equipment: - Test performed in closed vessels due to significant volatility of test substance: - Test performed in open system: - Details of trap for CO2 and volatile organics if used: - Other:SAMPLING- Sampling frequency: - Sampling method: - Sterility check if applicable: - Sample storage before analysis: - Other: CONTROL AND BLANK SYSTEM- Inoculum blank: - Abiotic sterile control: - Toxicity control: - Other: STATISTICAL METHODS:TEST CONDITIONS- Composition of medium: - Additional substrate: - Solubilising agent (type and concentration if used):- Test temperature: - pH: - pH adjusted: yes/no - CEC (meq/100 g):- Aeration of dilution water: - Suspended solids concentration: - Continuous darkness: yes/no - Other: TEST SYSTEM- Culturing apparatus: - Number of culture flasks/concentration: 2- Method used to create aerobic conditions: - Method used to create anaerobic conditions: - Measuring equipment: not mentioned- Test performed in closed vessels due to significant volatility of test substance: - Test performed in open system: - Details of trap for CO2 and volatile organics if used: - Other:SAMPLING- Sampling frequency: 3 day intervals- Sampling method: % of ThOD- Sterility check if applicable: - Sample storage before analysis: - Other: CONTROL AND BLANK SYSTEM- Inoculum blank: inoculated culture medium- Abiotic sterile control: - Toxicity control: 7 mg/L test material plus 1.5 mg/L sodium benzoate with inoculum- Other: 3 mg/L sodium benzoate with inoculumSTATISTICAL METHODS:

Test performance:

The toxicity control attained 62% degradation after 28 days confirming the test material was not toxic to the sewage treatment micro-organisms used in the study.

Key result

Parameter:

% degradation (O2 consumption)

Value:

>= 50

Sampling time:

28 d

Details on results:

The toxicity control, containing both test item and sodium benzoate, attained 62% degradation after 28 days, confirming that the test material was not toxic to the sewage tratment micro-organisms used in the stuy.

Results with reference substance:

Sodium benzoate attained 98% degradation after 28 days

Conclusions:

The test substance attained 50% degradation after 28 days and therefore is inherently biodegradable but does not meet the ready biodegradable criteria under the guideline.

Executive summary:

The study following OECD 301D was performed on the test item, to assess the ready biodegradability.

Dissolved oxygen measurements for the test and standard material solutions together with the controls on 0, 3, 6, 9, 12, 15, 18, 21 and 28 days were performed.

The test item attained 50% degradation after 28 days and therefore, cannot be considered as ready biodegradable under the strict term and conditions of OECD 301D.

The toxicity control, containing both test item and sodium benzoate, attained 62% degradation after 28 days, confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.

Sodium benzoate attained 98% degradation after 28 days, thereby confirming the suitability of the test method and culture conditions.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

other: Read across from another member of the category

Adequacy of study:

key study

Study period:

2020-08-06 / 2020-09-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

GLP compliance:

yes

Inoculum or test system:

activated sludge, non-adapted

Remarks:

Inoculum of the aqueous phase of non-adapted activated sludge. Source: Municipal sewage treatment plant, 31137 Hildesheim, Germany

Parameter:

% degradation (CO2 evolution)

Value:

98

Sampling time:

28 d

Remarks on result:

other:

Evaluation
The theoretical production of carbon dioxide (ThCO2) of the test item and functional control is calculated by the carbon content and the molecular formula, respectively.

ThCO2 [mgCO2/mg] = 3.67 · TOC [mgC/mg test item]

ThCO2 [mgCO2/mg] = (C- Atoms molecularweightof CO2) / molecularweightof referenceitem

The produced CO2 is calculated by:

1 mL HCl (c = 0.05 mol/L) = 1.1 mg CO2
The net amount of CO2 produced is calculated by correcting the results of the test item and functional control for endogenous CO2 production of the inoculum controls.
The biodegradation is calculated from the ratio theoretical CO2 production to net CO2 production in the following equation:

Degradation [%]: netCO2* 100 / ThCO [mgCO/3L]

Software:  Excel, MICROSOFT CORPORATION, SigmaPlot (Windows), SPSS CORPORATION
All data were computer generated and rounded for presentation from the fully derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures.

