Benzenesulfonic acid, 4-C15-16-sec-alkyl derivs.

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-12-12 until 2014-01-02 and 2014-01-16 until 2014-02-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The work involved two non-GLP OECD 301F studies with the aim to minimize inhibition of the inoculum by means of testing several concentrations of the test item and using silica gel to enable biodegradation.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

yes

Remarks:

The first OECD 301F study being conducted as typical; the second OECD 301F study was extended past the usual 28-day experiment to 42 days in order to demonstrate the inherent biodegradable status.

GLP compliance:

no

Remarks:

The laboratory complies the general requirements for the competence of testing and calibration laboratories.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of activated sludge: Escatawpa Wastewater Treatment Plant (ESC), Mississippi. secondary biological wastewater treatment system utilizing the activated sludge process.
- Laboratory culture: not applicable
- Method of cultivation: not applicable
- Storage conditions: not applicable
- Storage length: not applicable (settling immediately started)
- Preparation of inoculum for exposure:
The sludge after arrival is settled and decanted with mineral water added back. This settling procedure is repeated twice more and aerated over night prior to use. The day of the test, the air is turned off and inoculate settled in an Imhoff cone. The settled concentrate is drained into beaker and solids concentration determined.
- Concentration of sludge: 30mg/L of suspended solids (SS)

Duration of test (contact time):

>= 28 - <= 42 d

Initial conc.:

10 mg/L

Based on:

test mat.

Initial conc.:

20 mg/L

Based on:

test mat.

Initial conc.:

40 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: according to OECD 301F guideline
- Additional substrate: no (test 1December 2013) and yes (silica gel in test 2 January 2014).
- Solubilising agent (type and concentration if used): In test 2, approximately 500 mg of silica gel (Aldrich, Merck grade 9385) with a mesh size of 230-400 angstrom was weighed out and added to all test bottles (including blanks, reference compound and test material) after addition of the test compound and before the inoculate concentrate is added.
- Test temperature: 22±1 °C
- pH: not specified
- Suspended solids concentration: 30 mg/L
- Continuous darkness: not specified

TEST SYSTEM
- Culturing apparatus: 1-liter vessels
- Number of culture flasks/concentration: triplicates for test 1 and quadruplicates in test 2
- Measuring equipment: Coordinated Environmental Services (CES) Ltd (Cornwell, UK) aerobic electrolytic respirometer unit consisting of 20 one-liter
vessels fitted with heads containing O2 and CO2 sensors.
- Measurement frequency: CO2 generation and O2 uptake data is recorded by the instrument every six hours during the 28-day test.
- Other: Stirring is adjusted to 300-400rpm.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Blank 1-liter vessels are prepared identical to the samples (i.e. test medium, inoculate, and silica gel when present).
- Reference substance and control: Sodium benzoate, silica gel when present is also included in the reference 1-liter vessels.
- Abiotic sterile control: not applicable
- Toxicity control: no

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

>= 36.36 - <= 45.88

Sampling time:

28 d

Remarks on result:

other: (test 1, December 2013, 3 concentrations tested)

Key result

Parameter:

% degradation (O2 consumption)

Value:

>= 33.43 - <= 51.75

Sampling time:

28 d

Remarks on result:

other: (test 2, January 2014, 3 concentrations tested)

Key result

Parameter:

% degradation (O2 consumption)

Value:

>= 45.55 - <= 63.08

Sampling time:

42 d

Remarks on result:

other: (test 2, January 2014, 3 concentrations tested)

Key result

Parameter:

COD

Value:

ca. 2.643 other: mg COD/mg sample (test item, test 1 in December 2013)

Key result

Parameter:

COD

Value:

ca. 1.826 other: mg COD/mg sample (sodium benzoate, test 1 in December 2013)

Key result

Parameter:

COD

Value:

ca. 2.695 other: mg COD/mg sample (test item, test 2 in January 2014)

Key result

Parameter:

COD

Value:

ca. 1.752 other: mg COD/mg sample (sodium benzoate, test 2 in January 2014)

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

SOLOTERRA® 117H can be classified as “inherently biodegradable”. The second study was extended to 42-days to demonstrate that it can achieve 60% ThOD. The two tests clearly show that there is toxicity/inhibition to the test microbes. This results in considerable deficit for the %ThOD profile curves and likely impedes the ability to demonstrate “ready biodegradable” classification.

