Butyl toluene-4-sulphonate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 July 2019 to 29 August 2019

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)

Version / remarks:

17 July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Version / remarks:

29 December 1992

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Relative density: 1.12

Concentrations were adjusted for the purity and percent carbon of the test material. The percent carbon content was determined by the Test Facility in triplicate, using a total carbon analyser (Shimadzu TOC-V-CPH), to be 56.08 %.
Since the test material is insoluble in water at the tested concentration, a stock solution was not prepared and the test material was instead added directly to the test vessels. Four aliquots (71.7 μL) of the test material were placed directly into the four test vessels receiving test material. This volume of test material provided 45 mg of carbon per vessel after accounting for density of 1.12 g/mL and percent organic carbon of 56.08 %, which provided a concentration of 15 mg/L carbon.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Approximately 8 L of activated sludge was obtained from the Wareham Wastewater Treatment Plant, Wareham, Massachusetts on 30 July 2019, which receives primarily domestic waste.
- Pre-treatment: the sludge was passed through a 2-mm sieve and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, the sludge was washed with mineral medium, the contents were centrifuged again, and the supernatant was discarded. The moisture content of the activated sludge was determined (using a Sartorius MA 37-1US automated moisture analyser) to be 95.65 % and the percent solids was determined to be 4.35 %.
- Preparation of inoculum for exposure: An inoculum solution with 15 mg suspended solids/mL was prepared by diluting 69.00 g of sludge to 200 mL with mineral medium, stirred with a PTFE-coated magnetic stir bar at approximately 22 ± 2 °C, and aerated until used.

Duration of test (contact time):

28 d

Initial conc.:

15 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Remarks:

calculated from inorganic carbon in traps

Details on study design:

TEST CONDITIONS
- Composition of medium: 1 L of mineral salts medium contained:
10 mL phosphate buffer solution (8.53 g/L KH2PO4, 21.75 g/L K2HPO4, 26.64 g/L Na2HPO4, 0.50 g/L NH4Cl)
1 mL calcium chloride solution (27.50 g/L CaCl2)
1 mL magnesium sulphate solution (22.50 g/L MgSO4·7H2O)
1 mL ferric chloride solution (0.26 g/L FeCl3·6H2O and one drop of concentrated hydrochloric acid)
The water contained no more than 10 % of the total carbon content introduced by the test material
- Test temperature: 22 ± 2 °C
- pH: 7.43
- pH adjusted: no
- Suspended solids concentration: activated sludge concentration of 30 mg solids/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 4 L glass bottles with rubber stoppers into which stainless-steel needles (with a Luer-Lok connection and two pieces of glass tubing) were inserted. The stainless-steel needle served as a sampling port for solution samples. A rubber policeman cap was used to cover the top of the sample port. The glass tubing provided the inlet and outlet ports for air exchange.
- Number of culture flasks/concentration: six test vessels were established: two for the test material, two inoculum blanks, one sodium benzoate procedural control, and one toxicity control. An additional vessel was established in the same manner as the test material vessels, but was only used for pH measurements and sampling for TIC and total carbon (TC) on day 0. This vessel was not attached to KOH traps and was disposed of following pH, TIC, and TC determination.
- Method used to create aerobic conditions: CO2-free air was pumped under positive pressure through a hydration flask before entering the test system.
- Details of trap for CO2 and volatile organics if used: The outlet port of each system was connected to two CO2 effluent gas traps, the first consisting of 200 mL of 0.2 N potassium hydroxide (KOH) and the second trap containing 100 mL of 0.2 N KOH.
- Test performed in closed vessels: yes

TEST METHODOLOGY
On day -1, each 4.0 L vessel was established by adding 2991 mL (procedural control and toxicity control) or 2994 mL (inoculum blanks and test material replicates) of mineral medium. A 6 mL aliquot of the activated sludge inoculum was added to each vessel, for a total volume of 3000 mL per vessel after dosing on day 0. The six test vessels were attached to a CO2-free compressed air gas tank and aerated under positive pressure. The vessels were mixed and purged with CO2-free air until day 0 to remove any residual inorganic carbon in the test system prior to test initiation. At test initiation (day 0), test material replicate vessels (A and B) and the pH/TIC vessel were dosed. The total fortification was 15 mg C/L in the test suspension vessels and the pH/TIC vessel.
The toxicity control vessel, which was fortified in the same manner as the test material vessels, also received 4.5 mL of the 10 mg C/mL sodium benzoate stock solution for a total fortification of 30.0 mg C/L (test and reference substance) and total volume of 3000 mL in the toxicity control vessel. The sodium benzoate procedural control was fortified with 4.5 mL of the sodium benzoate stock solution for a final concentration of 15 mg C/L and total volume of 3000 mL.