Results
Carbon Content of the Test Item
Based on the carbon content a ThCO2 of 1.22 mg CO2/mg test item was calculated. A test

concentration of 31 mg/L (29 mg a.i./L), corresponding to a carbon content of 10.3 mg C/L in

the test vessels was selected.

 

 

CO2-Production and Biodegradation
The total amount of CO2 produced in 28 days was analysed by titration in 12 measurements.

The 28 day-values are shown in comparison to the readily degradable functional control in summarized form in Table 2 attached.

The results of the net CO2-production and biodegradation rate of each measurement are given in Table 3 to Table 4 attached.

 

The adaptation phase of the functional control changed within 4 days into the degradation phase (biodegradation > 10 %). The course of the biodegradation was fast and the functional control reached the pass level of 60 % within 8 days and a maximum biodegradation of 100 % on day 28. The validity criterion for biodegradation > 60 % after 14 days was fulfilled.

In the toxicity control containing both test item and reference item a biodegradation of 78 % was determined within 14 days, with 92 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

The biodegradation of the test item is shown graphically in Figure 1 attached in comparison to the readily degradable functional control and the toxicity control. The mean of replicates reached the 10 % level (beginning of biodegradation) within 6 days. Both test item replicates reached the 60 % pass level within 11 days. The mean biodegradation on day 28 was 98 %. The 10-day window was fulfilled.

Water Parameters
On day 28 the pH-value of all solutions was measured prior to acidification. The results are given in Table 5 attached.

 

Conclusions:

Readily biodegradable

Executive summary:

The ready biodegradability of the test item was determined with a non-adapted activated sludge over a test period of 28 days in the Modified Sturm Test. The study was conducted according to OECD 301 B at the test facility. The test item was tested at a concentration of 31 mg/L (29 mg a.i./L) with 2 replicates corresponding to a carbon content (TOC) of 10.3 mg C/L in the test vessels. The test vessels were incubated at low light conditions and at a temperature
of 22 ± 2 °C.
The biodegradation of the test item was followed by titrimetric analysis of the quantity of CO2 produced by the respiration of bacteria. The biodegradation was stopped on day 28 by acidification of the test solutions. The last titration was made on day 29 after residual CO2 had been purged from the test solutions over a period of 24 hours. The percentage CO2 production was calculated in relation to the theoretical CO2 production (ThCO2) of the test item. The biodegradation was calculated for each titration time.
To check the activity of the test system sodium benzoate was used as functional control. The percentage degradation of the functional control reached the pass level of 60 % within 8 days and a maximum biodegradation of 100 % on day 28.
In the toxicity control containing both test and reference item (sodium benzoate) a biodegradation of 78 % was determined within 14 days, with 92 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.
The mean of replicates reached the 10 % level (beginning of biodegradation) within 6 days. Both test item replicates reached the 60 % pass level within 11 days. The mean biodegradation on day 28 was 98 %. The 10-day window was fulfilled.

Reference 4

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

other: Read across from anologue substance

Adequacy of study:

weight of evidence

Study period:

1976

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Publication lacking some details; use of adapted sludge

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.6 (Degradation: Chemical Oxygen Demand)

Deviations:

yes

Remarks:

use of adapted sludge; no replication

GLP compliance:

not specified

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, adapted

Details on inoculum:

Innoculum from a sewage plant.
Preparation - daily 200 mL is separated fromthe 1L stock. After settling, the liquid is diluted with tap water, 600 mg/L starch or glucose, 600 mg/L peptone and 25 mL phosphate buffer (pH 7.2) plus the test compound.
The concentration of test compound was gradually increased to 200 mg/L as COD after 20 days
Mineral solution (medium) was 27.5 mg CaCl2, 22.5 mg MgSO4, 0.25 mg ferric chloride, 50 mg ammomium sulphate, 20 mL of phosphate buffer (pH 7.2) and 100 mL tap water in distilled water.

