Quaternary ammonium compounds, C18-22 (even numbered) alkyltrimethyl, chlorides; Docosyltrimethylammonium chloride; C18-22 TMAC

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From February 26, 2009 to April 14, 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide 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): Activated sludge was obtained from three locations on 26 February 2009; Totnes Sewage Treatment Works (STW), Devon, UK, Buckland STW, Newton Abbot, Devon UK, and Countess Weir STW, Exeter, Devon, UK. These STWs all treat sewage of predominantly domestic origin. At the laboratory, the activated sludge was kept aerated at room temperature.

- Preparation of inoculum for exposure: The solids concentrations of the three sludges was determined on the day of collection. Appropriate volumes of each sludge were mixed in order to give similar concentrations of suspended solids from each sewage treatment works in the final inoculum. The sludge was then diluted in medium to give a total sludge solids concentration in the test of 30 mg/L. This solution was added to test vessels and aerated at test temperature for five days until required for use.

Duration of test (contact time):

28 d

Reference substance:

benzoic acid, sodium salt

Remarks:

Radiolabelled

Key result

Parameter:

% degradation (CO2 evolution)

Value:

80

Sampling time:

28 d

Remarks on result:

other: maximum degradation

Details on results:

In the benzoic acid vessel more than 60% degradation was achieved within the 10 day window as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms. By day 35 approximately 83% of the applied radioactivity was measured as [14C]carbon dioxide.

Substantial mineralisation of [14C] radio labelled test substance was measured in all the test vessels. The addition of LAS made no discernable difference to the observed degradation. The concentration of [14C] radiolablled test substance was below that which would cause inhibition to the activated sludge bacteria, so there was no amelioration of inhibition by the LAS. Benzoic acid was shown to be readily biodegradable, with at least 60% of the applied radioactivity being converted to carbon dioxide by day 7. please see below table for details.

	[14C] radio labelled test substance	[14C] radio labelled test substance + LAS	Ref. substance, [14C]benzoic acid
Lag time	Days 0 to 3	Days 0 to 3	Days 0 to 1
Degradation time	Days 3 to 28	Days 3 to 28	Days 1 to 25
Maximum level of degradation	80%	75%	83%
Readily biodegradable, in compliance with criteria of Regulation (EC) 648/2004	yes	yes	yes

Mass Balance:

The mass balance was calculated using the [¹⁴C]carbon dioxide evolution values measured during the study and the additional sampling on Day 35. The total radioactivity recovered from each vessel was between 89 and 102% of the applied radioactivity, with the exception of one of the benzoic acid vessels, where a mass balance of 136% was measured. The sodium hydroxide from this vessel’s carbon dioxide trap had been accidentally added to the sodium hydroxide from one of the test vessels on day 21. The amount of [¹⁴C]carbon dioxide evolved from this vessel between days 14 and 21 was calculated from the difference between the measurements done on days 14 and 21, but because much more radioactivity was applied to the [14C] radio labelled test substance vessels than to the benzoic acid (approximately 100 times more), this calculated value was subject to large errors. Therefore, the results from only one of the benzoic acid vessels was used for results calculations.

Radioactivity measured in sludge solids

At the end of the study approximately 18% of the applied radioactivity was measured in the sludge solids from the [14C] radio labelled test substance vessels, and approximately 11% in the sludge solids from the [14C] radio labelled test substance plus LAS vessels.

In the benzoic acid reference substance vessels approximately 15% of the applied radioactivity was measured in the sludge solids.

Radioactivity measured in the filtrate

At the end of the study less than 5% of the applied radioactivity was measured in the filtrate from any of the test or reference substance vessels.

 

Radioactivity evolved as [¹⁴C]carbon dioxide

A significant amount of mineralisation was measured in all the vessels. In the benzoic acid vessel more than 60% degradationwas achieved within the 10 day window as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms. By day 35 approximately 83% of the applied radioactivity was measured as [¹⁴C] carbon dioxide.

