Quaternary ammonium compounds, di-C8-10-alkyldimethyl, chlorides

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Duration of test (contact time):

28 d

Initial conc.:

13.7 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

MICROBIAL ACCLIMATION PHASE
A 2-week inoculum buildup period was allocated for the adaptation of the microbial inoculum to the test substance. The acclimation medium was prepared by adding 1 L of test water to a 3-L Erlenmeyer flask. One milliliter of each test medium stock solution A, B, and C was added to the flask. One gram of soil no. BD-005A, 2 mL of activated sludge, and 50 mL of raw domestic sewage were also added to the flask. This mixture was stirred for 15 minutes, then filtered through glass wool into another flask and left standing for 1 hour. After 1 hour, the mixture was filtered again through glass wool. To the filtrate, the following were added: 0.025 g Casamino acid (Difco Laboratories; Detroit, MI), 0.025 g of yeast extract (Difco Laboratories; Detroit, :MI), and 1 mL of DDAC stock solution (2.806 mg/mL). The contents of the flask were mixed and put on the shaker at approximately 100 rpm in the dark. On each of days 7 and 11, 2 mL of DDAC stock solution were added to the flask. The total amount of carbon added to the flask from the DDAC was 10 mg .

DEFINITIVE TESTING PHASE
After the 2-week acclimation phase, nine 2-L Erlenmeyer flasks were prepared. To each of the control flasks, 897 mL of test water and 1 mL of each of the three test medium stock solutions were added. To each of the test, reference and sterile test control (STC) flasks, 896 mL of test water and 1 mL of each of the three test medium stock solutions were added. The acclimated microbial inoculum was filtered and 100 mL of the filtrate were added to each flask.
The test and reference flasks were dosed with carbon at 10 mg/L. To achieve this, 1 mL total of DDAC stock solution (13.7 mg DDAC/mL) was added to each of the test flasks.
The test substance was added incrementally on days 0, 2, 4, and 7 with 100, 200, 300, and 400 p.L of DDAC stock solution, respectively. One milliliter of dextrose stock solution (25.0 mg dextrose/mL test water) was added to each of the reference flasks on day O. No additional carbon was added to the control flasks. DDAC was added incrementally to the test flasks in order to prevent toxicity to the microbial population. The STC flask was dosed similar to the test flasks and also received 0.050 g of HgCl2 in order to inhibit microbial growth and determine background endpoint levels. The final volume of each flask was 1000 mL.
All flasks were sparged for 5 minutes with CO2-free air. After sparging, 10 mL of 0.2 N KOH were added to the culture tube reservoir. Flasks were placed on the shaker at 100 rpm in the dark.
Duplicate samples (7 mL) of the medium for dissolved organic carbon (DOC) analysis were taken after adequate mixing had occurred. The test systems were incubated at 21 to 24 °C, and the temperature was monitored continuously. On day27, approximately 3 mL of a 20 % H2S04 solution were added to each flask in order to liberate all CO2 from the test medium. The flasks were placed back on the shaker. The test was terminated on day 28.

Reference substance:

other: Dextrose

Parameter:

% degradation (CO2 evolution)

Value:

80.92

Sampling time:

28 d

Details on results:

The soil used during the acclimation phase of this study was classified as a loam. The number of colony forming units of the acclimated inoculum was 3.0E06 CFU/mL at start of the test.
By day 28 the % TCO2 evolution for the three DDAC test flasks was 85.55, 79.09, and 78.11 % with a mean of 80.92 %. By day 14, CO2 evolution from DDAC was similar to that from dextrose. The amount of CO2 evolution from DDAC at the earlier time points was lower than that from dextrose because DDAC was added to the test flasks incrementally. The amount of CO2 produced from the sterile test control flasks was minimal.
DDAC exhibited 85.07, 85.31, and 83.0 % TDOC removal with a mean of 84.46 % for the three test flasks throughout the 28-day testing period.

Results with reference substance:

Dextrose exhibited %TCO2 evolutions of 76.49 and 85.35 % with a mean of 80.92 % for the two flasks over 28 days and 83.71 and 86.85 % TDOC removal with a mean of 85.28%.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

In order for a test substance to be considered ultimately biodegradable, threshold values of 60 % ThCO2 and 70 % ThDOC removal must be obtained. These threshold values were obtained with dextrose, indicating that the microbial inoculum was viable and active. These criteria were also obtained with DDAC in the same time frame. Therefore, DDAC was determined to be ultimately biodegradable.

Executive summary:

A study was carried out according to EPA OTS 796.3100 (Aerobic Aquatic Biodegradation) using Didecyldimethylammonium chloride (DDAC).