Duration of test (contact time):

>= 120 h

Initial conc.:

>= 0 - <= 200 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

not specified

Details on study design:

Test conducted in glass flasks.
One flask for test substance + inoculum + medium
One flask for analine + inoculum + medium
One flask for inoculum + medium
No aeration
Test temperature 20 +/- 3C

Reference substance:

aniline

Parameter:

other: COD

Value:

98.7

Sampling time:

120 h

Details on results:

rate of biodegradation equivalent to 8.4 mg COD/g/h

Results with reference substance:

94.5.% biodegradation. Rate equivalent to 19.0 mg COD/g/h

Interpretation of results:

inherently biodegradable

Conclusions:

Inherently biodegradable

Executive summary:

Toluene 4-sulphonic acid was evaluated for biodegradability following official EU method C.6 (Degradation: Chemical Oxygen Demand). The tested substance is biodegradable with a COD of 98.7 % at 120 h under the conditions of this test.

Reference 5

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

other: Read across from analoguer substance

Adequacy of study:

weight of evidence

Study period:

1988

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Publication with minimal detail available

Principles of method if other than guideline:

No guideline indicated; activated sludge degradability test

GLP compliance:

not specified

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, industrial, adapted

Initial conc.:

100 mg/L

Based on:

other: TOC

Parameter followed for biodegradation estimation:

TOC removal

Reference substance:

not specified

Parameter:

% degradation (TOC removal)

Value:

90

Remarks on result:

other: no more details have been reported

Details on results:

Sampling time and standard deviation were no specified

Sampling time was no specified

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

inherenthly biodegradable

Executive summary:

Toluene 4-sulphonic acid was evaluated for biodegradability (no guideline). The tested substance is biodegradable with a TOC removal of 90 % h under the conditions of this test.

Reference 6

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

other: Read across from analogue substance

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Secondary source - literature review with some details and references for numerous studies

Principles of method if other than guideline:

Six published studies; see results

GLP compliance:

not specified

Inoculum or test system:

activated sludge (adaptation not specified)

Remarks on result:

other: See below

Details on results:

A test of PTSA's biodegradability (100 mg/L) with non-adapted activated sludge (30 mg/L) as inoculum, at 20C +/- 1 and pH 7, biological oxygen demand was measured by respirometer over a period of 14 days (BOD14). Following a lag period of 50-60 hours and a degredation time of 125 to 135 hours, at a rate constant of 0.041 - 0.045 h-1, PTSA was found to be 79-80% biodegradable (Urano and Kato, 1986).

In a modified OECD screening test and in a coupled units test (both without further detail) the degree of sodium toluene sulphonate degradation was determined by measurements of DOC. The results were 99.9% and 88 +/- 3.14%, respectively (Schoberl and Huber, 1988). Accordingly, sodium toluene sulphonate is classified as being readily biodegradable.

In the OECD test for inherent biodegradability, according to test guideline 302B (Zahn-Wellens Test) at a concentration of 720 mg/L PTSA monohydrate was eliminated to 95% after 15 days (Wellens, 1979), while PTSA from the 65% aqueous solution at a concentration of 307.5 mg/L was eliminated to almost 100% after 5 days (Wellens, 1983).

In another study (Zahn-Wellens Test) PTSA monohydrate was degraded to 90% after 10 days and 94% after 20 days while the 65% solution was degraded to 75% after 25 days. When the test with the 65% aqueous solution was repeated with an adapted activated sludge, over 90% degradation was achieved after 10 days (Wellens, 1981).

In a recently published paper (1990) Wellens reports on the course of biodegradation of PTSA in the Zahn-Wellens test according to OECD Guideline 302B and DIN-Norm 38412 (L12). The results showed 97% degradation after a maximum of 12 days incubation, whereby following an adaptation phase of 1-3 days, 85% degradation took place within 5-9 days.