 

The following phases (approximate) were observed for benzoic acid:

Phase	Time period
Lag phase	days 0 to 1
Degradation phase	days 1 to 25
10-day window	days 1 to 11

 

where;

Lag phase	Is the period from inoculation until the degradation has increased to 10%
Degradation phase	Is the time from the end of the lag phase to the time when 90% of the maximum level of degradation has been reached
10-day window	The 10 days immediately following the attainment of 10% degradation

 

In the [14C] radio labelled test substance vessels a significant amount of the applied radioactivity was evolved as [¹⁴C] carbon dioxide during the study, reaching between 75 and 80% in the vessels with and without LAS, with no discernable difference between the two treatments. Over 60% biodegradation was observed within 28 days, as shown in Figure 4, so [14C] radio labelled test substance can be classified as “readily biodegradable”, based on the criteria for Regulation (EC) 648/2004.

 

The following phases (approximate) were observed, for [14C] radio labelled test substance with and without LAS: 

Phase	Time period
Lag phase	days 0 to 3
Degradation phase	days 3 to 28
10-day window	days 3 to 13

Mass balance (recovered radioactivity shown as measured value (Bq) and percentage of applied)

Vessel contents	Applied radio-activity (Bq)	Recovered radioactivity
		Carbon dioxide traps		Filtrate		Sludge solids		Total
		Bq	%	Bq	%	Bq	%	Bq	%
[14C] radio labelled test substance	3140462	2502356	79.7	124376	3.96	593141	18.9	3219873	103
	3140462	2342399	74.6	129043	4.11	565757	18.0	3037199	96.7
[14C] radio labelled test substance plus LAS	3191703	2376303	74.5	135814	4.26	369177	11.6	2881294	90.3
	3191703	2381277	74.6	159218	4.99	301235	9.4	2841730	89.0
[^14C]Benzoic acid	40370	-^a	-^a	1652	4.09	7929	19.6	-^a	-^a
	40370	33610	83.3	1752	4.34	4553	11.3	39915	98.9

 

a =No result, due to carbon dioxide trap sampling error

Carbon dioxide evolution (% of applied radioactivity)

Vessel contents	Day 3	Day 7	Day 14	Day 21	Day 28	Day 35
[14C] radio labelled test substance	7.1	26	48	61	72	80
	11	26	48	52	67	75
[14C] radio labelled test substance plus LAS	8.4	27	51	54	68	74
	8.9	28	49	54	68	75
[^14C]Benzoic acid	52	65	75	107	110	112
	47	60	70	69	80	83

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the study conditions, the test substance was considered as readily biodegradable in a CO2 evolution test.

Executive summary:

A study was conducted to determine the ready biodegradability of the read across substance, ‘C22 TMAC (active: 99.9%)', using CO2 evolution test, according to OECD Guideline 301B, in compliance with GLP. Activated aerobic sludge was prepared with domestic origin three sewage plants and in order to have a concentration of total sludge solids of about 30 mg/L in the final 3 L of inoculated mixture. In the study, duplicate test vessels were prepared for positive control (blend of [14C] benzoic acid and benzoic acidat 0.2 mg/L) and read across substance ([14C] C22-ATQ at0.2 mg/L).Additional duplicate vessels were prepared which contained [14C] C22-ATQ at a nominal concentration of 0.2 mg/L and LAS at 0.17 mg/L. The purpose of these vessels was to show if the presence of LAS would ameliorate any inhibition of bacteria caused by [14C] C22-ATQ.The read across and reference substances were assessed for the rate and extent of biodegradation when exposed to Domestic activated sludge over a period of 35 d. Before and after dosing triplicate 1 mL aliquots of all the dosing solutions were taken for analysis by LSC (Liquid scintillation counting). For each dosing solution the mean of all six replicates was used to calculate the amount of radioactivity in the solutions, and the radio activity dosed to the test vessels. At the sampling points (3 h, Days 3, 7, 14, 21 and 28) the contents of the carbon dioxide trap nearest the test vessel was transferred to pre-weighed NalgeneTM bottle. This was re-weighed and triplicate 0.5 mL aliquots removed for LSC analysis. On Day 35 (the end of the exposure phase) the contents of both traps were added to the bottle and samples taken for LSC analysis. At the end of the study the test solutions were filtered through Whatman No 4 filter paper and weight of the filtrate measured. The filter papers were combusted to quantify radioactivity associated with the solid sludge. At the end of the study approximately 18% of the applied radioactivity was measured in the sludge solids from [C14] read across substance vessel, and approximately 11% in the sludge solids from the read across substance plus LAS vessels. In the reference substance benzoic acid vessels approximately 15% of the applied radioactivity was measured in the sludge solids. In the [14C] read across substance vessels a significant amount of applied radioactivity was evolved as [14C] carbon dioxide during the study, reaching 74 to 80% in the vessels with and without LAS, with no discernible difference between the two treatments within 28 d. In the reference substance, benzoic acid vessel more than 60% biodegradation was achieved with in the 10 d window as expected. All validity criteria for the acceptability of the test were met. Under the study conditions, the read across substance was considered as readily biodegradable (Noble, 2009). Based on the results of the read across study, the test substance, C18-22 TMAC, is also considered as readily biodegradable.

Description of key information

Based on the results of the read across study, the test substance, C18-22 TMAC, is considered as readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

A study was conducted to determine the ready biodegradability of the read across substance, ‘C22 TMAC (active: 99.9%)', using CO2 evolution test, according to OECD Guideline 301B, in compliance with GLP. Activated aerobic sludge was prepared with domestic origin three sewage plants and in order to have a concentration of total sludge solids of about 30 mg/L in the final 3 L of inoculated mixture. In the study, duplicate test vessels were prepared for positive control (blend of [14C] benzoic acid and benzoic acidat 0.2 mg/L) and read across substance ([14C] C22-ATQ at0.2 mg/L).Additional duplicate vessels were prepared which contained [14C] C22-ATQ at a nominal concentration of 0.2 mg/L and LAS at 0.17 mg/L. The purpose of these vessels was to show if the presence of LAS would ameliorate any inhibition of bacteria caused by [14C] C22-ATQ.The read across and reference substances were assessed for the rate and extent of biodegradation when exposed to Domestic activated sludge over a period of 35 d. Before and after dosing triplicate 1 mL aliquots of all the dosing solutions were taken for analysis by LSC (Liquid scintillation counting). For each dosing solution the mean of all six replicates was used to calculate the amount of radioactivity in the solutions, and the radio activity dosed to the test vessels. At the sampling points (3 h, Days 3, 7, 14, 21 and 28) the contents of the carbon dioxide trap nearest the test vessel was transferred to pre-weighed NalgeneTM bottle. This was re-weighed and triplicate 0.5 mL aliquots removed for LSC analysis. On Day 35 (the end of the exposure phase) the contents of both traps were added to the bottle and samples taken for LSC analysis. At the end of the study the test solutions were filtered through Whatman No 4 filter paper and weight of the filtrate measured. The filter papers were combusted to quantify radioactivity associated with the solid sludge. At the end of the study approximately 18% of the applied radioactivity was measured in the sludge solids from [C14] read across substance vessel, and approximately 11% in the sludge solids from the read across substance plus LAS vessels. In the reference substance benzoic acid vessels approximately 15% of the applied radioactivity was measured in the sludge solids. In the [14C] read across substance vessels a significant amount of applied radioactivity was evolved as [14C] carbon dioxide during the study, reaching 74 to 80% in the vessels with and without LAS, with no discernible difference between the two treatments within 28 d. In the reference substance, benzoic acid vessel more than 60% biodegradation was achieved with in the 10 d window as expected. All validity criteria for the acceptability of the test were met. Under the study conditions, the read across substance was considered as readily biodegradable (Noble, 2009). Based on the results of the read across study, similar biodegradation potential can be expected for the test substance, C18-22 TMAC.

[Type of water: freshwater]