SAMPLING
The pH/TIC vessel was only established for pH measurement, TIC, and TC analysis at the start of the study. The minimum/maximum temperature of the environmental chamber was recorded daily during the week (Monday through Friday) throughout the exposure period using a digital minimum/maximum thermometer. Minimum and maximum temperatures recorded on Mondays represented the temperature range for Saturday and Sunday.
On test days 1, 2, 5, 7, 9, 12, 14, 16, 19 and 23, a 7 mL sample was removed from the first KOH carbon dioxide trap on each test system and analysed for CO2 evolution. On day 28, 1 mL of concentrated hydrochloric acid was added to each test vessel following pH measurements to terminate biological activity. Aeration was continued to drive any residual inorganic carbon from the test vessels. After overnight aeration, 7 mL samples were removed from the first and second traps for analysis of CO2 evolution.

CALCULATIONS
The amount of carbon dioxide evolved from each test system was adjusted by subtracting the mean CO2 production value from the inoculum blank controls. The percent biodegradability for each test system was calculated using the following equation and is expressed as cumulative percent biodegradation (or percent of theoretical CO2 production).
% Ultimate Biodegradability = [mg CO2 produced / (mg TOC added × 3.667)] × 100
where: 3.667 is the molecular weight conversion factor for carbon to carbon dioxide

Reference substance:

benzoic acid, sodium salt

Remarks:

reported purity of 100.0 % (Total organic carbon was calculated to be 58.29 % based on the empirical formula: C7H5O2Na)

Test performance:

- Test Conditions: The temperature ranged from 19.3 to 22.0 °C. The pH of the pH check vessel on day 0 was measured to be 7.46. The pH values of the test vessels ranged from 7.07 to 7.41 at the end of the study on day 28.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

66.31

Sampling time:

28 d

Details on results:

- CO2 Evolution: The total inorganic carbon measured in the KOH traps was used to calculate the cumulative CO2 evolved from the test vessels. The cumulative CO2 value from the blank control test vessels (averaged) at day 28 were 14.66 mg/L. The cumulative CO2 value from the test material test vessels (averaged) and the toxicity control test vessel at day 28 were 51.14 and 87.82 mg/L, respectively. The mean cumulative net percent CO2 production (blank control values subtracted), or percent ultimate biodegradation, values for the test material and toxicity control were calculated to be 66.31 and 66.50 %, respectively.

Results with reference substance:

The cumulative CO2 value from the procedural control test vessel at day 28 was 57.82 mg/L. The mean cumulative net percent CO2 production (blank control values subtracted), or percent ultimate biodegradation, value for the procedural control was calculated to be 78.45 %.

Cumulative Net Percent CO2 Evolved from the Test Vessels (Ultimate Biodegradation)

Day	Test material (mean ± SD)	Procedural Control	Toxicity Control
1	2.58 ± 2.57	1.88	2.96
2	2.48 ± 1.71	20.87	13.16
5	8.42 ± 0.72	45.02	27.27
7	28.25 ± 0.98	50.61	33.04
9	43.12 ± 6.11	55.66	38.89
12	56.40 ± 12.06	61.44	47.11
14	56.96 ± 12.44	66.02	50.76
16	62.44 ± 9.63	68.71	53.58
19	64.24 ± 8.17	70.56	56.50
23	64.11 ± 6.70	72.65	58.61
28	66.31 ± 4.82	78.45	66.50

Study Acceptance Criteria

Acceptability Criterion	Study Performance	Criterion Met
Toxicity Control should be ≥ 25 % degradation (based on CO2 evolution data) within 14 days for the test substance to be considered non-toxic.	Evolved CO2 in the toxicity control at day 14: 50.76 %	Yes
Reference substance should exhibit ≥ 60 % biodegradation by day 14.	Net percent CO2 in the procedural control at day 14: 66.02 %	Yes
Difference between replicates A and B should not exceed 20 % at the plateau (approximately test day 14) or at test termination (test day 28).	The difference between the replicates at the plateau (test day 12) was 17.05 % and at test termination (test day 28) was 6.82 %. (NOTE: The larger difference at day 12 is due to a longer lag phase in the first replicate which may be attributed to the poor solubility of the test substance.)	Yes
The inorganic carbon content of the test substance suspension in the mineral medium at the beginning of the test must be < 5 % of the total carbon.	Inorganic carbon content of the test substance suspension in the mineral medium at the beginning of test was 4.95 % of the total carbon.	Yes
The evolution of CO2 in the blank controls should be ≤ 40 mg/L.	Blank control mean: 14.66 mg/L	Yes

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the conditions of the study, the test material attained 66.31 % degradation after 28 days. The test material can therefore be considered readily biodegradable. Furthermore, it achieved a mean of 60 % biodegradation on test day 14, which was within a 10-day window of reaching 10 % (which occurred between test day 5 and 7).

Executive summary:

The ready biodegradability of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 301.B and EU method C.4-C and under GLP conditions.

This study was performed to determine the potential for ultimate biodegradation of the test material in an aerobic aqueous medium by the carbon dioxide evolution method; the amount of carbon dioxide (CO2) released upon biodegradation of the test material and a reference substance, sodium benzoate, was measured to assess the potential for ultimate biodegradation. Two blank controls (containing inoculum), a procedural control (containing inoculum and reference substance), and a toxicity control (containing inoculum, reference substance, and test material) were established to account for background CO2 production, viability of the inoculum, and the toxicity of the test material, respectively. Test flasks were incubated aerobically in the dark for a period of 28 days.

The mean percent biodegradation observed in an aqueous test medium fortified with the test material at 15 mg C/L was 66.31 % on test day 28. Biodegradation of 50.76 % was observed in the toxicity control on test day 14, demonstrating that the test substance is not toxic to the inoculum (<25 % on test day 14 is considered toxic). Biodegradation in the procedural control was 66.02 % on test day 14, thus meeting the “pass” criteria of the test (reaching 60% biodegradation by test day 14). This rapid biodegradation of sodium benzoate confirmed the presence of an active microbial population and system integrity.

The difference between test material replicates A and B did not exceed 20 % at the plateau (approximately test day 12) or at test termination (test day 28). The inorganic carbon content of the test substance suspension in the mineral medium at the beginning of the test was determined to be <5 % of the total carbon (actual 4.95 % total carbon). The control blanks did not exceed 40 mg evolved CO2 per litre (blank control mean was 14.66 mg CO2/L).

Under the conditions of the study, the test material attained 66.31 % degradation after 28 days. The test material can therefore be considered readily biodegradable. Furthermore, it achieved a mean of 60 % biodegradation on test day 14, which was within a 10-day window of reaching 10 % (which occurred between test day 5 and 7).

Description of key information

Under the conditions of the study, the test material attained 66.31 % degradation after 28 days. The test material can therefore be considered readily biodegradable. Furthermore, it achieved a mean of 60 % biodegradation on test day 14, which was within a 10-day window of reaching 10 % (which occurred between test day 5 and 7).

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

The ready biodegradability of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 301.B and EU method C.4-C and under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

This study was performed to determine the potential for ultimate biodegradation of the test material in an aerobic aqueous medium by the carbon dioxide evolution method; the amount of carbon dioxide (CO2) released upon biodegradation of the test material and a reference substance, sodium benzoate, was measured to assess the potential for ultimate biodegradation. Two blank controls (containing inoculum), a procedural control (containing inoculum and reference substance), and a toxicity control (containing inoculum, reference substance, and test material) were established to account for background CO2 production, viability of the inoculum, and the toxicity of the test material, respectively. Test flasks were incubated aerobically in the dark for a period of 28 days.

The mean percent biodegradation observed in an aqueous test medium fortified with the test material at 15 mg C/L was 66.31 % on test day 28. Biodegradation of 50.76 % was observed in the toxicity control on test day 14, demonstrating that the test substance is not toxic to the inoculum (<25 % on test day 14 is considered toxic). Biodegradation in the procedural control was 66.02 % on test day 14, thus meeting the “pass” criteria of the test (reaching 60% biodegradation by test day 14). This rapid biodegradation of sodium benzoate confirmed the presence of an active microbial population and system integrity.

The difference between test material replicates A and B did not exceed 20 % at the plateau (approximately test day 12) or at test termination (test day 28). The inorganic carbon content of the test substance suspension in the mineral medium at the beginning of the test was determined to be <5 % of the total carbon (actual 4.95 % total carbon). The control blanks did not exceed 40 mg evolved CO2 per litre (blank control mean was 14.66 mg CO2/L).

Under the conditions of the study, the test material attained 66.31 % degradation after 28 days. The test material can therefore be considered readily biodegradable. Furthermore, it achieved a mean of 60 % biodegradation on test day 14, which was within a 10-day window of reaching 10 % (which occurred between test day 5 and 7).