Executive summary:

The purpose of these studies was to establish the ready biodegradability of the product SOLOTERRA® 117H (Benzenesulfonic acid, 4-C15-16-sec-alkyl derivs.) as an indication of its environmental fate. Tests of ready biodegradability are by definition stringent tests that provide limited opportunity for acclimation and biodegradation to occur. A positive result in a test of ready biodegradability is an indication the test substance will undergo rapid and ultimate biodegradation in the environment. However, a negative result in a test of ready biodegradability does not necessarily mean that the test substance will not be biodegraded under relevant environmental conditions but that additional testing may be needed. The work involved two studies; the first being conducted as typical; the second attempts to take into account and control toxicity/inhibition as well as extending past the usual 28-day experiment to demonstrate inherent biodegradable status. The two experimental start dates for the study was 12 December 2013 to 2 January 2014 and 16 January to 27 February 2014 (second test was extended to 42-days).

The biodegradation test was performed on SOLOTERRA® 117H in triplicates for test one and quadruplicates in test two using a 20 channel Coordinated Environmental Services (CES) Ltd (Cornwell, UK) aerobic electrolytic respirometer unit following the OECD 301F guideline. A CES electrolytic respirometer unit consists of 20 one-liter vessels fitted with heads containing O2 and CO2 sensors. The system measures the amount of CO2 generated by means of a cuvette filled with 2% sodium hydroxide and a conductivity probe that determines the change in conductivity as CO2 is absorbed from the headspace gases of the test vessel. A differential pressure sensor head is designed to measure the amount of O2 used by respiring microorganisms. It achieves this by detecting the difference in pressure during the uptake of oxygen and generation and caustic scrub of CO2 and then supplying O2 back to the headspace using an electrolytic copper sulfate cell.

The electrolytic cell contains a chamber filled with copper sulfate solution. Oxygen is generated in this chamber by passing an electrical charge (initiated by the test vessel head pressure change sensor) between electrodes. It is connected to the headspace of the sample vessel by a vent. Valves are used to balance the pressure and return generated O2 to the headspace of the test vessel. The data is handled by a PC which continuously records the amount of CO2 (mg) absorbed and O2 (mg) generated.

The 1-liter vessels are placed in the water bath (22±1 degree C) and screwed into the respirometer head/cap. The electrolytic respirometer unit with magnetic stirrer belt system is activated slowly. Once all bottles are in place, the speed of the stirring is adjusted to 300-400 rpm. The instrument undergoes a 60 minute initialization /equalization before data collection begins. CO2 generation and O2 uptake data is recorded by the instrument every six hours.

The blank 1-liter vessels are prepared identical to the samples (i.e. test medium, inoculate, and silica gel when present) except that they did not contain the test substance. To minimize the number of blanks required in the test, the silica gel when present is also included in the benzoate reference 1-liter vessels. The oxygen demand results for SOLOTERRA® 117H and the benzoate reference are determined by blanking out the oxygen demand contribution from the blank inoculate vessels that may or may not contain the added silica gel.

The average biodegradation obtained in the first study for the following test item concentrations of 10 mg/L, 20 mg/L, and 40 mg/L were 36.36%, 45.88%, and 44.75%, respectively, within 28 days.

In the extended second study, the average biodegradation after 28 days of 33.43%, 44.86%, and 51.75% and after 42 days of 45.55%, 57.68%, and 63.08%, for the respective test item concentrations of 10 mg/L, 20 mg/L, and 40 mg/L, were achieved.

The validity of the biodegradation test was confirmed by meeting the following criteria as specified in the OECD 301 F Test Guidelines.

• The delta of the max./min. of replicate %ThOD (COD) values at the plateau of the data, at the end of the test or at the end of the 10-day window, as appropriate, is less than 20%.

• The extent of biodegradation of the benzoate reference compound has reached the pass level of 60% ThOD (COD) on or before day 10-day window.

• The total O2 consumption in inoculum blanks with or without silica did not exceed 60 mg/L in 28 days. The maximum O2 consumed was < 30 mg/L.

• As the combined ThOD exceeded 25%, the test chemical SOLOTERRA® 117H can’t be assumed to be inhibitory based on the guidelines. However, there is an observed lag period, a reduction in respiration as compared to the controls that occurs. This resulted in a negative deficit for the %ThOD. The protocol criterion is not reflective of true inhibition in the case of SOLOTERRA® 117H.

• The sterility controls of similar compounds conducted in earlier studies demonstrated that biodegradation and not chemical oxidation was occurring in this test.

Based on the results of the two performed OECD 301F studies, the test item Benzenesulfonic acid, 4 -C15 -16 -sec-alkyl derivs. can be considered inherently biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2013-10-23 until 2013-11-21

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The ready biodegradation study with Benzenesulfonic acid, 4-C15-16-sec-alkyl derivs., carried out according to OECD guideline 301B, resulted in no/negligible biodegradation after 28 days. Although all validation criteria are met in regard to the guideline, based on our experience and experts’ judgment, we can consider the obtained result as not reliable. The reasons are obvious; the underlying study shows significant methodological deficiencies, as the substance properties and possible inhibition was not addressed. Hence, the test system is unsuitable. Despite the biodegradation of > 25% in the toxicity control within 14 days, we can see an indication of toxicity. We will test the biodegradation behavior of Benzenesulfonic acid, 4-C15-16-sec-alkyl derivs. in another biodegradation study at a lower concentration dispersed on silica gel to reduce toxicity and enable degradation by the microorganisms.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

(Temporary breakdown in the aeration < 1 day occurred between day 25 and 26. Evaluation.)

