N,N'-bis(5,5-dimethyl-1,3,2-dioxaphosphinane-2-oxide-2-yl)ethane-1,2-diamine

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:

January 10, 2013 to February 7, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Version / remarks:

OECD Guidelines for Testing Chemicals, No.301C, July 17, 1992, “Ready Biodegradability: Modified MITI Test (I)”

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Japanese Testing Standards

Version / remarks:

“Biodegradation study of chemical substances by microorganisms” stipulated in “Order Prescribing the Tests Relating to the New Chemical Substances” (Yaku-shoku-Hatsu No. 0331-7, Heisei 23.03.29 Seikyoku No. 5, Kanpoki-Hatsu No. 110331009, dated March 31, 2011; Yaku-shoku-Hatsu No. 0402-1, Heisei 24.03.28 Seikyoku No. 2, Kanpoki-Hatsu No. 120402001, partially revised on April 2, 2012)

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

No further details specified in the study report.

Oxygen conditions:

not specified

Inoculum or test system:

activated sludge, adapted

Details on inoculum:

Activated sludge
In accordance with the test method, sludge were sampled from 10 locations in Japan (surface waters containing surface soils at rivers, lakes, ponds, and inland seas, and return sludge of sewage plants), prepared and controlled as activated sludge in Kurume Laboratory. This activated sludge was used for the test (sampling time: November, 2012; start date of use: December 20, 2012). For the test, the activated sludge was used at approximately 19 hours after addition of synthetic sewage (prepared by dissolving glucose, peptone and potassium dihydrogen phosphate into purified water and adjusting pH to 7.0+/-1.0).

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Implementation of test
Preparation of the test
Determination of volume of additive activated sludge
The concentration of the suspended material in the activated sludge was measured in accordance with the following method. By considering this concentration, the amount of the activated sludge, which was added into each test vessel, was determined to be 2.57 mL.
Measurement method: Section 14.1 of JIS K 0102-2008
Date of measurement: January 7, 2013
Result of measurement: 3500 mg/L

Preparation of mineral media
Each 3 mL of solution A, B, C and D, whose compositions are prescribed in section 21, JIS K 0102-2008, was diluted with purified water (Takasugi Pharmaceutical Co., Ltd., Japanese Pharmacopeia grade) to make 1 L respectively in order to prepare total 4 L of mineral media and then the pH of the mineral media was adjusted to 7.0.

Validation of activity of activated sludge
It was confirmed by using aniline that the activated sludge was sufficiently active.

Preparation of the test solution
Test solutions were prepared as the following method for 6 test vessels.

Addition of the test substance and aniline
1) Water + Test substance (1 vessel, as vessel [1])
To give 100 mg/L of the test substance concentration, 300 mL of purified water and 30 mg of the provided test sample were placed in the test vessel. The provided test sample was accurately weighed with an analytical electronic balance.
2) Sludge + Test substance (3 vessels, as vessel [2], [3] and [4])
To give 100 mg/L of the test substance concentration, the mineral medium (amount of 300 mL minus added volume of the activated sludge (2.57 mL)) and 30 mg of the provided test sample were placed in the test vessels. The provided test sample was accurately weighed with an analytical electronic balance.
3) Sludge + Aniline (1 vessel, as vessel [6])
To give 100 mg/L of aniline concentration, the mineral medium (amount of 300 mL minus added volume of the activated sludge (2.57 mL)) and 29.5 μL (30 mg) of aniline were placed in the test vessel. Aniline was dispensed using a micro syringe.
4) Sludge blank (1 vessel, as vessel [5])
The mineral medium (amount of 300 mL minus added volume of the activated sludge (2.57 mL)) was placed in the test vessel.

Inoculation of the activated sludge
The activated sludge was added to each test solution of 2), 3) and 4) to make the concentration of the suspended solid at 30 mg/L.

Apparatus and conditions of cultivation
Apparatus for test solution cultivation
Closed system oxygen consumption measuring apparatus
Thermostatic Bath (including measurement unit): AI-0001 (Asahi Techneion Co.,Ltd.)
Data processor: OM7000A (Ohkura Electric Co., Ltd.)
Test vessel: Glass Culture Bottle
Carbon dioxide absorbent: Soda Lime (for carbon dioxide absorption, Wako Pure Chemical Industries, Ltd.)

