Dodecyl(2-hydroxy-3-sulphonatopropyl)dimethylammonium

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:

07-07-2016 to 12-09-2016 (revised report: 24-01-2020)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Remarks:

The procedure used was the "Method for Testing the Biodegradability of Chemical Substances by Microorgainsms" that is stipulated in Japanese law for the testing of new chemical substances.

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

The procedure used was the "Method for Testing the Biodegradability of Chemical Substances by Microorganisms" that is stipulated in Japanese law for the testing of new chemical substances. In addition to the core cumulative oxygen uptake measurements, the method also requires specific analysis for residues of the test substance and (solubility permitting) dissolved organic carbon at the end of incubation.

Deviations:

no

Principles of method if other than guideline:

The test method in this study corresponds to OECD test guideline 301C (Ready Biodegradability: Modified MITI Test (I)) and accounted for the validity criteria required by this guideline.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The composition of the test substance was as follows:
test item: 37.3%
water: 56.2%
NaCl: 6.5%

The 37.3% 'test item' component comprised the following organic constituents:

"Main component" 13197-76-7 31.0% C17H37NO4S mol wt 351.55
"Di-ol" 35396-47-5 2.8% C3H7NaO5S mol wt 178.15
"Disulfonate" 163709-39-5 1.7% C3H6Na2O7S2 mol wt 264.20
"Cation" 103947-09-7 1.0% C17H38ClNO2 mol wt 323.93
"Chlorohydrin" 126-83-0 0.8% C3H6ClNaO4S mol wt 196.59

Based on the individual constituents and their respective abundance, the theoretical oxygen demand was 66.6 mg/mg, calculated from the composite empirical formula C225H491ClN12.8Na3.72O65.5S16.0 and assuming nitrogen was oxidised to nitrate (NO3).

The identity of the test substance held in storage at the test facility was confirmed by infrared absorption spectrometry before and after the study. The initial spectrum recorded at the test facility matched the IR spectrum provided by the study sponsor. The final spectrum showed no evidence of instability in storage during the in-life phase of the biodegradation test.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

The inoculum was prepared according to standard Japanese practice, originating as a composite of samples obtained from 10 specified locations that included surface water and sediment from rivers, lakes and inland seas and return sludge from sewage treatment plants. The samples were collected during May 2016 and the composite inoculum was maintained by feeding with a synthetic sewage that comprised glucose, peptone and KH2PO4 dissolved in purified water. The batch of activated sludge formed in this way was first used on 206-06-22. It was used to inoculate this study on the experimental starting date, 2016-07-12.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

Additionally, concentrations of the test substance and its breakdown products were determined at the end of the test.

Details on study design:

The study design comprised six test vessels. Vessel No.1 contained the test substance (100 mg/L) in water, but with no added inoculum. Vessels No 2, 3 & 4 each contained the test substance (100 mg/L) in inoculated test medium. Vessel No. 5 contained inoculated medium with no further addition and served as the control. Vessel No. 6 contained the reference substance, aniline (100 mg/L) in inoculated medium. All inoculated media contained activated sludge at a concentration corresponding to 30 mg suspended solids/L.

Reference substance:

aniline

Remarks:

(Wako Pure Chemical Industries, Lot number KPF0517)

Preliminary study:

Not performed.

Test performance:

All vessels contained a working volume of 300 mL and the contents were continually stirred. CO2 was absorbed from the sealed headspaces and the vessels were connected as a closed system to an automatic electrolytic respirometer (Ohkura Electric) that measured biochemical oxygen demand (BOD) continuously during the 28-day incubation. The vessels were maintained in a water bath held at 25 ± 1°C.

Key result

Parameter:

% degradation (O2 consumption)

Value:

63

St. dev.:

0

Sampling time:

28 d

Parameter:

% degradation (DOC removal)

Value:

65

St. dev.:

1.5

Sampling time:

28 d

Parameter:

% degradation (test mat. analysis)

Value:

96

Sampling time:

28 d

Remarks on result:

other: The percentage biodegradation of the test item was calculated using the sum of residual amounts of each component, because five components of the test item were determined individually by LC-MS

Parameter:

% degradation (test mat. analysis)

Remarks:

Main component determined by LC-MS

Value:

100

Sampling time:

28 d

Remarks on result:

other: No intact main component was detected at the end of the incubation, indicating that complete primary degradation had occurred during the test.