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: municipal sewage treatment plant ('Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands)
- Laboratory culture: no
- Method of cultivation: not applicable
- Storage conditions: used immediately (after a 49 minutes settling period)
- Preparation of inoculum for exposure: supernatant liquid of settled sludge was used as inoculum at the amount of 10 ml/L; inoculum and Milli-RO water were added to each bottle and aerated with synthetic air overnight to purge the system of CO2 prior to the test start
- Pretreatment: no
- Concentration of sludge: 4.5 g suspended solids/L in concentrated sludge
- Water filtered: not mentioned

Duration of test (contact time):

ca. 28

Initial conc.:

12 mg/L

Based on:

other: TOC

Initial conc.:

23 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Stock solutions:
A) 8.50 g KH2PO4, 21.75 g K2HPO4, 67.20 g Na2HPO4.12H2O, 0.50 g NH4Cl /1L Milli-Q water
B) 22.50 g MgSO4.7H2O /1L Milli-Q water
C) 36.40 g CaCl2.2H2O /1L Milli-Q water
D) 0.25 g FeCl3.6H2O /1L Milli-Q water
- Composition of medium: 10 ml of solution A and 1 ml of each of the solutions B - D /1L Milli-Q water
- Additional substrate: no
- Solubilising agent (type and concentration if used): no
- Test temperature: 20.9 - 21.8°C
- pH: 7.5 - 7.8 (all bottles at start and test end)
- pH adjusted: yes, at test start with 1 M HCl (Merck, Darmstadt, Germany) in blanks and positive control A
- Suspended solids concentration: 4.5 g suspended solids/L in concentrated sludge
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2 litre all-glass brown coloured bottles
- Number of culture flasks/concentration: 2 flasks per concentration
- Method used to create aerobic conditions: synthetic air (CO2 < 1 ppm) for constant aeration during the test period
- Synthetic air: Mixture of oxygen (20%) and nitrogen (80%) was passed through 0.0125 M Ba(OH)2 solution, synthetic air was sparged through the scrubbing solution at a rate of approx. 1-2 bubbles per second (ca. 30-100 ml/min)
- Method used to create anaerobic conditions: not applicable
- Measuring equipment: Titration of remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany)
- Details of trap for CO2: three bottles filled with 100 ml 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle

SAMPLING
- Sampling frequency: Days 2, 5, 7, 9, 14, 19, 23, 27, 29 (Days 2, 5, 7, 9, 14 for positive and toxicity controls)
- Sampling method: CO2-absorber bottle nearest to the test bottle used for titration, remaining absorber bottles were moved one position towards test bottle and fresh CO2-absorber bottle was placed at the end of the trap series
- Sterility check if applicable: not applicable
- Sample storage before analysis: not applicable

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes (2 flasks)
- Abiotic sterile control: no
- Toxicity control: yes (1 flask)
- Positive control: yes (2 flasks)

STATISTICAL METHODS: no statistics performed

Reference substance:

acetic acid, sodium salt

Remarks:

(12 mg TOC/l in final test medium)

Parameter:

% degradation (CO2 evolution)

Value:

ca. 6

Sampling time:

28 d

Remarks on result:

other: 12 mg TOC/L

Details on results:

The TOC concentration of the 1 g/l Benzenesulfonic acid, mono, C15-C16-alkyl derivs. solution was determined to be 519.3 mg/l (=51.93%). The ThCO2 of Benzenesulfonic acid, mono, C15-C16-alkyl derivs. was calculated to be 1.90 mg CO2/mg. The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.
The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of Benzenesulfonic acid, mono, C15-C16-alkyl derivs.. In the toxicity control more than 25% biodegradation occurred within 14 days (32%, based on ThCO2). Functioning of the test system was checked by testing the reference substance sodium acetate, which showed a normal biodegradation curve.