Interpretation of results:

readily biodegradable

Conclusions:

readily biodegradable

Executive summary:

Toluene 4-sulphonic acid was evaluated for biodegradability following different OECD 301 and 302 guidelines. All the results reported in the publication showed ready biodegradability.

Reference 7

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

other: Read Across from analogue substance

Adequacy of study:

supporting study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge:Municipal sewage treatment plant, 31137 Hildesheim, Germany
- Laboratory culture: no
- Storage conditions: not mentioned
- Storage length: 2 days (test 1) resp. 1 day (test 2)
- Preparation of inoculum for exposure: The activated sludge was washed twice with chlorine free tap water
- Pretreatment: After the second washing the settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration with CO2 free air for two days before test start.
- Concentration of sludge: 4.45 mL/L of this mixture were used to initiate inoculation (25.1 mg/L dw).

Duration of test (contact time):

28 d

Initial conc.:

10.1 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test Method
Application: once at test start
Test vessels: 5000 mL, brown glass
Volume of the test medium: 3000 mL
Test medium: mineral salts medium acc. to OECD 301 B / CO2 Evolution Test
Test temperature: Nominal 22 ± 2 °C, actually measured 19 – 22.5 °C
Dispersion treatment: Continuous stirring
Aeration: 30 - 100 mL/min
Photoperiod: Low light conditions (brown glass bottles)

The necessary amounts of ultrapure water, mineral salts medium the test vessels and inoculum were placed in each incubation vessel. The vessels were aerated for 24 h with CO2 free air. After 24 h the CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles, each containing 100 mL of a 0.0125 mol/L Ba(OH)2 solution.
The test and reference item were weighed out. The test item was weighed into small beakers. A defined amount of ultrapure water was added to the test item. The test item dispersions and the reference item were transferred to the respective incubation vessels with ultrapure water. The vessels were made up to 3 L with ultrapure water and connected to the system for the production of CO2 free air.
On day 28, 1 mL 37 % HCl was added to each of the vessels. Aeration was continued for further 24 h and the quantity of CO2 released was determined

The biodegradation of the test item was followed by titrimetric analysis of the quantity of CO2 produced by the respiration of bacteria. The biodegradation was stopped on day 28 by acidification of the test solutions. The last titration was made on day 29 after residual CO2 had been purged from the test solutions over a period of 24 hours. The percentage CO2 production was calculated in relation to the theoretical CO2 production (ThCO2) of the test item. The biodegradation was calculated for each titration time.

Equipment:
DOC-Analysator Multi N/C 3100, ANALYTIK JENA
pH-Meter, Multi 350i, WTW
Thermohygrograph, LUFFT
Ultrasonic bath, SONOREX, BANDELIN
Analytical balance, SARTORIUS
Balance, KERN
Dispensette, BRAND
Digital Buret, continous RS, VITLAB
Medo compressor, FA. REBIE
Magnetic stirrer Mono, VARIOMAG
Multipette X-Stream, EPPENDORF
Various Pipette

Reference substance:

benzoic acid, sodium salt

Preliminary study:

not performed

Test performance:

Chronological test description
- Pre-treatment of the non-adapted activated sludge
- Preparation of the test solutions and aeration for 24h with CO2 free air
- Weighing out of the test item (experimental starting) and weighing out of the reference item
- Application
- Incubation
- Determination of CO2 by titration
- Determination of the pH-values on day 28 and acidification
- Last titration on day 29
- Evaluation of the data

The room temperature was recorded continuously throughout the test.
Determination of CO2 was carried out by titration subsequent to complete adsorption of the released CO2 in an alkaline solution (0.0125 mol/L Ba(OH)2). For each titration the first gas wash
bottle was removed and a new bottle was connected to the last one.
Back titration of the residual Ba(OH)2 with 0.05 N HCl was carried out three times a week during the first ten days and thereafter twice weekly.
On day 28 the pH of all solutions was measured prior to acidification.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

98

Sampling time:

28 d

Details on results:

The mean of replicates reached the 10 % level (beginning of biodegradation) within 4 days. Both test item replicates reached the 60% pass level within 8 days. The mean biodegradation on day 28 was 98 %. The 10-day window was met

Results with reference substance:

To check the activity of the test system sodium benzoate was used as functional control. The
percentage degradation of the functional control reached the pass level of 60 % within 8 days and
a maximum biodegradation of 90 % on day 28.