Parameter:

% degradation (test mat. analysis)

Remarks:

"Di-ol" component of test substance determined by LC-MS

Value:

100

St. dev.:

0

Sampling time:

28 d

Remarks on result:

other: No intact "Di-ol" component was detected at the end of the incubation.

Parameter:

% degradation (test mat. analysis)

Remarks:

"Disulfonate" component determined by LC-MS

Value:

37

Sampling time:

28 d

Remarks on result:

other: The mean recovery of "Disulfonate" on Day 28 was 37%, relative to the concentration measured in vessel No. 1 containing test item in water without inoculum.

Parameter:

% degradation (test mat. analysis)

Remarks:

"Cation" component determined by LC-MS

Value:

100

St. dev.:

0

Sampling time:

28 d

Remarks on result:

other: No intact "Cation" component was detected at the end of the incubation.

Parameter:

% degradation (test mat. analysis)

Remarks:

"Chlorohydrin" component determined by LC-MS

Value:

46

St. dev.:

2.1

Sampling time:

28 d

Remarks on result:

other: The mean recovery of "Chlorohydrin" on Day 28 was 54%, relative to the concentration measured in vessel No 1 containing test item in water without inoculum.

Results with reference substance:

Aniline degradation (based on ThODNH3, assumng no nitrification) was 71%, 89%, 90% and 91% after 7, 14, 21 and 28 days, respectively.

Table 1: Biodegradation of A-20HD based on biochemical oxygen demand (BOD)

Test vessel contents	Vessel No.	7^thday		14^thday		21^stday		28^thday		Average
		BOD (mg)	Deg. (%)	BOD (mg)	Deg. (%)	BOD (mg)	Deg. (%)	BOD (mg)	Deg. (%)	Deg. (%)
(Water + test item)	[1]	0.5	-	0.7	-	0.7	-	0.7	-	-
Sludge + test item	[2]	15.1	17	37.5	48	46.5	61	48.0	63	63
	[3]	15.3	17	37.4	48	46.5	61	48.3	63
	[4]	15.1	17	40.3	52	46.7	61	48.3	63
Control blank	[5]	3.8	-	5.4	-	5.9	-	6.1	-	-
Sludge + aniline	[6]	55.0	71	69.5	89	71.0	90	71.6	91	-

 

Table 2: Biodegradation of A-20HD based on dissolved organic carbon (DOC)

Test vessel contents	Vessel No.	DOC measured on day 28 (mgC/L)	Residual amount of DOC (mgC)	Residue (%)	Biodeg. (%)	Average biodeg. (%)
(Water + test item)	[1]	52.86	15.9	99	-	-
Sludge + test item	[2]	18.50	5.6	35	65
	[3]	18.00	5.4	34	66	65
	[4]	19.64	5.9	37	63
Control blank	[5]	n.d.	-	-	-	-

 

Table 3: Analytical results of test solutions after 28 days

		Water + test item	Sludge + test item			Theoretical amount
		Vessel No. 1	Vessel No. 2	Vessel No. 3	Vessel No. 4
BOD	mg	0.7	41.9	42.2	42.2	66.6
Residual amount and percentage residue of DOC	mgC	15.9	5.6	5.4	5.9	16.0
	%	99	35	34	37	-
Residual amount and percentage residue of Main component (by LC-MS)	mg(1)	25.1	0	0	0	24.9
	%	101	0	0	0	-
Residual amount and percentage residue of Di-ol (by LC-MS)	mg(2)	2.10	0	0	0	2.25
	%	93	0	0	0	-
Residual amount and percentage residue of Disulfonate (by LC-MS)	mg(3)	1.32	0.86	0.86	0.88	1.37
	%	96	63	63	64	-
Residual amount and percentage residue of Cation (by LC-MS)	mg(4)	0.939	0	0	0	0.804
	%	117	0	0	0	-
Residual amount and percentage residue of Chlorohydrin (by LC-MS)	mg(5)	0.656	0.325	0.364	0.344	0.643
	%	102	51	57	53	-
Total residual amount and percentage residue of test item (Sum of (1) to (5))	mg	30.1	1.2	1.2	1.2	30.0
	%	100	4	4	4	-
Detection of converted products (by LC-MS)	-	Detected	Four components detected	One component detected	Four components detected	-