Culture conditions
Cultivation temperature: 25 ± 1oC
Cultivating duration: 28 days (protected from light)
Stirring method: Rotary stirring by a magnetic stirrer

Observation
During incubation period, conditions of the test solutions were visually observed every day.

Measurement of Biochemical Oxygen Demand (BOD)
During incubation period, BOD values of the test solutions were continuously measured by the closed system oxygen consumption measuring apparatus. Temperature in the thermostatic bath of the closed system oxygen consumption measuring apparatus was recorded every day.

Analysis of the test solution
After the completion of incubation period, qualitative analysis for the test substance in the test solution was conducted to confirm presence of transformed products. Because the test substance, which was a reaction product containing several components, had a large difference of amount between the main component and the sub components, a quantitative analysis of all the components could not be conducted in the same analytical condition.
Therefore, amounts of the main component and sub components were separately measured.
Concentrations of each component are expressed as a concentration of the test sample without consideration of component composition. Based on the results of the preliminary examination that was conducted before the main test, the test substance was considered not to dissolve into test solution completely. Therefore, analysis of dissolved organic carbon (DOC) was not conducted. pH of the test solution of “Water + Test Substance” and “Sludge + Test substance” was measured.

Pretreatment of test solution
The test solution of “Water + Test Substance”, “Sludge + Test Substance” and “Sludge Blank” were pretreated in accordance with the following scheme for preparing samples for Liquid Chromatography-Mass Spectroscopy (LC-MS) for analysis of the test substance (main component), samples for Liquid Chromatography-Tandem Mass Spectroscopy (LC-MS/MS) for analysis of the test substance (sub components), and samples for LC-MS for qualitative analysis of transformed products.

Calculation method for percentage biodegradation
Percentage biodegradation was calculated based on the following formula and the calculated value was rounded to whole number to display.
Because the test substance, which was a reaction product, had a large difference of content between the main component and the sub components, a quantitative analysis of all the components could not be conducted in the same analytical condition. Therefore, for obtaining biodegradation (for sub components, percentage of decrease amount), the main component and sub components were separately calculated.
For sub components, since percentage of residual amount of the measured substance in “Water + Test Substance” was below 90% (64%), the percentage biodegradation of the measured substance (biodegradation of the measured substance by a direct analysis) was not calculated, but the percentage of decrease amount to the adding quantity of the test substance was calculated instead.

Biodegradation by BOD
Biodegradation (%) = (BOD-B)/TOD x 100
BOD: Biochemical oxygen demand of “Sludge + Test substance” (measured value: mg)
B: Biochemical oxygen demand of “Sludge Blank” (measured value: mg)
TOD: Theoretical oxygen demand which is required when the test substance is completely oxidized (calculated value: mg)
TOD was calculated using the molecular formula of the main component, C12H26N2O6P2, and a form of nitrogen was assumed to be nitrous acid (NO2).

Biodegradation by degradation of specific test substances
Biodegradation of the main component
Biodegradation (%) = (Sw-Ss)/Sw x 100
Ss: Residual amount of the measured substance in “Sludge + Test Substance” (measured value: mg)
Sw: Residual amount of the measured substance in “Water + Test Substance” (measured value: mg)

Percentage of decrease amount of sub components
Percentage of decrease amount (%) = (Sw-Ss)/Sw x 100
Ss: Residual amount of the measured substance in “Sludge + Test Substance” (measured value: mg)
Sw: Added volume of the measured substance (mg)

Reference substance:

aniline

Key result

Parameter:

% degradation (O2 consumption)

Value:

>= 3 - <= 4

Sampling time:

28 d

Details on results:

Biodegradation after 28 days is as follows.
Because the test substance, which was a reaction product, had a large difference of content between the main component and the sub components, a quantitative analysis of all the components could not be conducted in the same analytical condition. Therefore, for obtaining biodegradation (for sub components, percentage of decrease amount), the main component and sub components were separately calculated.
For sub components, since percentage of residual amount of the measured substance in “Water + Test Substance” was below 90% (64%), the percentage biodegradation of the measured substance (biodegradation of the measured substance by a direct analysis) was not calculated, but the percentage of decrease amount to the adding quantity of the test substance was calculated instead.

Biodegradability of total test substance
Concentrations of sub components in the provided test sample are unknown. However, based on the proportion of the main component and sub component 1 (half type) (main component: 98.82%, half type: 1.18%, according to information from the sponsor), the main component content was set to be 98.82% and the sub components content (total amount of 6 components) was set to be 1.18%, and these % contents were used for correcting residual amounts and a theoretical value so that a total biodegradability of the test substance was calculated.
As a result, the biodegradation of the test substance (total value) was -5 to -3%, biodegradation by BOD was 3 to 4%, and therefore no biodegradability was noted. Since the test substance contained quaternary carbon structures which were hardly biodegraded and were derived from the reacted raw material into its structural formula, it was considered that the test substance was not biodegraded.

Confirmation of test conditions

Standard values for validity of the test and the values obtained from this test are shown in the following table. Since all values of this test met with standard values, this test was validated. A form of nitrogen was assumed to be ammonia for calculation of biodegradation by BOD of aniline.

		Value in the test	Standard value
Difference between the highest and lowest values of biodegradation (or percentage of decrease amount)	Biodegradation by BOD	1%	<20%
	Biodegradation of the test substance (ore percentage of decrease amount)	Main: 2% Sub: 7%
Biodegradation of BOD of aniline	After 7 days	74%	>40%
	After 14 days	98%	>65%
BOD of “Sludge Blank”	After 28 days	8.2 mg	<60 mg/L (<18 mg)

 

Condition of the test solution

Conditions of the test solutions are shown in the following table:

	Test solution	Condition (by visual observation)	pH
At the start of incubation	Water + Test Substance	The test substance was not dissolved. The test solution was colourless.	--
	Sludge + Test Substance	The test substance was not dissolved. The test solution was colourless.	--
At the end of incubation	Water + Test Substance	No undissolved substance was observed. The test solution was colourless	[1] 5.9
	Sludge + Test Substance	No undissolved substance except sludge was observed. No growth of sludge was observed. The test solution was colourless.	[2] 7.0 [3] 7.1 [4] 7.0

 

Analysis result of test solution

The results of analysis after 28 days are shown below

		Water + Test Substance	Sludge + Test Substance			Theoretical Amount
		[1]	[2]	[3]	[4]
BOD^*4	mg	2.4	1.6	2.3	2.0	54.0^*5
Amount and rate of residual test substance (main component) (LC-MS)	mg	28.8	29.7	30.2	30.1	30.6^*6
	%	96	99	101	100	-
Amount and rate of residual test substance (sub components) (LC-MS/MS)^*7	mg	19.1	17.6	15.5	17.0	30.0^*6
	%	64	59	52	57	-
Detection of transformed products (LC-MS)	-	Not detected				-

*4 Values in “Sludge + Test substance” are shown after subtracting the value of Sludge Blank.

*5 Calculated from the molecular formula of the main component of the test substance.

*6 Since concentrations of the main component and sub components are unknown, theoretical value of 30.0 mg was used for calculation.

*7 Calculated using total peak area on chromatogram.

 

Biodegradation

Biodegradation after 28 days

			Sludge + Test substance
			[2]	[3]	[4]	Mean
Biodegradation by BOD		%	3	4	4	4
Test substance	Biodegradation of the main component (LC-MS/MS)	%	-3	-5	-5	0 (-4)^*8
	Percentage of decrease amount of sub components (LC-MS/MS)	%	41	48	43	44

^*8Since the mean value of biodegradation was calculated as a negative value, it is expressed as “0” and the calculated value was indicated in parentheses.

 

Biodegradation of total test substance

		Water + Test Substance	Sludge + Test Substance			Theoretical Amount
		[1]	[2]	[3]	[4]
Residual amount of the main component^*9	mg (1)	28.5	29.3	29.8	29.7	29.6^*10
Residual amount of the sub components^*11	mg (2)	0.225	0.208	0.183	0.201	0.354^*12
Residual amount and residual rate of the total test substance ((1)+(2))	mg	28.7	29.6	30.0	29.9	30.0
	%	96	99	100	100	--
Percentage biodegradation of the total test substance	%	--	-3	-5	-4	--

^*9Residual amount of the main component (shown in analysis table) x 0.9882 (which is a content rate of the main component).

^*10Theoretical amount of the main component (30.0 mg) (shown in analysis table) x 0.9882 (which is a content rate of the main component).

^11*Residual amount of sub components (shown in analysis table) x 0.0118 (which is a content rate of the cub components).

^*12Theoretical amount of the main component (30.0 mg) (shown in analysis table) x 0.0118 (which is a content rate of the sub components).

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the condition of this study, most of the test substance was persistent and not biodegradable. Some sub components were transformed and the amounts of transformed products are considered to be small (below 1% of the total of test substance).

Executive summary:

Test conditions

Test substance concentration: 100 mg/L

Activated sludge concentration: 30 mg/L (as suspended material concentration)

Test solution volume: 300 mL

Incubation temperature for test solution: 25+/-1ºC

Incubation period for test solution: 28 days (protected from light)

 

Measurement and analysis for calculation of biodegradability (percentage of decrease amount)

a) Measurement of biochemical oxygen demand (BOD) by closed system oxygen consumption measuring apparatus

b) Quantitative analysis of test substance (main component) by liquid chromatography – mass spectrometry (LC-MS)

c) Quantitative analysis of test substance (sub components) by liquid chromatography – tandem mass spectrometry (LC-MS/MS)

 

Test results

			Sludge + test substance
			[2]	[3]	[4]	Mean
BOD biodegradation		%	3	4	4	4
Test substance^*2	Main component biodegradation (LC-MS)	%	-3	-5	-5	0 (-4)^*1
	Sub components percentage of decrease amount^*3 (LC-MS)	%	41	48	43	44

^*1Since the mean value of biodegradation was calculated as a negative value, it was expressed as “0” and the calculated value was indicated in parentheses.

^*2Because the test substance, which was a reaction product, had a large difference of content between the main component and the sub components, a quantitative analysis of all the components could not be conducted in the same analytical condition. Therefore, for obtaining biodegradation (for sub components, percentage of decrease amount), the main component and sub components were separately calculated.

^*3Since percentage of residual amount of the measured substance in “Water + Test Substance” was below 90% (64%), the percentage biodegradation of the measured substance (biodegradation of the measured substance by a direct analysis) was not calculated, but the percentage of decrease amount to the adding quantity of the test substance was calculated instead.

 

Conclusion

Under the condition of this study, most of the test substance was persistent and not biodegradable.

Some sub components were transformed, but the amounts of transformed products are considered to be small (below 1% of the total amount of the test substance).

Description of key information

Under the condition of this study, most of the test substance was persistent and not biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

Test conditions

Test substance concentration: 100 mg/L

Activated sludge concentration: 30 mg/L (as suspended material concentration)

Test solution volume: 300 mL

Incubation temperature for test solution: 25+/-1ºC

Incubation period for test solution: 28 days (protected from light)

 

Measurement and analysis for calculation of biodegradability (percentage of decrease amount)

a) Measurement of biochemical oxygen demand (BOD) by closed system oxygen consumption measuring apparatus

b) Quantitative analysis of test substance (main component) by liquid chromatography – mass spectrometry (LC-MS)

c) Quantitative analysis of test substance (sub components) by liquid chromatography – tandem mass spectrometry (LC-MS/MS)

 

Test results

			Sludge + test substance
			[2]	[3]	[4]	Mean
BOD biodegradation		%	3	4	4	4
Test substance^*2	Main component biodegradation (LC-MS)	%	-3	-5	-5	0 (-4)^*1
	Sub components percentage of decrease amount^*3 (LC-MS)	%	41	48	43	44

^*1Since the mean value of biodegradation was calculated as a negative value, it was expressed as “0” and the calculated value was indicated in parentheses.

^*2Because the test substance, which was a reaction product, had a large difference of content between the main component and the sub components, a quantitative analysis of all the components could not be conducted in the same analytical condition. Therefore, for obtaining biodegradation (for sub components, percentage of decrease amount), the main component and sub components were separately calculated.

^*3Since percentage of residual amount of the measured substance in “Water + Test Substance” was below 90% (64%), the percentage biodegradation of the measured substance (biodegradation of the measured substance by a direct analysis) was not calculated, but the percentage of decrease amount to the adding quantity of the test substance was calculated instead.

 

Conclusion

Under the condition of this study, most of the test substance was persistent and not biodegradable.

Some sub components were transformed, but the amounts of transformed products are considered to be small (below 1% of the total amount of the test substance).