Evaluation
The theoretical production of carbon dioxide (ThCO2) of the test item and functional control is calculated by the carbon content and the molecular formula, respectively.

ThCO2 [mgCO2/mg] = 3.67 · TOC [mgC/mg test item]

ThCO2 [mgCO2/mg] = (C- Atoms molecularweightof CO2) / molecularweightof referenceitem

The produced CO2 is calculated by:

1 mL HCl (c = 0.05 mol/L) = 1.1 mg CO2 (3)
The net amount of CO2 produced is calculated by correcting the results of the test item and functional control for endogenous CO2 production of the inoculum controls.
The biodegradation is calculated from the ratio theoretical CO2 production to net CO2 production in the following equation:

Degradation [%]: netCO2* 100 / ThCO [mgCO/3L]

Software:  Excel, MICROSOFT CORPORATION, SigmaPlot (Windows), SPSS CORPORATION
All data were computer generated and rounded for presentation from the fully derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures.

Results
Carbon Content of the Test Item
Based on the carbon content a ThCO2 of 1.028 mg CO2/mg test item was calculated. A test concentration of 36 mg/L, corresponding to a carbon content of 10.1 mg C/L in the test vessels was selected.

Colony Forming Units of the Inoculum
Colony forming units (CFU) of the inoculum for the Modified Sturm Test were determined prior to test start by standard dilution plate count: approx. 1.5 x 109 CFU/L, corresponding to approx. 0.67 x 107 CFU/L in the test vessel.

CO2-Production and Biodegradation
The total amount of CO2 produced in 28 days was analysed by titration in 12 measurements.
The 28 d-values are shown in comparison to the readily degradable functional control in summarized form in Table 2 attached.
The results of the net CO2-production and biodegradation rate of each measurement are given in Table 3 to Table 4 attached.

The adaptation phase of the functional control changed within 4 days into the degradation phase (degradation > 10 %). The course of the degradation was rapid and the functional control reached the pass level of 60 % within 6 days and a maximum biodegradation of 90 % on day
28. The validity criterion degradation > 60 % after 14 days is fulfilled.
In the toxicity control containing both test item and reference item a biodegradation of 78 % was determined within 14 days, with 89 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.
The biodegradation of the test item is shown graphically in Figure 1 attached in comparison to the readily degradable functional control and the toxicity control. The mean of replicates reached the 10 % level (beginning of biodegradation) within 4 days. Both test item replicates did reach the 60 % pass level within 8 days. The mean biodegradation on day 28 was 98 %. The 10-day window was
met.

Water Parameters
On day 28 the pH-value of all solutions was measured prior to acidification. The
results are given in Table 5 attached.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

readily biodegradable

Executive summary:

Hydroxybenzene sulphonic acid was evaluated for biodegradability following official guideline OECD 301B (Ready biodegradability).The ready biodegradability of the test item was determined with a non-adapted activated sludge over a test period of 28 days in the Modified Sturm Test.The test item was tested at a concentration of 36 mg/L with 2 replicates corresponding to a carbon content (TOC) of 10.1 mg C/L in the test vessels. The test vessels were incubated at low light conditions and at a temperature of 22 ± 2 °C.
The biodegradation of the test item was followed by titrimetric analysis of the quantity of CO2 produced by the respiration of bacteria. The degradation was stopped on day 28 by acidification of the test solutions. The last titration was made on day 29 after residual CO2 had been purged from the test solutions over a period of 24 hours. The percentage CO2 production was calculated in relation to the theoretical CO2 production (ThCO2) of the test item. The biodegradation was
calculated for each titration time.
To check the activity of the test system sodium benzoate was used as functional control. The percentage degradation of the functional control reached the pass level of 60 % within 8 days and a maximum biodegradation of 90 % on day 28.
In the toxicity control containing both test and reference item a biodegradation of 78 % was determined within 14 days, with 89 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.
The mean of replicates reached the 10 % level (beginning of biodegradation) within 4 days. Both test item replicates reached the 60% pass level within 8 days. The mean biodegradation on day 28 was 98 %. The 10-day window was met.
Under the test conditions the test item is readily biodegradable with the 28 day period of the study.

Reference 8

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

other: Read Across from analogue substance

Adequacy of study:

supporting study

Study period:

2020/08/06 - 2020/09/04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: municipal wastewater treatment plant Hildesheim, Germany
- Laboratory culture: no
- Storage conditions: not mentioned
- Storage length: 2 days (test 1) resp. 1 day (test 2)
- Preparation of inoculum for exposure: The activated sludge was washed twice with chlorine free tap water. by settling the sludge, decanting the supernatant and resuspending the sludge in aerated tap water.
- Pretreatment: aerated with CO2-free air for two days before treatment.
- Concentration of sludge: 4.03 mL/L of this mixture were used to initiate inoculation (25.0 mg/L dw).

Duration of test (contact time):

28 d

Initial conc.:

45 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test Method
Application: once at test start
Test vessels: 5000 mL, brown glass
Volume of the test medium: 3000 mL
Test medium: mineral salts medium acc. to OECD 301 B / CO2 Evolution Test
Test temperature: Nominal 22 ± 2 °C, actually measured 20.5 – 22 °C
Dispersion treatment: Continuous stirring
Aeration: 30 - 100 mL/min
Photoperiod: Low light conditions (brown glass bottles)

The necessary amounts of ultrapure water, mineral salts medium the test vessels and inoculum were placed in each incubation vessel. The vessels were aerated for 24 h with CO2 free air. After 24 h the CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles, each containing 100 mL of a 0.0125 mol/L Ba(OH)2 solution.
The test and reference item were weighed out. The test item was weighed into small beakers. A defined amount of ultrapure water was added to the test item. The test item dispersions and the reference item were transferred to the respective incubation vessels with ultrapure water. The vessels were made up to 3 L with ultrapure water and connected to the system for the production of CO2 free air.
On day 28, 1 mL 37 % HCl was added to each of the vessels. Aeration was continued for further 24 h and the quantity of CO2 released was determined

The biodegradation of the test item was followed by titrimetric analysis of the quantity of CO2 produced by the respiration of bacteria. The biodegradation was stopped on day 28 by acidification of the test solutions. The last titration was made on day 29 after residual CO2 had been purged from the test solutions over a period of 24 hours. The percentage CO2 production was calculated in relation to the theoretical CO2 production (ThCO2) of the test item. The biodegradation was calculated for each titration time.

Equipment:
pH-Meter, Multi 350i, WTW
Thermohygrograph, LUFFT
Ultrasonic bath, SONOREX, BANDELIN
Analytical balance, SARTORIUS
Balance, KERN
Dispensette, BRAND
Digital Buret, continous RS, VITLAB
Medo compressor, FA. REBIE
Magnetic stirrer Mono, VARIOMAG
Multipette X-Stream, EPPENDORF
Various Pipette

Reference substance:

benzoic acid, sodium salt

Remarks:

20 mg/L DOC

Preliminary study:

not performed

Test performance:

- Pre-treatment of the non-adapted activated sludge test description
- Preparation of the test solutions and aeration for 24h with CO2 free air
- Weighing out of the test item (experimental starting) and weighing out of the reference item
- Application
- Incubation
- Determination of CO2 by titration
- Determination of the pH-values on day 28 and acidification
- Last titration on day 29
- Evaluation of the data