 

Table 4: Calculated value of DOC of residual components

		Sludge + test item
		Vessel No. 2	Vessel No. 3	Vessel No. 4
Residual amount of Disulfonate	mg	0.86	0.86	0.88
	mgC (1)	0.12	0.12	0.12
Residual amount of Chlorohydrin	mg	0.325	0.364	0.344
	mgC (2)	0.0596	0.0667	0.0630
Produced amount of converted product A	mg	13.0	13.0	13.0
	mgC (3)	4.25	4.25	4.25
Residual amount of DOC [calculated value: (1) + (2) + (3)]	mgC	4.4	4.4	4.4
Residual amount of DOC (measured value)	mgC	5.6	5.4	5.9

 

Table 5: BOD calculated from biodegraded components of test item

			Sludge + test item
			Vessel No. 2	Vessel No. 3	Vessel No. 4
The carbon chain part of Main component (C12H25)	Theoretical amount	mg	12.0	12.0	12.0
	Corresponding amount of BOD	mg (1)	41.4	41.4	41.4
Di-ol	Decrease amount	mg	2.25	2.25	2.25
	Corresponding amount of BOD	mg (2)	1.41	1.41	1.41
Disulfonate	Decrease amount	mg	0.51	0.51	0.49
	Corresponding amount of BOD	mg (3)	0.22	0.22	0.21
Cation	Decrease amount	mg	0.804	0.804	0.804
	Corresponding amount of BOD	mg (4)	2.14	2.14	2.14
Chlorohydrin	Decrease amount	mg	0.318	0.279	0.299
	Corresponding amount of BOD	mg (5)	0.194	0.170	0.183
BOD [calculated value: (1)+(2)+(3)+(4)+(5)]]		mg	45.4	45.3	45.3
BOD (measured value)		mg	41.9	42.2	42.2

 

Validity criteria fulfilled:

yes

Remarks:

The differences between extremes in terms of test substance biodegradation were 0% (BOD and LC-MS) and 3% (DOC). The reference substance reached 71% and 89% degradation after 7d and 14d, respectively, based on ThODNH3.

Interpretation of results:

readily biodegradable

Conclusions:

The biodegradability of dodecyl(2-hydroxy-3-sulphonatopropyl)dimethylammonium was determined in accordance with OECD TG 301C (MITI-I test) to fulfil the national requirement under Japanese law for the notification of new chemical substances. Under the test conditions, most of the test item (about 60% of the organic matter) was biodegraded and four converted products (A to D) were generated. Two minor constituents, "Disulfonate" and "Chlorohydrin", were only partly degraded and residues of these two substances were detected at the end of the test. According to OECD TG 301C, the criterion of ready biodegradability is that the percentage biodegradation by BOD reaches 60% on Day 28. Since the mean measured BOD reached 63% ThOD after 28 days, the test item is considered readily biodegradable.

Executive summary:

The biodegradability of dodecyl(2-hydroxy-3-sulphonatopropyl)dimethylammonium was determined in accordance with the version of OECD TG 301C (MITI-I test) that is the standard method used under Japanese law for the notification of new chemical substances in Japan.  The test substance was applied at 100 mg/L in the presence of a composite activated sludge inoculum at a concentration of 30 mg suspended solids/L.  Biochemical oxygen demand (BOD) was recorded continuously during the 28-day incubation and was related to the theoretical demand (ThOD) that corresponds to mineralisation of all components to their highest oxidation state, based on a composite empirical formula representing the sum of all components. In addition, measurements of dissolved organic carbon (DOC) were made in all vessels at the end of the test and the media were also analysed by LC-MS to detect residues of constituents initially present and undegraded breakdown products and intermediates. The test design comprised six vessels. Three were used to follow the biodegradation of the test substance, a fourth contained inoculated test medium with no further addition and served as a control blank, a fifth contained 100 mg aniline (reference compound)/L in inoculated test medium and the sixth contained test substance (100 mg/L) in purified water with no inoculum and served as a source of reference concentration measurements at Day 28 for calculations of % biodegradation based on DOC and LC-MS measurements.

