bis(diethylamino)-N,N-diethylmethaniminium chloride

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 10, 2003 to January 7, 2004

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)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Remarks:

OECD GLP

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Fresh activated sludge was obtained from the municipal waste treatment facility at Newburyport, Massachusetts. This facility treats predominantly domestic waste. Viability of the microorganisms was confirmed after inoculation of the test solutions and activity was checked by means of the positive control. The sludge was used on the day it was collected and it was aerated for 4 hours prior to use. A subsample (approximately 1 L) of the activated sludge was homogenized for approximately 2 minutes with a mechanical blender, settled for approximately 30 minutes, and centrifuged in 6 tubes for approximately 30 minutes. The supernatant was decanted, pooled, and added to each test vessel to provide sufficient volume for a 1% inoculum. Carry-over of sludge solids, which would interfere with the measurement of CO2 production, was avoided. Viability counts were performed on the supernatant at the start of the test and the inoculum contained 1 x 10^6 colony forming units per mL and 0.020 g/L total suspended solids.

Duration of test (contact time):

28 d

Initial conc.:

33.8 mg/L

Based on:

act. ingr.

Initial conc.:

20 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Basal Salts Medium (BSM) was used as dilution medium. The definitive degradability test was performed using six test vessels (2.5 L capacity amber glass bottles); two for inoculated BSM (the inoculum blank), one for the inoculated positive control solution, one for the toxicity control, and two for the inoculated replicate test solution. Each test vessel was filled with 1,235 mL of BSM and 15 mL of the activated sludge supernatant inoculum. This mixture was aerated for approximately 24 hours to purge the system of carbon dioxide. After the aeration period, 100 mL of 0.0125 M Ba(OH)2 was introduced into each of three CO2 absorber bottles connected in series to the exit air line of each vessel. Test substance was added to the appropriate vessels to begin the test. The test substance was tested at 20 mg/L total carbon. The test concentration was prepared in duplicate by bringing 0.0797 g of test substance to a total volume of 250 mL with BSM and mixing on a magnetic stir plate until dissolved. The pH of the solutions was 7.5. After mixing, the solutions were added to each of two replicate test vessels. The inoculum blanks, which contained no test substance, received only 250 mL of CO2-free BSM. The positive control, sodium benzoate, was prepared by adding 0.0515 g to 250 mL of CO2 purged BSM. This stock solution was then added to the test vessel. The toxicity control was prepared by bringing 0.0797 g test substance (based on correction for purity) and 0.0515 g sodium benzoate to a total volume of 250 mL with CO2 purged BSM. The solution was mixed on a magnetic stir plate for approximately 6 hours. The pH of the solution was 7.5. After mixing, the solution was added to the test vessel. The final volume in each test vessel was 1,500 mL.

CO2-free air was bubbled through the solutions at a rate of approximately 50 to 100 mL/minute. The CO2 produced in each test vessel reacted with the barium hydroxide and was precipitated as barium carbonate. Barium hydroxide was prepared by adding 4 g/L of Ba(OH)2.8H2O to up to 6 liters of deionized water and mixing until dissolved. The solution was then filtered and sealed in 1 L plastic bottles prior to use. The amount of CO2 produced by each measurement day was determined by titrating the remaining Ba(OH)2 with 0.05 N (0.05 M) HCl. The CO2 absorber bottle nearest the test vessel was removed on each measurement day for titration. The remaining two absorber bottles were each moved one place closer to the test vessel, and a new absorber bottle containing 100 mL of fresh 0.0125 M Ba(OH)2 was placed at the far end of the series. The 100 mL Ba(OH)2 solutions were titrated with 0.05 N (0.05 M) HCl, using phenolphthalein as an indicator. Titrations were made every one to three days during the first 10 days, then at least every five days until day 28.

The test was performed at a target temperature range of 22 ± 2°C (the temperature of a representative flask of water incubated among the test vessels was recorded daily during the test period). The pH of both BSM and stock solutions was measured at the start of the test. The pH was measured in all test vessels on day 27 and 1 mL of concentrated HCl was added to drive off inorganic carbonate. The test vessels were aerated overnight and final titrations were performed on day 28.

The total amount of CO2 produced by the test substance was determined for the test period and calculated as a percentage of the total CO2 that the test substance, based on its carbon content, could have theoretically produced. The inoculum blank served as a control to correct for CO2 produced through endogenous respiration of bacteria. The amount of CO2 produced by the test substance was determined by the difference (in mL of titrant) between the test and inoculum blank Ba(OH)2 traps. As titrations were performed with 0.05 N (0.05 M) HCl, each 1.0 mL of HCl corresponds to 1.1 mg of CO2 produced. The percent biodegradation for the test substance and reference substance were calculated.