The room temperature was recorded continuously throughout the test.
Determination of CO2 was carried out by titration subsequent to complete adsorption of the released CO2 in an alkaline solution (0.0125 mol/L Ba(OH)2). For each titration the first gas wash bottle was removed and a new bottle was connected to the last one.
Back titration of the residual Ba(OH)2 with 0.05 N HCl was carried out three times a week during the first ten days and thereafter twice weekly.
On day 28 the pH of all solutions was measured prior to acidification.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

99

Sampling time:

28 d

Details on results:

The mean of replicates reached the 10 % level (beginning of biodegradation) within 6 days. Both test item replicates reached the 60 % pass level within 11 days. The mean biodegradation on day 28 was 99 %. The 10-day window was fulfilled.

Results with reference substance:

The reference compound reached the pass level for ready biodegradablity.

Evaluation
The theoretical production of carbon dioxide (ThCO2) of the test item and functional control is calculated by the carbon content and the molecular formula, respectively.

ThCO2 [mgCO2/mg] = 3.67 · TOC [mgC/mg test item]

ThCO2 [mgCO2/mg] = (C- Atoms molecularweightof CO2) / molecularweightof referenceitem

The produced CO2 is calculated by:

1 mL HCl (c = 0.05 mol/L) = 1.1 mg CO2
The net amount of CO2 produced is calculated by correcting the results of the test item and functional control for endogenous CO2 production of the inoculum controls.
The biodegradation is calculated from the ratio theoretical CO2 production to net CO2 production in the following equation:

Degradation [%]: netCO2* 100 / ThCO [mgCO/3L]

Software:  Excel, MICROSOFT CORPORATION, SigmaPlot (Windows), SPSS CORPORATION
All data were computer generated and rounded for presentation from the fully derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures.

Results
Carbon Content of the Test Item
Based on the carbon content a ThCO2 of 0.829 mg CO2/mg test item was calculated. A test concentration of 45 mg/L (31.6 mg a.i./L), corresponding to a carbon content of 10.2 mg C/L in the test vessels was selected..

Colony Forming Units of the Inoculum
Colony forming units (CFU) of the inoculum for the Modified Sturm Test were determined prior to test start by standard dilution plate count: approx. 1.5 x 109 CFU/L, corresponding to approx. 0.67 x 107 CFU/L in the test vessel.

 

CO2-Production and Biodegradation
The total amount of CO2 produced in 28 days was analysed by titration in 12 measurements.

The 28 day-values are shown in comparison to the readily degradable functional control in summarized form in Table 2 attached.

The results of the net CO2-production and biodegradation rate of each measurement are given in Table 3 to Table 4 attached.

 

The adaptation phase of the functional control changed within 4 days into the degradation phase (biodegradation > 10 %). The course of the biodegradation was fast and the functional control reached the pass level of 60 % within 8 days and a maximum biodegradation of 100 % on day 28. The validity criterion for biodegradation > 60 % after 14 days was fulfilled.

In the toxicity control containing both test item and reference item a biodegradation of 83 % was determined within 14 days, with 93 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

The biodegradation of the test item is shown graphically in Figure 1 attached in comparison to the readily

degradable functional control and the toxicity control. The mean of replicates reached the 10 % level (beginning of biodegradation) within 6 days. Both test item replicates reached the 60 % pass level within 11 days. The mean biodegradation on day 28 was 99 %. The 10-day window was

fulfilled.

Water Parameters
On day 28 the pH-value of all solutions was measured prior to acidification. The
results are given in Table 5 attached.

 

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the test conditions the test item is classified as readily biodegradable (passing the 10d window) within the 28 day period of the study.

Executive summary:

Benzene sulphonic acid was evaluated for biodegradability following official guideline OECD 301B (Ready biodegradability). The tested substance is biodegradable with a COD of 98.7 at 120 h under the conditions of this test.