Biodegradation [%] calculated as the ratio between CO2 produced (cumulative) and the sum of the ThCO2

Day	2	5	7	9	14	19	23	27	29	29	29
test item (a) [%]	0	0	1	1	1	2	2	2	2	3	3
test item (b) [%]	0	1	2	3	4	5	5	6	7	8	8
reference (a)  [%]	5	36	53	63	73	-	-	-	-	-	-
reference (b)  [%]	4	33	48	57	69	-	-	-	-	-	-
tox control  [%]	1	12	18	24	32	-	-	-	-	-	-
mean blank [mg CO2/L]	3.3	8.6	13.4	18.2	23.2	29.3	38.6	45.8	52.1	54.7	56.7

 

Validity criteria fulfilled:

yes

Remarks:

In the toxicity control more than 25% biodegradation occurred within 14 days (32% on day 14, based on ThCO2).

Interpretation of results:

other: not readily biodegradable

Conclusions:

Benzenesulfonic acid, mono, C15-C16-alkyl derivs. was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Executive summary:

The study procedures described in this report were based on the OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-C and ISO 9439, 1999.

The batch of Benzenesulfonic acid, mono, C15-C16-alkyl derivs., a dark brown viscous liquid with a purity of 89.2%, was easily soluble in water. Therefore, the test media were prepared using a stock solution of 1 g/l in Milli-RO water. The TOC concentration of the clear light brown solution was determined to be 519.3 mg/l (51.93%). The Theoretical CO2 production (ThCO2) of Benzenesulfonic acid, mono, C15-C16-alkyl derivs. was calculated to be 1.90 mg CO2/mg. Benzenesulfonic acid, mono, C15-C16-alkyl derivs. was tested in duplicate at 12 mg TOC/l, corresponding to 23 ml of the stock solution/l (=23 mg/l). The exact amounts of the stock solution were added to the test medium, containing the microbial organisms, of test substance bottles A and B and the toxicity control (final volume: 2 litres). The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms. Test duration was 28 days (last CO2-measurement on the 29th day).

The study consisted of 7 bottles:

- 2 inoculum blanks (no test substance),

- 2 test bottles (Benzenesulfonic acid, mono, C15-C16-alkyl derivs.),

- 2 positive control (sodium acetate) bottles and

- 1 toxicity control (Benzenesulfonic acid, mono, C15-C16-alkyl derivs. plus sodium acetate).

The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of Benzenesulfonic acid, mono, C15-C16-alkyl derivs..

In the toxicity control, Benzenesulfonic acid, mono, C15-C16-alkyl derivs. was found not to inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion, Benzenesulfonic acid, mono, C15-C16-alkyl derivs. was designated as not readily biodegradable.

Description of key information

The ready biodegradability of Benzenesulfonic acid, 4-C15-16-sec-alkyl derivs. was assessed in various studies.

Based on the observations in an OECD 301B GLP study, two further 301F studies with slight modifications were conducted. The first OECD 301F study being conducted as typical; the second OECD 301F study attempted to take into account and control toxicity/inhibition as well as extending past the usual 28-day experiment to demonstrate inherent biodegradable status.

The initial ready biodegradation study with Benzenesulfonic acid, 4-C15-16-sec-alkyl derivs., carried out according to OECD guideline 301B, resulted in no/negligible biodegradation after 28 days. Although all validation criteria are met in regard to the guideline, based on our experience and experts’ judgment, it can be considered that the obtained result is not reliable. The underlying study shows significant methodological deficiencies, as the substance properties and possible inhibition was not addressed. Hence, the test system is unsuitable.
To address the shortcomings of the first biodegradation study, Benzenesulfonic acid, 4-C15-16-sec-alkyl derivs. was tested subsequently in further biodegradation studies according to OECD 301F guideline at different concentrations and in the latest setup also extended and dispersed on silica gel to reduce toxicity and enable degradation by the microorganisms.
The obtained results and observations made during the two studies show that Benzenesulfonic acid, 4-C15-16-sec-alkyl derivs. does induce inhibition to the inoculum, but can be considered inherently biodegradable as shown in the extended 301F study.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Additional information

Based on the OECD guidelines, Benzenesulfonic acid, 4 -C15 -16 -sec-alkyl derivs. is not inhibitory to the inoculum. In contrast, the negative %ThOD during the first 10 -14 days of the extended OECD 301F study clearly shows that the oxygen uptake in all three concentrations tested is being impacted and hence reduced below the background oxygen uptake. This phenomenon can only occur as a result of some form of microbial toxicity or inhibition. It appears to be lessened in the test where silica gel is present. It is more dramatic/exacerbated in the lower concentrations due to less total mg ThOD. During the second OECD 301F test, in the first 24 hours the % inhibition to respiration is greater than 80% at the 40 mg/L concentration. The EC50 for % inhibition appears to be less than 10 mg/L. In conclusion the silica gel did provide some reduction to the maximum negative %ThOD observed at all test concentrations.

Benzenesulfonic acid, 4-C15-16-sec-alkyl derivs. can be considered inherently biodegradable based on the results obtained in an extended OECD 301F test resulting in > 60% biodegradation after 42 days.