DDAC was evaluated for biodegradability in an aerobic aquatic shake flask test system by exposing DDAC to a mixed microbial inoculum for 28 days. Prior to the 28 day testing period, the mixed microbial inoculum was acclimated to DDAC for 14 days in a defined inorganic salts medium. A loam soil, activated sewage sludge, and influent raw sewage served as sources of the mixed microbial inoculum. Three flasks containing DDAC were utilized in this study. Two flasks containing a reference substance, dextrose, were utilized in order to monitor the viability of the microbial population. Three control flasks containing the microbial inoculum without test or reference substance were utilized in order to determine the endogenous microbial respiration. A sterile test control flask was utilized in order to monitor background CO2, The concentration of carbon from DDAC or dextrose was 10 mg/L. DDAC or dextrose provided the sole source of carbon for the respective systems. All test systems were analyzed for CO2 evolution on days 3, 7, 14, 21, and 28 and for dissolved organic carbon (DOC) on days 0, 3, 7, 14, 21, and 28. The biodegradability of DDAC and dextrose was calculated as percent theoretical CO2 (% ThCO2) evolution which was corrected for endogenous microbial activity for each sample time point. The percent theoretical DOC (%ThDOC) removal of DDAC and dextrose was calculated as % ThDOC removal which was corrected for endogenous values obtained on day 28. For DDAC on day 28, the mean %ThCO2 and the mean %ThDOC removal values were 80.92 % and 84.46 %, respectively. For dextrose on day 28, the mean %ThC02 and the mean %TDOC values were 80.92 and 85.28 %, respectively. These results indicate that the microbial inoculum was viable and that DDAC was ultimately biodegradable under the test conditions.

Description of key information

A reliable OECD 301B test guideline study (Klimisch 1) to determine the ready biodegradability for the read across substance, N,N-Didecyl-N,N-dimethylammonium carbonate (DDACarbonate) (NOACK, 2007), measured 10 % biodegradation (beginning of biodegradation) after 5 days and the pass level of 60 % after 12 days. The 2nd test item replicate reached the 10 % level after 4 days and the pass level of 60 % after 10 days. At test end a biodegradation rate of 99 % was reached. The positive control and toxicity controls both passed the test criteria, demonstrating the test system was functioning well and the test substance was not toxic to the microorganisms. The results indicate the test substance is readily biodegradable.

Another reliable OECD 301B test guideline study (Klimisch 1) to determine the ready biodegradability for the read across substance, N,N-Didecyl-N,N-dimethylammonium carbonate (DDACarbonate) (NOACK, 2008). Again, the positive control and toxicity controls both passed the test criteria, with percentage degradation of the positive control reaching the pass level of 60 % after 6 days, with 81 % after 14 days. In the toxicity control containing both test and reference item a biodegradation rate of 62 % occurred within 14 days and came to 97 % after 28 days. In the test vessels, the 10 % level (beginning of biodegradation) was reached after 11 days and the pass level of 60 % after 19 days. The mean biodegradation came to 96 % after 28 days.

In addition, four reliable studies are available for the read across substance (DDAC). A reliable study by Clariant (1992) included two separate experiments, one conducted according to OECD 301A (Die away test) and the other conducted according to OECD 302B guidelines (inherent biodegradability, Zahn Wellens). Activated sludge from a sewage treatment plant was used for the OECD 302B test, which after 28 days, was used as pre-adapted sludge for the Die-away study. In the Zahn-Wellens Test elimination of DDAC after 28 days was 95.5 % (related to 3 h-value). In the DOC Die-Away Test the maximum degradation was 90 % and fulfilled the 10-day window criteria. A further reliable study was carried out by Wildlife (1996) according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test) using DDAC. The results of this study indicated that both un-complexed and test substance complexed to bentonite clay were biodegradable when exposed to unacclimated microorganisms obtained from a receiving system representative of a site where these test substances may be expected to be discharged. Degradation of 77.5 % was attained for non-complexed test substance after 28 days, and slightly lower when DDAC was complexed with clay. A further Wildlife study (2001) conducted not according to guidelines, in which activated sludge pre-exposed to non-labelled DDAC, was exposed to radiolabelled DDAC for a 28 days period and the DDAC (plus metabolites) in the different phases (sludge, water and mineralisation) were measured. 14CO2 production reached 71.82 % at Day 1, 88.36 % at Day 7, and 93.30 % at Day 28, with low amounts of DDAC or metabolites measured in the sludge or water phases. The fourth study on DDAC by ABC Laboratories (1993) was conducted according to the EPA OPTS 796.3100 guideline, using a mix of inoculum from soil, activated sludge and influent sewage. Degradation of 80.92% was attained after 28 days.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable but failing 10-day window