The ready biodegradability of the test item was determined with a non-adapted activated sludge over a test period of 28 days in the Modified Sturm Test. The test item was tested at a concentration of 45 mg/L (31.6 mg a.i./L) with 2 replicates corresponding to a carbon content (TOC) of 10.2 mg C/L in the test vessels. The test
vessels were incubated at low light conditions and at a temperature of 22 ± 2 °C.
The biodegradation of the test item was followed by titrimetric analysis of the quantity of CO2 produced by the respiration of bacteria. The biodegradation was stopped on day 28 by acidification of the test solutions. The last titration was made on day 29 after residual CO2 had been purged from the test solutions over a period of 24 hours. The percentage CO2 production was calculated in relation to the theoretical CO2 production (ThCO2) of the test item. The biodegradation was calculated for each titration time.
To check the activity of the test system sodium benzoate was used as functional control. The percentage degradation of the functional control reached the pass level of 60 % within 8 days and a maximum biodegradation of 100 % on day 28.
In the toxicity control containing both test and reference item (sodium benzoate) a biodegradation of 83 % was determined within 14 days, with 93 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.
The mean of replicates reached the 10 % level (beginning of biodegradation) within 6 days. Both test item replicates reached the 60 % pass level within 11 days. The mean biodegradation on day 28 was 99 %. The 10-day window was fulfilled.

Description of key information

readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

The ASA Category comprises the following 5 aromatic sulphonic acids:

TSA, Toluene-4-sulphonic acid (EC 203-180-0, CAS 104-15-4)

XSA, (Xylenes and 4-ethylbenzene) sulphonic acid (EC 701-247-3, CAS -) Former EC 246-839-8

CSA, p-cumene sulphonic acid (EC 240-210-1, CAS 16066-35-6)

BSA, Benzene sulphonic acid (EC 202-638-7, CAS 98-11-3)

HBSA, 4-hydroxybenzensulphonic acid (EC No. 202-691-6, CAS No. 98-67-9)

The related HYDROTOPES Category comprises the following 6 substances which are considered to be similar to ASA:
STS - Sodium toluene 4-sulphonate (CAS 657-84-1, EC 211-522-5)
SXS - Sodium (xylenes and 4-ethylbenzene) sulfonates (EC 701-037-1)
NH4XS - Ammonium (xylenes and 4-ethylbenzene) sulfonates (EC 943-024-5)
SCS - Sodium p-cumenesulphonate (CAS 15763-76-5, EC 239-854-6)
KCS - Potassium p-cumenesulphonate (CAS 164524-02-1, EC 629-764-9)
NH4CS - Ammonium p-cumenesulphonate (CAS 680972-33-2, EC 811-484-5)
In addition CaXS (Calcium Xylenesulphonate, CAS 28088-63-3, EC 248-829-9) was evaluated for complete the assessment despite it is not registered under REACH.

Ready biodegradability was assessed with avaialble studies on Asa and Hydrotropes category members. Hydrotropes are completely dissociated in water and could be used in read across for the related acid form.
Biodegradability data are available for toluene, benzene and hydroxybenzene sulphonic acids. The ready biodegradability of BSA and HBSA was assessed with two new tests with a non-adapted activated sludge over a test period of 28 days in the Modified Sturm Test, following OECD 301B. Both test item replicates reached the 60 % pass level within 11 days. The mean biodegradation on day 28 was 99 %. Moreover, some publications are available for TSA, and even if they have some deficiencies, the results are consistent within the category. Therefore, they can be used to support and strengthen the ready biodegradability of ASA category. The publication available on TSA considered the test substance as ready biodegradable which is confirmed by the existing test on the related salts (Sodium Tolunene 4-sulphonate).
There are no data on CSA but there are two tests available on the related calcium and sodium salt following OECD 301B. Under the test conditions the test items are readily biodegradable with the 28 day period of the study. These test was considered not adequate since there are some deficiencies but the results can be considered consistent and therefore it can be used as supporting in the whole ASA category. There are no data on XSA but there is a new study performed on the related sodium salt following OECD 301B which showed ready biodegradability.
Based on the available information, ASA substances are considered to be ready biodegradable.