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Remarks on result:

other: Replicate 1

Parameter:

% degradation (CO2 evolution)

Value:

0.9

Sampling time:

28 d

Remarks on result:

other: Replicate 2

Details on results:

The positive control, sodium benzoate, yielded 100% of the theoretical CO2 during the test, demonstrating the adequacy of the inoculum and the toxicity control allowed 46% biodegradation after 14 days (48% biodegradation after 28 days), indicating that the test substance was not inhibitory to the activated sludge at the tested concentration (the test substance is considered to be inhibitory if there is less than 25% degradation within 14 days in the toxicity control). The inoculum blanks evolved an acceptable amount of CO2/L (11 mg CO2/L). No unusual variation in pH was noted in any test vessel. Temperature remained within the acceptable range of 22 ± 2°C, except on days 3, 4, and 21 when it was recorded as 19.8, 19.9, and 19.8 °C, respectively. Daily measured aeration flow rates were approximately 50 to 80 mL/minute in each test vessel.

A test substance is considered to be readily biodegradable if at least 70% removal of dissolved organic carbon (based on CO2 production) occurs in a 10-day window within the 28-day period of the test. Less carbon dioxide was evolved from test vessels containing the test sustance than from the inoculum blank, resulting in a mean of 0% biodegradation after subtraction of the blank to correct for background CO2 evolution. The lag phase, the 10-day window starting with the day the observed level of biodegradation first exceeded 10%, the degradation phase, and the slope of the degradation line could not be calculated because there was no biodegradation observed. These results (0% biodegradation) indicate that test substance was not readily biodegradable under test conditions.

Cumulative percent of theoretical carbon dioxide evolved during the ready biodegradability (biotic degradation) test with the test substance using the CO₂ evolution test (modified Sturm).

Day	Inoculum Blank			Positive Control (%)	Toxicity Control (%)	Treatment
	Rep. 1 (%)	Rep. 2 (%)	Mean (%)			Rep. 1 (%)	Rep. 2 (%)	Mean (%)
2	NA	NA	NA	37.3	19.6	1.4	0.1	0.8
3	NA	NA	NA	60.1	30.1	0.7	0.4	0.6
4	NA	NA	NA	70.7	35.1	0	0.6	0
6	NA	NA	NA	80.9	39.9	0	0.8	0
9	NA	NA	NA	88.7	43.1	0	2.1	0
14	NA	NA	NA	95.0	45.6	0	2.6	0.7
19	NA	NA	NA	98.1	46.9	0	1.0	0
21	NA	NA	NA	98.9	47.2	0	1.0	0
26	NA	NA	NA	100	47.9	0	0.8	0
27	NA	NA	NA	100	48.0	0	1.0	0
28	NA	NA	NA	100	48.2	0	0.9	0

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of this study, the active ingredient is considered not readily biodegradable.

Executive summary:

The ready biodegradability of the active ingredient was investigated using a modified Sturm test, in accordance with the standardised guidelines OECD 301B, under GLP conditions. 

Fresh activated sludge, was used as the source of unacclimated microorganisms for the test.  Biodegradability was determined by measuring the amount of carbon dioxide evolved from the chemical over the 28-day test period.  The degradation is defined as the CO2 produced by the substance, as a percentage of the theoretical CO2 it could have produced, calculated from the carbon content of the test substance.

The test was performed at a nominal concentration of 53.1 mg/L whole test substance (33.8 mg active ingredient).  The nominal carbon concentration of the replicate solutions was 20 mg/L.  This concentration and an inoculum blank were established in duplicate and a single positive control, sodium benzoate at 34.3 mg/L, was also established at a dissolved organic carbon concentration of 20 mg/L.  A single toxicity control, containing sodium benzoate at 34.3 mg/L and the test substance at 53.1 mg/L whole test substance, was also established at a combined dissolved organic carbon concentration of 40 mg/L.

After 28 days, the test substance at a nominal concentration of 20 mg/L dissolved organic carbon showed 0 % biodegradation.  These results indicate that the active ingredient was not readily biodegradable under test conditions.

Description of key information

Under the conditions of this study the active ingredient is considered not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

The ready biodegradability of the active ingredient was investigated using a modified Sturm test, in accordance with the standardised guidelines OECD 301B, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Fresh activated sludge, was used as the source of unacclimated microorganisms for the test.  Biodegradability was determined by measuring the amount of carbon dioxide evolved from the chemical over the 28-day test period.  The degradation is defined as the CO2 produced by the substance, as a percentage of the theoretical CO2 it could have produced, calculated from the carbon content of the test substance.

The test was performed at a nominal concentration of 53.1 mg/L whole test substance (33.8 mg active ingredient).  The nominal carbon concentration of the replicate solutions was 20 mg/L.  This concentration and an inoculum blank were established in duplicate and a single positive control, sodium benzoate at 34.3 mg/L, was also established at a dissolved organic carbon concentration of 20 mg/L.  A single toxicity control, containing sodium benzoate at 34.3 mg/L and the test substance at 53.1 mg/L whole test substance, was also established at a combined dissolved organic carbon concentration of 40 mg/L.

After 28 days, the test substance at a nominal concentration of 20 mg/L dissolved organic carbon showed 0 % biodegradation.  Under the conditions of this study the active ingredient is considered not readily biodegradable.