Quaternary ammonium compounds, benzyl C12-C16 (even numbered)-alkyldimethyl chlorides

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:

From March 16, 1992 to April 20, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

slight inoculum modification

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

other: Secondary activated sludge was used

Details on inoculum:

A minor deviation of the test procedures described in the guidelines was introduced: instead of an effluent/extract/mixture, activated sludge was used as an inoculum. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end the sludge (200 mg Dry Weight (DW)/L) was aerated for one week. The sludge was diluted to a concentration of 2 mg DW/L in the BOD bottles. This method was described in a proposal for harmonizing ready biodegradability test protocols.

Duration of test (contact time):

28 d

Initial conc.:

4 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

- The closed bottle test was performed according to the EEC, OECD and ISO Test Guidelines. Ten bottles containing only inoculum, 10 bottles containing test substance and inoculum, and 10 bottles containing sodium acetate and inoculum were used. The concentrations of the test compound and sodium acetate in the bottle were 4.0 and 6.7 mg/L, respectively. The inoculum was diluted to 2 mg DW/L in the closed bottles. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero time bottles were immediately analysed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated at 21°C in the dark.
- Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at Day 7, 14 , 21 and 28. One extension from the protocol of the closed bottle test was introduced.

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

63

Sampling time:

28 d

Details on results:

The test was conducted in the presence of silica gel due to toxicity to inoculum.
The validity of the test was demonstrated by an endogenous respiration of 1.3 mg/L at Day 28 . Furthermore, the differences of the replicate values at Day 28 were less than 20%.
The biodegradation percentage of the reference compound, sodium acetate, at Day 14 was 78.
The validity of the test was shown by oxygen concentrations being >0.5 mg/L in the bottles.

Results with reference substance:

The biodegradation percentage of the reference compound, sodium acetate, at Day 14 was 78.

Table 1. Biodegredation percentages (%)

	Test substance	Reference substance
0	0	0
7	1	69
14	50	78
21	66	86
28	63	88

Nitrification corrections:

Ammonium chloride is omitted from the medium to prevent oxygen consumption bynitrifying bacteria increasing the endogenous oxygen consumption in the BOD bottles. The reason for this omission is the goal to lower the endogenous respiration thereby increasing the accuracy of the assessment of the biodegradability. This goal is reflected in the validity criterion of less than 1.5 mg/L of oxygen consumption in the control bottles at day 28. Omission of ammonium should not hamper the biodegradation of organic compounds in the Closed Bottle test. The biodegradation of the reference substance (sodium acetate) does demonstrate that nitrogen is not limiting growth. The nitrogen introduced with the inoculum is sufficient to fulfill the nitrogen requirement of the microorganisms.

Nitrification of the nitrogen present in test substance itself could occur. This could be a reason for using the ThOD_NO3. Using the ThOD_NO3of 1.52 g/g would result in a biodegradation percentage of 57.4, not allowing classification as readily biodegradable.

	Molecular formula	MW	ThODNH3 (g/g)	ThODNO3(g/g)	Weight (%)
C12-16 ADBAC ( n= C12)	C21H38NCl	340	2.78	2.96	0.501
Water	H2O	18	0	0	0.488
Amines	C16H35N	241.46	3.18	3.45	0.011
The ThODNH3of the test substance is =					1.43
The ThODNO3of the test substance is =					1.52

 

Day	O2 consumption	BOD	ThODNH3	% biodegradation	ThODNO3	% biodegradation
7	0.1	0.025	1.43	1.8	1.52	1.6
14	2.8	0.7		49.1		46.0
21	3.7	0.925		64.9		60.7
28	3.5	0.875		61.4		57.4

Test conc: 4 mg/L

However, it is not obligatory to use ThOD_NO3for all nitrogen containing test substances. The choice of the ThOD used to calculate biodegradation percentages should not be based on possible formation of nitrite or nitrate. Tests of the OECD 301 series were developed to assess the biodegradability and mineralization of organic substances. Nitrogen containing substances are biodegraded in ready biodegradability tests by heterotrophic micro-organisms capable of utilizing these substances as carbon and energy source.  This usually results in the formation of biomass (growth), water, carbon dioxide and ammonium (mineralization).  The ammonium formed may subsequently be oxidized by nitrifying bacteria. These nitrifying bacteria utilizing ammonium as energy source and carbon dioxide as carbon source (autotrophic growth) are not involved in the biodegradation of nitrogen containing substances. Biodegradation percentages calculated with the ThOD_NH3do therefore represent the biodegradability and mineralization of most nitrogen containing substances. The formation of nitrite and nitrate during the degradation of organic substances is rare and only occurring when organic nitrogen is for example present in the form of a nitro group. Organic nitrogen is always liberated by microorganisms as ammonium when nitrogen is present as primary amine (amino group), secondary amine group, tertiary amine or quaternary ammonium group.

Biodegradration of the C12 -16 ADBACs and in general:

The test substance has a quaternary ammonium group. Benzyldimethylamine was found as first metabolite of alkylbenzyldimethylammonium salts degradation byAeromonas hydrophila, activated sludge and aPseudomonas spp. community (Patrauchan and Oriel, 2003; van Ginkel 2004; Tezelet al.,2012). Alkyl-N fission is therefore the most probable strategy to gain access to the carbon of the alkyl chain. The next step in the biodegradation of these quaternary ammonium compounds was the successive removal of the two methyl groups. Benzylamine formed, in turn, is converted into benzaldehyde and ammonium (Patrauchan and Oriel, 2003). In contrast to the alkylbenzyldimethylammonium salts biodegradation pathway reported by Patrauchan and Oriel, (2003) neither benzylmethylamine nor benzylamine (BA) were identified as metabolites by two enrichment cultures. Exposure of activated sludge to decylbenzyldimethyl­ammonium chloride probably selects for three microorganisms that utilize the alkyl chain, the aromatic moiety, and dimethylamine (van Ginkel, 2004). Kinetic assays demonstrated that benzylmethylamine and benzylamine were not intermediates of alkylbenzyldimethylammonium salts transformation by the enrichedPseudomonasspp. community (Tezelet al., 2012). Thus, benzyldimethylamine is thought to be transformed to dimethylamine and benzoic acid via debenzylation. Dimethylamine is degraded by the successive removal of both methyl groups resulting in the formation of ammonium (Large, 1971). Both the pure and mixed culture studies showed that the degradation of the alkyl chain of alkylbenzyldimethylammonium chloride results in the formation of water, carbon dioxide and ammonium (Figure).

 

Figure:Biodegradation pathway of alkylbenzyldimethylammonium salts

Alkylbenzyldimethylammonium salts are biodegraded by microorganisms first utilizing the alkyl chain. Subsequently the methyl and benzyl groups are removed. In conclusion calculation of the biodegradation percentages with the ThODNH3 is thought to be a more appropriate choice.Further, the registrant would like to point out that this study has been evaluated as a key and valid study under the biocide dossier by the RMS - Italy, which has been published by ECHA (RMS: Italy) in June 2015 (ECHA, 2015; refer to page 29). As per the assessment report, C12-16 ADBAC has been concluded to be readily biodegradable.The registrant will include the above details in the next dossier update.

References

• Ginkel CG van (2007). Ultimate biodegradation of ingredients of cleaning agents. In; Handbook of Cleaning Agents/Decontamination of Surfaces, Eds. I Johansson and P Somasundaran, Elsevier Amsterdam, The Netherlands Volume 2:655-694. Large P(1971). The oxidative cleavage of alkyl nitrogen bonds in microorganisms. Xenobiotica 1:457-467.
• Large P(1971). The oxidative cleavage of alkyl nitrogen bonds in microorganisms. Xenobiotica 1:457-467. Patrauchan MA and Oriel PJ (2003). Degradation of benzyldimethylalkylammonium chloride byAeromonas hydrophihlasp K. J Appl Microbiol 94:266-272.
• Patrauchan MA and Oriel PJ (2003). Degradation of benzyldimethylalkylammonium chloride byAeromonas hydrophihlasp K. J Appl Microbiol 94:266-272. TezelU,TandukarM, MartinezRJ,SobeckyPA, andPavlostathisSG(2012).Aerobic Biotransformation ofn-Tetradecylbenzyldimethylammonium chloride by an enrichedPseudomonasspp. communityEnviron.Sci. Technol.46(16):8714–8722.
• TezelU,TandukarM, MartinezRJ,SobeckyPA, andPavlostathisSG(2012).Aerobic Biotransformation ofn-Tetradecylbenzyldimethylammonium chloride by an enrichedPseudomonasspp. communityEnviron.Sci. Technol.46(16):8714–8722.

·        ECHA (RMS: Italy) 2015: Assessment Report: Alkyl (C12-16) dimethylbenzyl ammonium chloride, Product-type 8 (Wood preservative); Regulation (EU) No 528/2012 concerning the making available on the market and use of biocidal products, Evaluation of active substances. (Biocides assessment report), Approval ID: 0063-08; Asset No: EU-0005415-0000; Rapporteur Member State: Italy. Report date: June, 2015.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the conditions of the study, the biodegradation of the test substance was determined to be 63% at Day 28. The test substance was considered readily biodegradable.

Executive summary:

A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (50.1% active in water) in water according to OECD Guideline 301D (closed bottle test), in compliance with GLP. Secondary activated sludge was used in this experiment and the percentage of degradation (O2 consumption) was measured. Since the substance was toxic to microorganisms, it was tested in the presence of silica gel to reduce the concentration in the water phase. During the test period, the substance was released slowly from the silica gel. The validity of the test was demonstrated by an endogenous respiration of 1.3 mg/L at Day 28. Furthermore, the differences between the replicate values at Day 28 were less than 20%. The biodegradation of the reference substance, sodium acetate, at Day 14 was 78%. Finally, the validity of the test was shown by oxygen concentrations being > 0.5 mg/L in the bottles. Under the conditions of the study, the biodegradation of the substance was determined to be 63% at Day 28. The substance was considered readily biodegradable (van Ginkel and Stroo, 1992).

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January, 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Not GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

>106 CFU/mL; consisting of micro-organisms in mud collected from Station Epuration Wavre 2nd Stage, a household water treating plant.
Although the inoculum concentration is unbounded, however, this is not expected to affect the overall result as can be demonstrated based on fulfillment of the validity criteria for the control group. That is, the total CO2 evolution in the inoculum blank at the end of the test is 39.2 mg which is <40 mg/L medium, which is an evidence of the inoculum having a low concentration of microorganisms (not being excessive).

Duration of test (contact time):

28 d

Initial conc.:

5 other: mgC/L

Based on:

test mat.

Remarks:

Testing at low concentrations, was required due to the toxicity of the test substance towards the inoculum at higher concentrations (see section 6.1.7 of IUCLID)

Details on study design:

Test conditions:
- Test temperature: 22 ± 2°C
- pH: 7.3 at start and 7.8 at the end of study
- Aeration of dilution water: Air free from carbon dioxide was passed through the solutions using a flow of 50 to 100 mL/min
- Continuous darkness: Yes
- Other: Total organic carbon content: 62.8%
Test system:
- Culturing apparatus: 3-litre flasks mounted with an aeration tube on a magnetic stirrer
- Details of trap for CO2 and volatile organics if used: the CO2 produced in each flask was precipitated with Ba(OH2). The amount of CO2 produced was determined by titration.

Reference substance:

benzoic acid, sodium salt

Remarks:

20 mgC/L

Key result

Parameter:

% degradation (CO2 evolution)

Value:

95.5

Sampling time:

28 d

Details on results:

- The biodegradability in the test flask was determined to be 95.5% after 28 days (See Table 1 under 'Any other information on results inc. tables).
- The CO2 production in the blank (inoculum control) was 39.2 mg. The pH measured at the start and end of the test period was 7.3 and 7.8, respectively. The temperature throughout the test ranged from 20.8 to 21.4°C.

Results with reference substance:

The biodegradability in the reference flask was determined to be 88.9% after 28 days (See Table 1 under 'Any other information on results inc. tables and the graph under 'Illustration (picture/graph)'.

Table 1. Biodegradability values:

Day	Reference substance		Test substance
	% CO2Produced	% CO2 Total	% CO2Produced	% CO2 Total
0	0.00	0.00	0.0	0.0
1	14.16	14.16	1.2	1.2
3	28.54	42.69	1.0	2.2
6	21.20	63.89	9.5	11.6
8	8.77	72.67	16.0	27.7
10	3.72	76.39	20.2	47.9
13	3.26	79.65	17.4	65.3
15	2.53	82.18	7.5	72.8
17	1.47	83.65	5.1	77.9
20	2.31	85.95	3.9	81.8
22	1.15	87.11	1.6	83.5
24	0.25	87.36	4.4	87.8
28	0.16	87.52	1.8	89.6
29	1.35	88.87	5.8	95.5

For details on results of replicates of test substance and the graphs of test and reference substances, please refer to the attachment under 'Attached background material'.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the test conditions, the biodegradation of the test substance in water was determined to be 95.5 after 28 days (CO2 evolution). The test substance was considered to be readily biodegradable.