Aniline was degraded by 71% and 89% after 7 and 14 days, respectively, confirming the satisfactory activity of the inoculum, and was completely degraded (91%) by the end of the test. The test substance was extensively degraded during the test and mean measured BOD represented 63% of ThOD on Day 28. Contemporary mean DOC removal in the test substance vessels was 65%. LC-MS analysis of the test vessel contents revealed that the constituents had been degraded to varying degrees during the test. No intact "Main component" was found, however a substance denoted "Converted product A", which contained a sulphonyl group and a quaternary nitrogen - and must therefore have been formed as a product of the degradation of the Main component - was found in all three test substance vessels. Converted product A was assigned the presumed structure HN+(CH3)2CH2CH(OH)CSO3- and the identity 3 -(dimethylammonio)-2 -hydroxypropane-1 -sulfonic acid. Three other degradates (termed "Converted product B", "Converted product C" and "Converted product D") that were formed from the Main component by sequential alkyl chain shortening were present in two flasks, but absent in the third, where the elimination of the alkyl chain is presumed to have run to completion before the end of the test. No residues of the "Di-ol" or "Cation" constituents remained in any of the triplicate test substance vessels on Day 28, however residues of "Disulfonate" and "Chlorohydrin" were found at mean concentrations corresponding to 63% and 54% of those detected in the vessel containing only test substance and water, indicating that these constituents had degraded on average by 37% and 46%, respectively.

According to OECD TG 301C, the criterion of ready biodegradability is that the percentage biodegradation by BOD reaches 60% on Day 28. Since the mean measured BOD reached 63% ThOD after 28 days, the test item is considered readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1995-05-19 to 1995-06-16

Reliability:

4 (not assignable)

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:

no

Specific details on test material used for the study:

Identity: Betadet S-20.
Description: Colourless liquid.
Batch/Lot number: Not reported.
Considered to be the batch (7244) received by the same laboratory on 1995-05-01 for acute toxicity testing with D. magna (Point 6.1.3, Wetton (1995)). Purity: Not reported.

The Certificate of Analysis for batch 7244 (not included in the report) provides the following information:
Batch: 7244 (29/03/1995)
Appearance: Liquid, transparent, slightly yellowish, fluid
Dry matter: 44.5%
pH: 6.8
Sodium chloride: 6.4%

The principal constituent (included in the 44.5% 'dry matter') represented approximately 30% (w/w) of the tested batch.

Oxygen conditions:

aerobic

Inoculum or test system:

other: Secondary STP effluent.

Details on inoculum:

Effluent from the secondary (activated sludge) treatment stage at Belper STP, Derbyshire, UK, which treats sewage of predominantly domestic origin. Test media were inoculated at the rate of one drop of effluent per litre.

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The test design comprised the following four treatments:
1) blank control, consisting of inoculated test medium; 2) test substance Betadet S-20 dosed at 10 mg/L (nominally 4.3 mg ThOD/L) in inoculated test medium; 3) reference substance sodium benzoate dosed at 3 mg/L (nominally 5.0 mg ThOD/L) in inoculated test medium; 4) toxicity control comprising Betadet S-20 (10 mg/L) plus sodium benzoate (1.5 mg/L), giving a combined nominal ThOD of 6.8 mg O2/L, in inoculated test medium. The various media were dispensed to sufficient test vessels at initiation to facilitate measurements of residual dissolved oxygen (DO) concentrations in pairs of vessels at each of the following timepoints: 3, 6, 9, 12, 15, 21 and 28 days.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

Not relevant.

Test performance:

The % biodegradation values calculated for the reference compound sodium benzoate are based on a measured COD value of 1.67 mg O2/mg, which corresponds to the ThOD value that may be calculated from the molecular formula. The % biodegaradtion values presented for the reference substance are therefore considered to be reliable.