Executive summary:

A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (80% active in hydroalcoholic solution), in water according to OECD Guideline 301B (CO2 evolution test). Flasks containing inoculum from a household water-treating plant dosed with the equivalent of 5 mg C/L test or 20 mg C/L reference substances were maintained for 28 d. Testing at low concentrations, was required due to the toxicity of the test substance towards the inoculum at higher concentrations. Biodegradability was calculated from the released CO2 over time in the test and reference flasks compared to the blank control (a flask prepared without test or reference substance). CO2 production in the blank (inoculum control) was 39.2 mg. Biodegradability in the reference flask was determined to be 88.9% after 28 d. Under the test conditions, the biodegradation of the test substance in water was determined to be 95.5% after 28 d (CO2 evolution). The test substance was considered to be readily biodegradable (van Dievoet, 2005).

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Remarks:

Activated sludge domestic unadapted in river water, sea water, ditch water and soil (loam)

Details on inoculum:

Secondary activated sludge and primary settled sewage were obtained from a wastewater treatment plant (WWTP) in Duiven, The Netherlands. This WWTP is an activated sludge plant treating predominantly domestic wastewater. The activated sludge used as inoculum for the Closed Bottle test was preconditioned to reduce the endogenous respiration rates. To this end, the sludge from this WWTP (200 mg dry weight/l) was aerated for one week. The sludge was diluted to a concentration of 2 mg dry weight/L in the BOD bottles. The ditch water samples were collected near Zevenaar, the Netherlands and river water samples were obtained from the river IJssel near Arnhem, the Netherlands. These samples were used immediately. Seawaterwas collected from coastal water near West Kapelle, the Netherlands. The seawater was aged for two weeks to reduce the concentration of biodegradable compounds present in the seawater. Loam was collected at Heino in the Netherlands. Stones and plant fragments were removed by hand. After collection, the soil was air-dried for approximately 2 days and passed through a 2 mm sieve. A small portion of the soil was dried in an oven (104°C) to a constant weight in order to determine the dry weight and thereby the initial moisture content. A portion of the soil was sent to a soil analysis laboratory (Bedrijfslaboratorium voor Grond en Gewasonderzoek, Oosterbeek, the Netherlands) for characterization. In terms of texture, the loam was composed of 35% sand, 49% silt and 15% clay. The organic matter content was 1.4% and the pH was 7.6. The maximum field moisture capacity was 34%.

Duration of test (contact time):

3 d

Initial conc.:

2 other: mg dry weight/L

Details on study design:

Closed Bottle test:
The biodegradability was determined as follows: Test substance was added to either seawater,water from the river IJssel, ditch water or mineral medium inoculated with activated sludge such that its concentration was 2 mg dry weight/l. The oxygen decrease in the bottles as a function of time was measured using a special funnel. This funnel fitted exactly into the bottle and served as an overflow reservoir permitting multiple measurements in one bottle. In some cases, this device was also used to measure the biological oxygen demand continuously by connecting the oxygen monitor to a recorder. Biochemical oxygen demands (BOD) pf the test substances were corrected by substacting the BOD of the control. The biodegradability was claculated by dividing the corrected BOD by the chemical oxygen demand (COD).

Reference substance:

benzoic acid, sodium salt

Remarks:

Controls with benzoate revealed that there was no appreciable difference with respect to the lag phase with unadapted sludge and sludge acclimatized to cocobenzyldimethylammonium chloride.

Key result

Parameter:

% degradation (O2 consumption)

Value:

60

Sampling time:

3 d

Details on results:

- The test substance biodegradation was >60% within three days in the closed bottle test inoculated with this unacclimatized sludge. Hence it should be classified as readily biodegradable.
Compared to test substance, tallow benzyldimethylammonium chloride was less easily biodegradable in closed bottle tests. Differences were observed with respect to the length of the lag phase and the slope of the biodegradation curve. The differences are caused by the toxicity of tallowbenzyldimethylammonium chloride in the closed bottle test as shown by the inhibition of the endogenous respiration. The flat slope obtained with tallowbenzyldimethylammonium chloride in the closed cottle test suggests that this compound is degraded at low rates. However, the biodegradation rate of alkylbenzyldimethylammonium salts with longer alkyl chains is probably governed by the rate of desorption from the sludge or glass wall of the bottles. From the literature, alkylbenzyldimethylammonium chlorides with C10 to C14 alkyl chains were shown to be readily biodegradable (Masuda, 1976). The underlying causes for the failure to demonstrate the readily biodegradability of alkylbenyldimethylammonium salts with C16 to C18 alkyl chains are the toxicity of these compounds and their limited bioavailability.
- In soil, a 64% biodegradation of the test substance was reached after 70 days, this percentage indicates complete mineralization in soil (the test substance biodegradation rate is limited by the desorption rate from the soil).
- In the closed bottle test, the biodegradation of the test substance was 71, 69 and 60% in seawater, ditch water and river water, respectively (half lives of 0.3, 0.1 and 0.1 d respectively).
The closed bottle test results with adapted and unadapted sludge demonstrate that exposure of sludge to test substance only results in higher levels of cocobenzyldimethylammonium chloride-, N, N-dimethylbenzylamine- and dimethylamine-degrading microorganisms. Exposure to the test substance probably selected three microorganisms that utilize the alkyl chain, the aromatic moiety and the dimethylamine sequentially.

- The results clearly illustrate that the test substance was completely converted into water, carbon dioxide, and ammonia. Additional proof of the complete oxidation is given by the high BOD/ThOD ratio obtained in the closed bottle test inoculated with both acclimatized and unacclimatized sludge.

- On the basis of the low half-lives calculated, biodegradation of test substance was shown to be an environmentally significant process. It can be degraded in rivers, ditches, seawater, soil, and activated sludge plants. Accumulation of intermediate products is not expected.

Interpretation of results:

readily biodegradable

Conclusions:

Under the study conditions, the biodegradation of the test substance was determined to be >60% within three days in the closed bottle test inoculated with unacclimatized sludge. The test substance was therefore considered readily biodegradable.

Executive summary:

A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (49-52% active in water) in water according to OECD Guideline 301D (closed bottle test). Half-lives were determined using inoculum from various aquatic sources. The test substance was added to either seawater or water from the river IJseel, ditch water or mineral medium inoculated with activated sludge such that its concentration was 2 mg/dry weight/L. The oxygen decrease in the bottles as a function of time was measured using a special funnel. This funnel fitted exactly into the bottle and derverd as an overflow reservoir permitting multiple measurements in one bottle. Biochemical oxygen demands (BOD) of the test substances were corrected by subtracting the BOD of the control. The biodegradability was calculated by dividing the corrected BOD by the chemical oxygen demand (COD). The biodegradation of the test substance was 71, 69 and 60% in seawater, ditch water and river water, with half -lives of 0.3, 0.1 and 0.1 d, respectively. Under the study conditions, the biodegradation of the test substance was determined to be >60% within three days in the closed bottle test inoculated with unacclimatized sludge. The test substance was therefore considered readily biodegradable (van Ginkel, 1996).

Reference 4

Endpoint:

biodegradation in water: screening tests

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From October 11, 1992 to January 11, 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See 'Principles of method if other than guideline'

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 A (Inherent Biodegradability: Modified SCAS Test)

Deviations:

yes

Remarks:

See 'Principles of method if other than guideline'

Principles of method if other than guideline:

- Deviations OECD 301 D: Ammonium chloride was omitted from the medium to prevent nitrification. The closed bottle test was prolonged by measuring the course of the oxygen decrease in the bottles of Day 28 using a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing the BOD bottle. The test substance was toxic to microorganisms. Therefore, it was tested in the presence of silica gel to reduce the concentration in the water phase. During the test period, the test substance should be released slowly from the silica gel (0.5 g/bottle). Although no additional oxygen consumption was expected, controls with silica gel were carried out as well (10 bottles containing test substance, inoculum and silica gel).
- Deviations OECD 302 A: Fill and draw procedure was only six times per week instead of daily. To maintain a constant pH in the SCAS unit, phosphate buffer was added six times per week. Effluent samples were filtered using cellulose nitrate filters with pores of 8 µm to remove sludge particles.

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

other: The study was conducted, in parallel with a SCAS test, where sludge from the SCAS test unit on Day 0 (corresponding to non-adapted) and Day 21 (adapted) were fed into separate test vessels for the closed bottled test.

Duration of test (contact time):

>= 28 - <= 56 d

Initial conc.:

2 mg/L

Based on:

test mat.

Key result

Parameter:

% degradation (O2 consumption)

Value:

52

Sampling time:

28 d

Remarks on result:

other: inoculated with sludge from the SCAS unit (Day 0); 62% biodegradation at Day 56

Key result

Parameter:

% degradation (O2 consumption)

Value:

77

Sampling time:

28 d

Remarks on result:

other: inoculated with activated sludge from the SCAS unit (Day 21)

Remarks:

The biodegradation in the closed bottle tests did increase due to the acclimatisation of the microorganisms in the SCAS test unit.

Details on results:

- OECD 302A:
Before the addition of the test substance, the effluent non purgeable organic carbon (NPOC) values obtained from the test substance unit and the control unit were comparable and constant. After the first addition (Day 0), the removal of 69% was immediately accomplished due to the adsorption on the sludge, dilution and/or biodegradation. Next, the NPOC values of the units decreased and subsequently remained constant. The percentage removal after 4 weeks over 3 consecutive measurements was 99. Additional closed bottle tests were performed to draw conclusion on the removal mechanism in the SCAS test.
- OECD 301D:
In the closed bottle tests inoculated with sludge taken on Days 0, and 21 from the SCAS test unit, biodegradation of the test substance took place. The test substance was biodegraded at 52% on Day 28 in the closed bottle test inoculated with sludge from the SCAS test (Day 0). In the prolonged closed bottle test the test substance was biodegraded at 62% on Day 56. The biodegradation in the closed bottle tests did increase due to acclimatization of the microorganisms in the SCAS test unit. The test substance was biodegraded at 77% on Day 28 in the closed bottle test inoculated with sludge sampled on Day 21.

The test substance was degraded in waste water treatment plant after a short acclimatization period (5 d). In an acclimatized waste water treatment plant, the test substance was probably totally mineralized. Total mineralization was demonstrated by increased biodegradation percentages in the closed bottle test inoculated with sludge taken on Day 21 compared to the closed bottle test inoculated with unacclimatized sludge. The algal growth inhibition test showed that the effluent of Day 1 was slightly toxic. This toxicity was probably caused by the test substance and/or biodegradation products present in the effluent of the test unit of the SCAS test. The results of the toxicity test of Day 23 demonstrated that acclimatized waste water treatment plants detoxified the test substance by biodegradation.

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

Under the study conditions, the test substance was considered to be inherently biodegradable.

Executive summary:

A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (50% active in water) in water according to OECD Guidelines 301D and 302A (closed bottle test / modified SCAS test), in compliance with GLP. The experiment was carried out using a combination of an inherent and a ready biodegradability test. To predict the effects of possible biodegradation products, the toxicity of effluents from semi-continuous activated sludge (SCAS) units was assessed. The test substance caused no reduction of the biodegradation of non-purgeable organic carbon (NPOC) present in primary settled sewage. Therefore, it was considered to be non-inhibitory to activated sludge. During the test period, 99% of the substance was removed from the wastewater by adsorption and/or biodegradation. In a second step, the distinction between biodegradation and adsorption was evaluated in closed bottle tests inoculated with approximately 2 mg/L of activated sludge collected on Days 0 and 28 from the SCAS unit fed with the test substance. With the Day 0 SCAS sample, the test substance was biodegraded by 52% within 28 d and by 62% within 56 d. The biodegradation in the closed bottle tests did increase due to the acclimatisation of the microorganisms in the SCAS test unit. The test substance was biodegraded at 77% on Day 28 in the closed bottle test inoculated with sludge sampled on Day 28. The closed bottle test results demonstrated that the test substance was removed by biodegradation in the SCAS test. Under the study conditions, the test substance was considered to be inherently biodegradable (van Ginkel, 1993). This study was primarily carried out to determine the biodegradation pathway of alkylbenzyldimethylammonium salts and not to assess the ready biodegradability; therefore, the study has been used only as a supporting study.

Reference 5

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From December 12, 1986 to February 04, 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

GLP compliance:

no

Remarks:

But includes statement of inspections by GLP quality assurance officer.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

The inoculum is taken from an activated sludge plant, the municipal waste water treatment plant in Duiven (NL). The sludge is preconditioned during a week: a sludge suspension of 1 g/L is aerated in the dilution water. This modification is introduced to reduce high residual respiration rates.

Duration of test (contact time):

42 d

Initial conc.:

2.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Performance of the test:
The biodegradability test was carried out according to OECD Test Guideline 301D: Closed Bottle Test. The method was modified according to the recommendations of ECETOC (1985) or Blok et al. (1985). Modifications concern the inoculum, the composition of the dilution water and the analyses. The density of the inoculum in the test was 3 mg/L. On Day 0, 14, 28 and 42 the concentration of oxygen was measured. On Day 28, nitrite and nitrate concentrations were measured. The dilution water was the medium as prescribed by the test guideline without ammonia. This modification was introduced to minimize the consumption of oxygen for the nitrification process. Dark glass bottles of about 280 mL with glass stoppers were filled completely with a suspension of preconditioned activated sludge (3 mg/L) in dilution water and a concentration of the test substance equivalent to about 6 mg ThOD/L (Theoretical Oxygen Demand). The test was carried out in triplicate and at every observation time measurements of oxygen and pH were carried out in a new series of three bottles.

Reference substance:

acetic acid, sodium salt

Test performance:

Adequate reference substance performance.
Adequate blank performance.

Key result

Parameter:

% degradation (O2 consumption)

Value:

65

Sampling time:

28 d

Details on results:

- A toxicity control was carried out with 4.3 and 12.8 mg of test substance and was found not to be toxic at these concentrations although a slight inhibition was seen at 12.8 mg/L.
- Ready biodegradable

Validity criteria fulfilled:

no

Interpretation of results:

readily biodegradable

Conclusions:

Under the conditions of the study, the test substance was considered readily biodegradable.

Executive summary:

A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (50.15% active in hydroglycolic solution) in water according to OECD Guideline 301D (closed bottle test).The method was adapted according to the recommendations of ECETOC (1985) or Blok et al. (1985). Modifications concerned the inoculum, the composition of the dilution water and the analyses. The inoculum was taken from an activated sludge plan, the municipal waste water treatment plant in Duiven (NL). The sludge was preconditioned by aeration, to reduce high residual respiration rates. The density of the inoculum in the test was 3 mg s.s./L. On Days 0, 14, 28 and 42, the concentration of oxygen was measured. On Day 28, nitrite and nitrate concentrations were measured. The dilution water was the medium as prescribed by the test guideline without ammonia. This modification was introduced to minimize the consumption of oxygen for the nitrification process. Dark glass bottles of about 280 mL with glass stoppers were filled completely with a suspension of pre-conditioned activated sludge (3 mg/L) in dilution water and a concentration of the test substance equivalent to about 6 mg ThOD/L (Theoretical Oxygen Demand). The test was carried out in triplicate and at every observation time measurements of oxygen and pH were conducted in a new series of three bottles. The test concentration was 4.3 mg/L, therefore the COD in the test suspension was 5.2 mg O2/L. After 4 weeks, the nitrite and nitrate concentrations were measured to be <0.1 and <1.5 mg/L respectively. The extent of biodegradation, calculated as the BOD related to the COD for test substance is about 65% after 2, 4 and 6 weeks. All validity criteria were fulfilled: i.e., inoculum blank indicated >1.5 mg dissolved oxygen/L after 28 days; the residual concentration of oxygen in the test bottles were >0.5 mg/L; difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, was less than 20%; and toxicity control showed >25% degradation. Therefore, under the conditions of the study, the test substance was considered readily biodegradable (Balk, 1987).

Reference 6

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From November 06, 1991 to December 05, 1991

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, adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): acclimated activated sludge, Avondale Sewage Treatment Plant, Avondale.
- Method of cultivation and preparation of the inoculum mixture: acclimated activated sludge was obtained from the final day of a previous SCAS assay conducted with the test substance at a concentration of 10 mg active substance per litre. Approximately 150 mL of mixed liquor was collected from each duplicate units, composited and homogenized at medium speed in a blender for 2 minutes. The homogenised sample was poured into a beaker and allowed to settle for 30 mins. The supernatant was decanted and added to the flasks at a conc. of 1% (v/v). On the same day the sludge was collected a standard plate count (SPC) was performed on the inoculum. The plates were incubated at test temperature. The result was 2.3 E06 CFU/mL
- Inoculum addition: 20 mLof the above inoculum mixture was added to all test flasks.

Duration of test (contact time):

28 d

Initial conc.:

5 mg/L

Based on:

act. ingr.

Initial conc.:

10 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test conditions:
- Composition of medium: Momodified BOD water
- Test temperature: 22.1- 23.0°C
Test system:
- Culturing apparatus: four glass four-litre Erlenmeyer flasks containing two litres of modified biochemical oxygen demand (BOD) water
- Number of culture flasks/concentration: 1
- Method used to create aerobic conditions: Tte test flasks were placed on a rotary shaker, connected to the scrubbing train and aerated over-night to purge the system of background CO2.
- Details of trap for CO2 and volatile organics if used: the CO2 produced in each flask reacted with 0.024 N Ba(OH)2 and precipitated as BaCO3. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 N standardized hydrochloric acid (HCl).
- Other: after 28 d, the contents of the flasks were acidified with concentrated sulfuric acid (H2SO4) and aerated overnight. One final titration was performed.
Sampling:
- Sampling frequency: on Day 2, 5, 8, 11, 14, 17, 20, 23, 28.
Control and blank system:
- Inoculum blank: one flask containing test medium and inoculum
- Reference control: one flask containing only d-glucose at a conc. of 20 mg active/L
Statistical methods:
- Gauss-newton method

Reference substance:

other: d-glucose at a concentration of 20 mg/L

Key result

Parameter:

% degradation (CO2 evolution)

Value:

84

Sampling time:

28 d

Remarks on result:

other: at 5 mg/L concentration

Key result

Parameter:

% degradation (CO2 evolution)

Value:

82.6

Sampling time:

28 d

Remarks on result:

other: at 10 mg/L concentration

Details on results:

- Biodegradability in the test flasks was determined to be 84.0% and 82.6% within 28 d at 5 and 10 mg/L of test substance respectively.
- Biodegradation in the blank was 0%.
- Final SOC test concentrations were 0.7 mL/L (5 mg/L) and 0.6 mL/L (10 mg/L) compared to 0.4 mL/L with the control and 2.2 mL/L with d-glucose.

Results with reference substance:

Biodegradability in the reference flask was determined to be 80.6% in 28 d.

Table 1. % TCO2 over time

Day	d-glucose	Test substance 5 mg a.i./L	Test substance 10 mg a.i./L
2	20.6	9.3	1.8
5	39.2	43.8	39.1
8	50.6	58.5	54.2
11	61.0	75.4	67.9
14	64.9	85.0	76.0
17	69.2	88.5	80.2
20	73.4	87.2	81.6
23	77.0	85.6	81.9
28	80.1	85.0	82.3
28	80.6	84.0	82.6

Table 2. Final results

Test substance	Concentration (mg a.i./L)	Final %TCO2	Final SOC (mL/L)
Control (blank)	0	0	0.4
d-glucose	20	80.6	2.2
Test substance	5	84.0	0.7
Test substance	10	82.6	0.6

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable

Conclusions:

Under the conditions of the study, the test substance was considered to be readily biodegradable.

Executive summary:

A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (80.8% active in ethanol) in water according to OECD Guideline 301B (CO2 evolution test), in compliance with GLP. Flasks containing acclimated inoculum (at 10 mg/L) from a previous SCAS assay were dosed with 5 and 10 mg a.i./L of the test substance or 20 mg/L of the reference substance (d-glucose) and were maintained for 28 d. Biodegradability was calculated from the CO2 released over time in the test and reference flasks relative to that which was released in the blank control (a flask prepared without test or reference substance). The results indicated that 84.0 and 82.6% CO2 was produced in vessels dosed with 5 and 10 mg/L test substance, respectively, compared to 0% with the control and 80.6% with d-glucose. Final suspended organic carbon (SOC) concentrations were 0.7 mL/L (5 mg/L) and 0.6 mL/L (10 mg/L) compared to 0.4 mL/L with the control and 2.2 mL/L with d-glucose. Under the conditions of the study, the substance was considered to be readily biodegradable (Corby, 1992a).

Reference 7

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From October 30, 1991 to November 06, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 A (Inherent Biodegradability: Modified SCAS Test)

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): acclimated activated sludge, Avondale Sewage Treatment Plant, Avondale.
- Preparation of the SCAS units: the sludge was screened through a 2 mm sieve to remove large clumps. The total suspended solids (TSS) level was determined and based on this reading the sludge was distributed among the SCAS units such that when the volume in each unit was adjusted to 1.5L with tap water the suspended solids level was approx. 2500 mg/L.
- Pre-acclimation period: the units were aerated for 23 and half hours at a rate adequate to maintain solids suspension. At the end of this period, the air was turned off and the sludge was allowed to settle for 30 mins. One liter of effluent was drawn off and replaced with 1L of influent consisting of 10 mL synthetic sewage and 990 mL tap water to bring back to 1.5L. The air was turned on. This process was repeated on a daily basis.
- Sludge acclimation period: all the units were fed 10 mL of synthetic sewage and 990 mL of tap water daily for a minimum of 4d prior to initiation of the test substance acclimation period.
- Sludge distribution: on the day where the addition of the test substance was to be started, the sludge was settled, composited, and re-distributed among the units so that each unit contained a uniform sample of the sludge.

Duration of test (contact time):

7 d

Initial conc.:

10 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

other: SOC removal

Details on study design:

Test conditions:
- Composition of medium: synthetic sewage. It was prepared using the following composition: d-glucose: 30g; nutrient broth: 20g; K2HPO4: 13g; tap water: 1L. 20 mL of synthetic sewage was added to each unit on the 1st two days of the pre-acclimation period to help maintain suspended solids at 2500 mg/L.
- Temperature: 21.9-23.2°C
- Suspended solids concentration: 2500 mg/L.
- Lighting: during daily maintenance, the SCAS units were exposed to only room lighting and not exposed to direct sunlight.
- Other:
Test substance acclimation period: the test substance was added to the test units incrementally for a 7-d acclimation period. The control units are treated in the same way except that they do not receive any of the test substance.
Testing period: the test substance was added to the test units at the specified test concentration for an additional 7d following the acclimation period. The control units were treated on the same way.

Test system:
- Culturing apparatus: SCAS aeration chambers containing 1.5L of the activated sludge
- Number of culture flasks/concentration: 2

Sampling:
- Sampling frequency: daily (i.e., on Day 1, 2, 3, 4, 5, 6 and 7)
- Effluent analysis: one liter of effluent was withdrawn daily from each unit and saved for analysis. The effluent was replaces with comparable volume of influent of synthetic sewage. An aliquot of each effluent sample was centrifuged. A subsample of centrate was acidified with conc. H2SO4, purged with N2 and submitted for soluble organin chemical analysis (SOC).

Control and blank sampling system:
- Inoculum blank: yes, two control units were maintained only on synthetic sewage

Statistical method: Analysis of variance (ANOVA)

Key result

Parameter:

other: % SOC removal

Value:

100

Sampling time:

7 d

Details on results:

- The percentage of carbon removed varied from 98.22% to 106.51% over a 7-d period between the two test vessels. The average percent SOC removal for test substance was >100%.
- The mean SOC value in mg/L was 4.1±0.3 for control 1 and 4±0.3 for control 2.

Table 1. % Carbon removed

Days	Test 1	Test 2
1	100.59	102.96
2	98.22	106.51
3	100.00	104.73
4	98.22	100.59
5	100.59	102.96
6	101.18	104.73
7	102.96	102.96

Validity criteria fulfilled:

not specified

Interpretation of results:

inherently biodegradable

Conclusions:

Under the conditions of the study, the average percent SOC removal of the test substance was >100% indicating it to be inherently biodegradable.

Executive summary:

A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (80.8% active in ethanol) in water according to OECD Guideline 302A (Modified SCAS test), in compliance with GLP. Four chambers containing activated sludge were aerated and the suspended solid contents were adjusted to 2500 mg/L. The test substance at 1000 mg a.i./L was added to two test units for a 7 d acclimation period. This consisted of incremental additions until the final test concentration of 10 mg a.i./L was reached. Two additional units did not receive test substance and served as controls. The testing period was an additional 7 d following the acclimation period. Throughout the study, all units were fed synthetic sewage. Effluents withdrawn from each unit were analysed for SOC. Under the conditions of the study, the average percent SOC removal was >100% indicating that the test substance was inherently biodegradable (Corby, 1992b).

Reference 8

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Remarks:

Literature data

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Documentation insufficient for assessment.

Principles of method if other than guideline:

High performance liquid chromatography and gas chromatography mass spectrometry analyses have been used to study the degradation pathway.

GLP compliance:

not specified

Inoculum or test system:

activated sludge (adaptation not specified)

Aeromonas hydrophila sp. K, an organism isolated from soil is capable of utilising alkylbenzyldimethylammonium salts as sole source of carbon and energy. High performance liquid chromatography and gas chromatography mass spectrometry
analyses have been used to study the degradation pathway. During alkylbenzyldimethylammonium chloride biodegradation,
formation of benzyldimethylamine, benzylmethylamine, benzylamine, benzaldehyde and benzoic acid occurs. Formation
of benzyldimethylamine suggests that the cleavage of Calkyl-N bond occurs as the first step of alkylbenzyldimethylalkylammonium chloride metabolism [Patrauchan and Oriel, 2003]. A series of Closed Bottle
tests inoculated with unadapted micro-organisms and micro-organisms adapted to decylbenzyldimethylammonium
chloride also suggests cleavage of the Calkyl-N bond [van Ginkel, 2004]. The alkyl chains of the quaterrnary ammonium
salts are liberated as alkanals [van Ginkel, 2004]. Alkanals are subsequently channelled into the  -oxidation cycle.
Dimethylbenzylamine is degraded by other micro-organisms [Patrauchan and Oriel, 2003]. This biodegradation pathway
i.e. channelling all alkyl chains with varying length into -oxidation cycle allows intrapolation and extrapolation of
ready biodegradability test results. For a number of alkylbenzyldimethylammonium salts biodegradation percentages
of >60 have been obtained after 28 d in ready biodegradability test.
Alkylbenzyldimethylammonium salts with alkyl chains ranging from C8 to C18 are therefore readily biodegradable. Read
across of biodegradation data is not restricted to ready biodegradation tests.

Interpretation of results:

readily biodegradable

Conclusions:

Based on the literature data, the test substance is considered readily biodegradable.

Executive summary:

A literature review was conducted to determine the biodegradability of the test substance, C12 -16 ADBAC (purity not specified). Van Ginkel et al 2004 and Patrauchan et al 2003 publication were reviewed. Based on the literature data, the test substance is considered readily biodegradable (Van Ginkel, 2004 and Patrauchan, 2003).

Description of key information

A number of reliable studies have shown that the test substance is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

Freshwater:

Study 1: A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (50.1% active in water) in water according to OECD Guideline 301D (closed bottle test), in compliance with GLP. Secondary activated sludge was used in this experiment and the percentage of degradation (O2 consumption) was measured. Since the substance was toxic to microorganisms, it was tested in the presence of silica gel to reduce the concentration in the water phase. During the test period, the substance was released slowly from the silica gel. The validity of the test was demonstrated by an endogenous respiration of 1.3 mg/L at Day 28. Furthermore, the differences between the replicate values at Day 28 were less than 20%. The biodegradation of the reference substance, sodium acetate, at Day 14 was 78%. Finally, the validity of the test was shown by oxygen concentrations being > 0.5 mg/L in the bottles. Under the conditions of the study, the biodegradation of the substance was determined to be 63% at Day 28. The substance was considered readily biodegradable (van Ginkel and Stroo, 1992).

Study 2: A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (80% active in hydroalcoholic solution), in water according to OECD Guideline 301B (CO2 evolution test). Flasks containing inoculum from a household water-treating plant dosed with the equivalent of 5 mg C/L test or 20 mg C/L reference substances were maintained for 28 d. Testing at low concentrations, was required due to the toxicity of the test substance towards the inoculum at higher concentrations. Biodegradability was calculated from the released CO2 over time in the test and reference flasks compared to the blank control (a flask prepared without test or reference substance). CO2 production in the blank (inoculum control) was 39.2 mg. Biodegradability in the reference flask was determined to be 88.9% after 28 d. Under the test conditions, the biodegradation of the test substance in water was determined to be 95.5% after 28 d (CO2 evolution). The test substance was considered to be readily biodegradable (van Dievoet, 2005). This study was also submitted as part of the biocides dossier for product type-8 and was concluded by the authority to be a key and valid study (see below discussions; ECHA assessment report, 2015).

Study 3:A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (49-52% active in water) in water according to OECD Guideline 301D (closed bottle test). Half-lives were determined using inoculum from various aquatic sources. The test substance was added to either seawater or water from the river IJseel, ditch water or mineral medium inoculated with activated sludge such that its concentration was 2 mg/dry weight/L. The oxygen decrease in the bottles as a function of time was measured using a special funnel. This funnel fitted exactly into the bottle and derverd as an overflow reservoir permitting multiple measurements in one bottle. Biochemical oxygen demands (BOD) of the test substances were corrected by subtracting the BOD of the control. The biodegradability was calculated by dividing the corrected BOD by the chemical oxygen demand (COD). The biodegradation of the test substance was 71, 69 and 60% in seawater, ditch water and river water, with half -lives of 0.3, 0.1 and 0.1 d, respectively. Under the study conditions, the biodegradation of the test substance was determined to be >60% within three days in the closed bottle test inoculated with unacclimatized sludge. The test substance was therefore considered readily biodegradable (van Ginkel, 1996).

Study 4: A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (50.15% active in hydroglycolic solution) in water according to OECD Guideline 301D (closed bottle test). The method was adapted according to the recommendations of ECETOC (1985) or Blok et al. (1985). Modifications concerned the inoculum, the composition of the dilution water and the analyses. The inoculum was taken from an activated sludge plan, the municipal wastewater treatment plant in Duiven (NL). The sludge was preconditioned by aeration, to reduce high residual respiration rates. The density of the inoculum in the test was 3 mg s.s./L. On Days 0, 14, 28 and 42, the concentration of oxygen was measured. On Day 28, nitrite and nitrate concentrations were measured. The dilution water was the medium as prescribed by the test guideline without ammonia. This modification was introduced to minimize the consumption of oxygen for the nitrification process. Dark glass bottles of about 280 mL with glass stoppers were filled with a suspension of pre-conditioned activated sludge (3 mg/L) in dilution water and a concentration of the test substance equivalent to about 6 mg ThOD/L (Theoretical Oxygen Demand). The test was carried out in triplicate and at every observation time measurements of oxygen and pH were conducted in a new series of three bottles. The test concentration was 4.3 mg/L, therefore the COD in the test suspension was 5.2 mg O2/L. After 4 weeks, the nitrite and nitrate concentrations were measured to be <0.1 and <1.5 mg/L respectively. The extent of biodegradation, calculated as the BOD related to the COD for test substance is about 65% after 2, 4 and 6 weeks. All validity criteria were fulfilled: i.e., inoculum blank indicated >1.5 mg dissolved oxygen/L after 28 days; the residual concentration of oxygen in the test bottles were >0.5 mg/L; difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or the end of the 10-d window, as appropriate, was less than 20%; and toxicity control showed >25% degradation. Therefore, under the conditions of the study, the test substance was considered readily biodegradable (Balk, 1987).

Study 5: A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (50% active in water) in water according to OECD Guidelines 301D and 302A (closed bottle test / modified SCAS test), in compliance with GLP. The experiment was carried out using a combination of an inherent and a ready biodegradability test. To predict the effects of possible biodegradation products, the toxicity of effluents from semi-continuous activated sludge (SCAS) units was assessed. The test substance caused no reduction of the biodegradation of non-purgeable organic carbon (NPOC) present in primary settled sewage. Therefore, it was considered to be non-inhibitory to activated sludge. During the test period, 99% of the substance was removed from the wastewater by adsorption and/or biodegradation. In a second step, the distinction between biodegradation and adsorption was evaluated in closed bottle tests inoculated with approximately 2 mg/L of activated sludge collected on Days 0 and 28 from the SCAS unit fed with the test substance. With the Day 0 SCAS sample, the test substance was biodegraded by 52% within 28 d and by 62% within 56 d. The biodegradation in the closed bottle tests did increase due to the acclimatisation of the microorganisms in the SCAS test unit. The test substance was biodegraded at 77% on Day 28 in the closed bottle test inoculated with sludge sampled on Day 28. The closed bottle test results demonstrated that the test substance was removed by biodegradation in the SCAS test. Under the study conditions, the test substance was considered to be inherently biodegradable (van Ginkel, 1993). This study was primarily carried out to determine the biodegradation pathway of alkylbenzyldimethylammonium salts and not to assess the ready biodegradability; therefore, the study has been used only as a supporting study.

Study 6: A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (80.8% active in ethanol) in water according to OECD Guideline 301B (CO2 evolution test), in compliance with GLP. Flasks containing acclimated inoculum (at 10 mg/L) from a previous SCAS assay were dosed with 5 and 10 mg a.i./L of the test substance or 20 mg/L of the reference substance (d-glucose) and were maintained for 28 d. Biodegradability was calculated from the CO2 released over time in the test and reference flasks relative to that which was released in the blank control (a flask prepared without test or reference substance). The results indicated that 84.0 and 82.6% CO2 was produced in vessels dosed with 5 and 10 mg/L test substance, respectively, compared to 0% with the control and 80.6% with d-glucose. Final suspended organic carbon (SOC) concentrations were 0.7 mL/L (5 mg/L) and 0.6 mL/L (10 mg/L) compared to 0.4 mL/L with the control and 2.2 mL/L with d-glucose. Under the conditions of the study, the substance was considered to be readily biodegradable (Corby, 1992a).

Study 7: A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (80.8% active in ethanol) in water according to OECD Guideline 302A (Modified SCAS test), in compliance with GLP. Four chambers containing activated sludge were aerated and the suspended solid contents were adjusted to 2500 mg/L. The test substance at 1000 mg a.i./L was added to two test units for a 7-d acclimation period. This consisted of incremental additions until the final test concentration of 10 mg a.i./L was reached. Two additional units did not receive the test substance and served as control. The testing period was an additional 7 d following the acclimation period. Throughout the study, all units were fed synthetic sewage. Effluents withdrawn from each unit were analysed for SOC. Under the conditions of the study, the average percent SOC removal was >100% indicating that the test substance was inherently biodegradable (Corby, 1992b).

A literature review was conducted to determine the biodegradability of the test substance, C12 -16 ADBAC (purity not specified). Van Ginkel et al 2004 and Patrauchan et al 2003 publication were reviewed. Based on the literature data, the test substance is considered readily biodegradable (Van Ginkel, 2004 and Patrauchan, 2003).

The Biocides assessment report on C12-16 ADBAC, published by the Italian authorities in June 2015, reported the above key studies and stated that“The reliability factor of US ISC study (van Dievoet, 2005)is 1. Therefore, the study by US ISC should be considered for the environmental risk assessment at product authorization stage. In conclusion, ADBAC/BKC is ready biodegradable being the 10-day window criterion met (OECD 301B). On the other hand, the EQC study (van Ginkel and Stroo, 1992) has a reliability factor of 2 because it cannot distinguish between the degradation of ADBAC/BKC and Propan-2-ol (solvent). If we follow the argument that Propan-2-ol is readily biodegradable and might contribute more to the oxygen consumption. This results in an overestimation of ADBAC/BKC, and the 14-day window criteria was not met (OECD 301D). Alkyl (C12-16) dimethylbenzyl ammonium chloride is readily biodegradable.”  

Sea water:

A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (49-52% active in water) in seawater and sediment according to OECD Guideline 306 (biodegradation in seawater). Three bottles containing only seawater and 3 bottles containing seawater and the test substance were used. The test substance was added at a concentration of 2 mg/L. The biodegradability was determined by following the course of the oxygen decrease in the bottles using a special funnel. The funnel fitted exactly in the bottle and served as an overflow reservoir permitting multiple measurements in one bottle. The oxygen concentration was measured on Days 0, 7, 14, 21. 28. 42, 56 and 84. The test substance was toxic to microorganisms and was therefore studied in the presence of silica gel to reduce the concentration in the water phase. During the test period, the substance should be released slowly from the silica gel (0.5 g/bottle). Although no additional oxygen consumption was expected, controls with silica gel were carried out as well. Under the study conditions, the test substance was biodegraded by 38 and 31% on Day 28 in the absence and the presence of silica gel, respectively. Since the test substance was biodegraded at 61% on Day 84 in the prolonged closed bottle test with silica gel, it is expected to be biodegraded in seawater (van Ginkel, 1994).

 

Therefore, based on the available information and in line with the biocides assessment report, the test substance is considered to be readily biodegradable.