The % biodegradation values calculated for the test substance Betadet S-20 are based on a measured COD value of 0.43 mg O2/mg, which is ca. 5x lower than the ThOD that may be estimated from the structural formula. The reliability of the % biodegradation values presented for Betadet S-20 in the table above is therefore uncertain and it cannot be ruled out that the values may be over-estimated by as much as x5. It is therefore not possible to make a reliable quantitative assessment of ready biodegradability based on the information presented in the test report. From a purely qualitative point of view it is evident that at least partial biodegradation did occur: the degradation of Betadet S-20 followed a sigmoid curve, with a phase of rapid oxidation that commenced after approximately 8 days and declined at about Day 15.

The toxicity control vessel contained half the concentration of sodium benzoate as was present in the reference medium where Na benzoate was dosed alone, and the same concentration of Betadet S-20 as was present in the test substance treatment. Because of the unreliablity of the COD value of the Betadet S-20 component, it is not possible to evaluate % biodegradation of the toxicity control mixture reliably. Nevertheless, the degradation curve for the toxicity control is biphasic and it is possible to match the second phase to the onset of degradation of the Betadet S-20 component as it coincides with the timing of the rapid degradation phase observed in the vessels dosed with the test substance alone. The first phase of the toxicity control degradation curve up to and including Day 6 is due to Na benzoate oxidation.

Parameter:

% degradation (O2 consumption)

Value:

78

Sampling time:

28 d

Results with reference substance:

Sodium benzoate was biodegraded by 66% within 3 days and biodegradation at the end of the test on Day 28 was 93%.

Validity criteria fulfilled:

yes

Remarks:

Biodegradation of the reference substance exceeded 60% of ThOD within 3 days and mean O2 uptake in the blank control treatment remained below 2 mg O2/L throughout the incubation.

Interpretation of results:

other: It is not possible to make a reliable quantitative evaluation of the biodegradation of Betadet S-20 or to draw a reliable conclusion on its ready biodegradability from this study.

Conclusions:

A study of the ready biodegradability of Betadet S-20 was performed according to the closed Bottle Test (OECD TG 301D). The study was dosed with Betadet S-20 at a concentration of 10 mg/L, based on a chemical oxygen demand (COD) value of 0.43 mg O2/mg determined by a bench-top digestion kit system, and the same COD value was used as the comparator for calculating percentage biodegradation from measurements of biochemical oxygen demand (BOD) recorded at intervals during the 28-day incubation. However, the COD value obtained for Betadet S-20 is ca. 5x lower than would be anticipated based on structural considerations, which suggests the COD determination achieved only partial oxidation. This would in turn mean that the biodegradation percentages calculated for Betadet S-20 are over-estimated by a similar margin. Betadet S-20 was reported to have achieved 78% biodegradation in 28 days and to have fulfilled the criteria for classification as readily biodegradable, however these findings must be considered to be unreliable in view of the uncertainty over the COD value. The test data do provide qualitative evidence of Betadet S-20 mineralisation under stringent Closed Bottle Test conditions, but they are presented here as supporting information only.

Unreliability of the biodegradability outcome notwithstanding, the results obtained in this study with the reference substance sodium benzoate and the assessment of the microbial toxicity of Betadet S-20 that may be inferred from the toxicity control that contained sodium benzoate and Betadet S-20 in combination are considered to be valid. These secondary aspects of the study are considered separately under Point 6.1.7.

Description of key information

The biodegradability of dodecyl(2-hydroxy-3-sulphonatopropyl)dimethylammonium was determined in accordance with the version of OECD TG 301C (MITI-I test) that is the standard method used under Japanese law for the notification of new chemical substances in Japan.  The test substance was applied at 100 mg/L in the presence of a composite inoculum at a concentration of 30 mg suspended solids/L.  Biochemical oxygen demand (BOD) was recorded continuously during the 28-day incubation and was related to the theoretical demand (ThOD) that corresponds to mineralisation of all components to their highest oxidation state, based on a composite empirical formula representing the sum of all components. In addition, measurements of dissolved organic carbon (DOC) were made in all vessels at the end of the test and the media were also analysed by LC-MS to detect residues of constituents initially present and undegraded breakdown products and intermediates.

According to OECD TG 301C, the criterion of ready biodegradability is that the percentage biodegradation by BOD reaches 60% on Day 28. Since the mean measured BOD reached 63% ThOD after 28 days, the test item is considered readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater