4,4'-methylenedianiline

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)

GLP compliance:

yes

Radiolabelling:

yes

Oxygen conditions:

aerobic/anaerobic

Inoculum or test system:

natural water / sediment

Details on source and properties of surface water:

- Details on collection (e.g. location, sampling depth, contamination history, procedure): Water samples were taken at two different locations: 1. Surface water samples from Love Point Lake, Kent Island, Maryland, USA and 2. Surface water samples from Choptank River, Denton, Maryland, USA.
- Storage conditions: under refrigerated conditions.
- Temperature (°C) at time of collection: 1.: 26.1; 2.: 25.5
- pH at time of collection: 1.: 8.45; 2.: 7.03
- Oxygen concentration (mg/l) initial: 1.: 7.6; 2.: 5.8
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): Love Point Lake: aerobic 143 CFU, anaerobic 370 CFU; Choptank River: aerobic: 1210 CFU, anaerobic 1087 CFU

Details on source and properties of sediment:

- Details on collection (e.g. location, sampling depth, contamination history, procedure): Sediment samples were taken at two different locations: 1. Surface water samples from Love Point Lake, Kent Island, Maryland, USA and 2. Surface water samples from Choptank River, Denton, Maryland, USA. For the aerobic sediments the sampling depth was 0 - 5 cm for Love Point Lake and 0 - 10 cm for Choptank river, for anaerobic sediments a sampling depth of 5 - 15 cm and 10 - 20 cm were chosen for Love Point lake and Choptank river, respectively.
- Storage conditions: At the sampling site the aerobic sediments were stored in buckets allowing air circulation by using loosely attached lids. The anaerobic sediment samples were collected in buckets which were then seald to exclude air. On the day of collection the samples were transferred to the laboratory and stored under refrigerated conditions.
- Textural classification (i.e. %sand/silt/clay): Love Point Lake aerobic: 34% sand, 46% silt and 20% clay; Love Point Lake anaerobic: 36% sand, 44% silt and 20% clay; Choptank river aerobic: 96% sand, 2% silt and 2% clay; Choptank river anaerobic: 98% sand, 2% silt and 0% clay.
- pH at time of collection: associated water had a pH of 8.45 for the Love Point Lake waters (aerobic and anaerobic samples) and 7.03 for Choptank River waters (aerobic and anaerobic samples).
- Organic carbon (%): Love Point Lake aerobic: 8.8, Love Point Lake anaerobic: 8.7; Choptank River aerobic: 0.55, Choptank River anaerobic: 0.55 (see also table 2)
- Redox potential (mv) initial/final: Love Point Lake: aerobic: initial: 110.2, final: 177.2; anaerobic: initial: -48.7, final: -144.2; Choptank River: aerobic: initial: 163.2, final: 239.2; anaerobic: initial: 146.7, final: 124.5 (see also table 3)
- Bulk density (g/cm³): Love Point Lake aerobic: 1.15, Love Point Lake anaerobic: 1.17; Choptank River aerobic: 1.88, Choptank River anaerobic: 1.85.
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): Microbial biomass in µg/g: Love Point Lake: aerobic: start of test: 2228.4, end of test: 2780.4; anaerobic: start of test: 1520.3, end of test: 1370.7; Choptank River: aerobic: start of test: 49.1, end of test: 81.4; anaerobic: start of test: 36.1, end of test: 36.8 (see also table 1)
- Sediment samples sieved: yes; overlying water and stones > 2 mm discarded

Duration of test (contact time):

100 - 102 d

Initial conc.:

7.56 - 7.71 other: µCi/vessel

Based on:

other: radioactivity

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: Approximately 100 g of aerobic or anaerobic sediment from Love Point Lake and approximately 150 g of aerobic or anaerobic sediment from Choptank River were placed in each transformation and each mineralization test vessel, resulting in sediment depths of 2.0 to 3.2 cm. The depths of the water layer ranged from 8.5 to 10.4 cm. The water to sediment ratio ranged from 3.0:1 to 3.6:1
- Test temperature: 20 ± 2 °C
- Continuous darkness: yes
- Other: The test vessels were acclimated prior to application of the test substance. Bubbling was observed in the test chambers and traps each working day; therefore, aerobic and/or anaerobic conditions were maintained throughout the test period.

TEST SYSTEM
- Culturing apparatus: Transformation test vessels were cylindrical, wide-mouth, 500-mL glass jars with PTFE-lined lids. Mineralization test vessels were 500-mL glass jars with ground-glass openings at the tops. The glass lids had mating groud-glass joints.
- Number of culture flasks/concentration: see table 4
- Method used to maintain aerobic conditions: gentle stream of air through the water layer
- Method used to maintain anaerobic conditions: gentle stream of nitrogen through the water layer
- Measuring equipment: gas traps

- Details of trap for CO2 and volatile organics if used: Effluent gases from each test chamber were passed through a series of four trap bottles, mixed with oxygen and passed through a quartz tube in a furnace set at 800 °C, and then passed through another series of three trap bottles. The trap bottles before the tube furnace consisted of an empty bottle followed by two bottles containining 100 mL of 1.5 N KOH solution and another empty bottle. The quartz tubes were packed with cupric oxide for the transformation of 14CH4 and other volatile 14C-gases to 14CO2. After the tube oven the gasses passed through the second series of trap bottles, consisting of an empty bottle followed by two bottles filled with 100 mL 1.5 N KOH solution.

CONTROL AND BLANK SYSTEM
- Abiotic sterile control: yes

Compartment:

other: Love Point Lake and Choptank River freshwater, aerobic conditions, live and sterile

DT50:

3.1 - 5.8 d

Type:

(pseudo-)first order (= half-life)

Temp.:

20 °C

Remarks on result:

other: R²: 0.9714 - 0.9817

Compartment:

other: Love Point Lake and Choptank River sediment water systems, aerobic conditions, live and sterile

DT50:

3.1 - 5.8 d

Type:

(pseudo-)first order (= half-life)

Temp.:

20 °C

Remarks on result:

other: No parent material was found in the extracts from aerobic sediments systems after 7-day incubation suggesting that the rate of parent material degradation in the whole systems is close to that in the water layers.

Compartment:

other: Love Point Lake and Choptank River freshwater, anaerobic conditions, live and sterile

DT50:

12.7 - 28.9 d

Type:

(pseudo-)first order (= half-life)

Temp.:

20 °C

Remarks on result:

other: R²: 0.8322 - 0.9234

Compartment:

other: Love Point Lake and Choptank River sediment water systems, anaerobic conditions, live and sterile

DT50:

5 - 21 d

Type:

other: assessed graphically

Temp.:

20 °C

Remarks on result:

other: In anaerobic sediment/water systems the decrease in the total content of parent material with incubation period did not follow first-order kinetics equation; the DT50 values were assessed graphically

Transformation products:

not measured

Details on transformation products:

Due to technical reasons no analytical verification of metabolites was possible. It was impossible to derive a sufficient amount of metabolites to characterize them, mainly due to the difficult test conditions (sediment matrix etc.). Further steps to determine metabolites are considered and discussed by the member companies.

Evaporation of parent compound:

yes

Volatile metabolites:

no

Residues:

yes

Details on results:

VOLATILIZATION
- Percentage of the applied radioactivity present as volatile organics at end of study:
- Both aerobic systems: 0.03 - 0.04 % of applied radioactivity
- Both anaerobic systems: 0.03 - 0.09 % of applied radioactivity

Percentages of distribution of radioactivity in aerobic and anaerobic mineralization systems at the end of incubation period (average values of two replicates):

 

		Incubation period [d]	Water layer	Extractable from sediment	Non-extractable from sediment	CO2 traps	Sorbent traps	Total Recovery
Love Point Lake, aerobic	Live	100	11.7	8.4	69.3	2.76	0.026	92
Love Point Lake, anaerobic	Live	102	18.6	32.9	85.2	0.64	0.03	137
Choptank River, aerobic	Live	100	6.1	12.0	90.2	5.66	0.043	114
Choptank River, anaerobic	Live	102	12.1	29.9	53.7	0.58	0.09	96.3

Percentages of distribution of radioactivity in aerobic transformation systems (average values of two replicates):

		Incubation period [d]	Water layer	Extractable from sediment	Non-extractable from sediment	CO2 traps	Sorbent traps	Total
Love Point Lake, aerobic	Live	0	93.2	-	1.9	-	-	95.1
		14	20.3	15.8	55.4	2.09	0.002	93.7
		56	5.8	7.3	79.0	4.49	0.005	96.6
		100	7.5	6.3	70.4	4.05	0.004	88.3
Love Point Lake, aerobic	Sterile	0	99.4	-	2.3	-	-	101.7
		14	53.0	8.35	29.4	0.75	0.02	91.6
		56	48.2	8.16	47.1	1.34	0.02	104.9
		100	27.8	5.59	73.2	1.60	0	108.2
Choptank River, aerobic	Live	0	98.2	-	1.0	-	-	99.2
		14	11.1	6.44	50.1	3.62	0	71.2
		56	2.6	3.61	74.6	6.09	0	86.9
		100	1.5	3.24	54.8	6.71	0.01	66.3
Choptank River, aerobic	Sterile	0	95.7	-	1.0	-	-	96.7
		14	41.2	10.8	28.8	0.00	0.79	81.5
		56	23.1	6.58	55.6	0.01	1.89	87.2
		100	11.8	3.88	50.5	0.02	2.04	68.3

Percentages of distribution of radioactivity in anaerobic transformation systems (average values of two replicates):

		Incubation period [d]	Water layer	Extractable from sediment	Non-extractable from sediment	CO2 traps	Sorbent traps	Total
Love Point Lake, anaerobic	Live	0	122	-	1.4	-	-	124
		12	59.8	29.1	31.0	0.49	0	120
		56	23.4	24.9	73.9	1.59	0.005	124
		102	6.9	12.0	110	3.99	0.008	132
Love Point Lake, anaerobic	Sterile	0	121	-	1.3	-	-	122
		12	53.8	30.9	34.7	0.11	0.006	120
		56	32.5	20.8	66.4	0.49	0.003	120
		102	22.0	12.7	101.2	0.54	0.004	136
Choptank River, anaerobic	Live	0	117	-	1.1	-	-	118
		12	31.3	26.1	49.1	3.11	0.004	110
		56	7.2	10.5	48.7	7.77	0.012	74.2
		102	3.0	9.8	73.4	5.87	0.013	92.1
Choptank River, anaerobic	Sterile	0	115	-	4.9	-	-	120
		12	62.1	27.8	24.9	0.31	0	115
		56	15.6	21.3	49.6	0.54	0.027	87.0
		102	18.4	39.8	59.0	1.02	0.024	118

Cumulative amounts of 14C gases evolved in mineralization systems:

Test system	Incubation period [d]	Cumulative amounts of gases evolved [% applied radioactivity]
		CO2	VOC	Total [14C]gases
Love Point Lake, anaerobic	12	0.13	0.006	0.13
	56	0.45	0.022	0.47
	84	0.50	0.031	0.53
	102	0.64	0.032	0.67
Choptank River, anaerobic	12	0.03	0.005	0.03
	56	0.27	0.066	0.34
	84	0.32	0.081	0.40
	102	0.58	0.090	0.67
Love Point Lake, aerobic	14	0.23	0.00	0.23
	56	1.83	0.01	1.85
	84	2.23	0.02	2.25
	102	2.76	0.03	2.78
Choptank River, aerobic	14	0.63	0.01	0.64
	56	3.41	0.03	3.44
	84	3.69	0.04	3.73
	102	5.66	0.04	5.70

Reference 2

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water

Details on source and properties of surface water:

- Details on collection (e.g. location, sampling depth, contamination history, procedure): Water samples were taken at two different locations: 1. Surface water samples from Love Point Lake, Kent Island, Maryland, USA and 2. Surface water samples from Choptank River, Denton, Maryland, USA
- Storage conditions: Samples were stored at room temperature and mixed on a shaker at approximately 80 rpm
- pH at time of collection: 1.: 7.7; 2.: 7.3
- Electrical conductivity: 1.: 20.8 mmhos/cm (= 2.1 mSiemens/cm); 2.: 2.49 mmhos/cm (= 0.25 mSiemens/cm)
- Dissolved organic carbon (mg/L): 1.: 5.4; 2.: 6.9
- Total phosphorus (mg/L): 1.: 1.0; 2.: 0.9
- Total plate count (colony forming units, CFU): 1.: Bacteria: 18 (48.4 hours), Actinomycetes: 23 (47.8 hours), Fungi: 0 (92.1 hours); 2.: Bacteria: 3210 (48.4 hours), Actinomycetes: 170 (47.8 hours), Fungi: 12 (48.5 hours)

Duration of test (contact time):

92 d

Initial conc.:

100 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: 2 L per treatment and replicate
- Test temperature: 19.0 - 21.1 °C
- pH: Love Point: 7.7; Choptank River: 7.3
- Aeration: CO2 free air at a rate of approximately 5 mL/minute
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 1-gallon amber glass jugs covered with aluminum foil. 2 L test volume for biotic and abiotic treatments
- Number of culture flasks/concentration: Two vessels for biotic and a single sterile vessel for abiotic test for each surface water type.
- Method used to create aerobic conditions: Aeration with CO2 free air
- Measuring equipment: evolving CO2 was trapped in 1.5 N KOH solution

- Details of trap for CO2 and volatile organics if used: In a row of 4 bottles a single blank bottle was directly connected to the test vessels. Subsequently 3 additional bottles were connected each on containing 100 mL of 1.5 N KOH solution.

SAMPLING
- Sampling frequency: Biotic and abiotic samples were taken after 0, 1, 4, 7, 16, 29, 60 and 92 days; evolved 14CO2 was sampled after 7, 16, 29, 60 and 92 days; dissolved 14CO2 was sampled after 19, 29, 60 and 92 days.
- Sampling method: refer to Details in analytical methods

CONTROL AND BLANK SYSTEM
- Abiotic sterile control: yes

% Degr.:

18.1

Parameter:

other: total cumulative 14CO2 production

Sampling time:

92 d

Remarks on result:

other: Love Point biotic incubations

% Degr.:

25.5

Parameter:

other: total cumulative 14CO2 production

Sampling time:

92 d

Remarks on result:

other: Choptank River biotic incubations

Compartment:

natural water: freshwater

DT50:

11.1 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Love Point surface water; r2: 0.879, DT90: 36.92 days

Compartment:

natural water: freshwater

DT50:

7.3 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Choptank River Surface Water; r2: 0.922; DT90: 24.36 days

Transformation products:

not specified

The parent compound ¹⁴C-MDA was not detected or was present at < 1% of the initial radioactivity at the termination of the study on day 92.

The peaks and retention times of the parent compound and the transformation products were:

Love Point Lake

Biotic samples (maximum values from two replicates)
Ethyl acetate extract
Peak number	More or less polar compared to parent	Retention time (min); approximately	Maximum of initial radioactivity on day (in brackets)
1	More	3.3	1.18 (60)
2	More	4.5	1.95 (60)
3	More	5.1	1.41 (92)
4	More	6.7	1.73 (92)
5	More	7.6	12.97 (60)
6	Parent compound	7.9	93.2 (0)
7	Less	8.2	8.94 (29)
8	Less	9.4	5.93 (60)
9	Less	10.4	1.64 (16)
Aqueous layer
Peak number	More or less polar compared to parent	Retention time (min); approximately	Maximum of initial radioactivity on day (in brackets)
1	More	2.7	5.26 (60)
2	More	3.7	24.3 (60)
3	More	4.6	20.2 (16)
4	Parent compound	8.0	1.47 (4)
Abiotic
Ethyl acetate extract
Peak number	More or less polar compared to parent	Retention time (min); approximately	Maximum of initial radioactivity on day (in brackets)
1	More	3.3	0
2	More	4.5	0
3	More	5.1	0
4	More	6.7	0
5	More	7.6	0
6	Parent compound	7.9	95.1 (0)
7	Less	8.2	0
8	Less	9.4	0
9	Less	10.4	0
Aqueous layer
Peak number	More or less polar compared to parent	Retention time (min); approximately	Maximum of initial radioactivity on day (in brackets)
1	More	2.7	0
2	More	3.7	4.75 (92)
3	More	4.6	15.4 (92)
4	Parent compound	8.0	0

 

Choptank River

Biotic samples (maximum values from two replicates)
Ethyl acetate extract
Peak number	More or less polar compared to parent	Retention time (min); approximately	Maximum of initial radioactivity on day (in brackets)
1	More	3.1	9.22 (60)
2	More	4.3	2.31 (29)
3	More	5.7	4.85 (92)
4	More	6.9	2.95 (29)
5	More	7.4	22.7 (60)
6	Parent compound	7.9	95.7 (0)
7	Less	8.3	3.91 (16)
8	Less	10.2	6.73 (92)
Aqueous layer
Peak number	More or less polar compared to parent	Retention time (min); approximately	Maximum of initial radioactivity on day (in brackets)
1	More	2.4	9.66 (16)
2	More	3.7	25.2 (60)
3	More	4.5	15.7 (16)
Abiotic
Ethyl acetate extract
Peak number	More or less polar compared to parent	Retention time (min); approximately	Maximum of initial radioactivity on day (in brackets)
1	More	3.1	0
2	More	4.3	0
3	More	5.7	0
4	More	6.9	0
5	More	7.4	0
6	Parent compound	7.9	94.6 (0)
7	Less	8.3	0
8	Less	10.2	0
Aqueous layer
Peak number	More or less polar compared to parent	Retention time (min); approximately	Maximum of initial radioactivity on day (in brackets)
1	More	2.4	0
2	More	3.7	15.3 (92)
3	More	4.5	28.5 (92)

Total cumulative ¹⁴CO₂ production in biotic samples at the end of the exposure period

Sample site	Cumulative % of initial radioactivity at the end of the exposure period
Love Point Lake	18.1
Choptank River	25.5

Description of key information

 The test substance rapidly disappears from surface waters with half-lives of maximum 11.1 days whereas the maximum half-life in sediments was determined to be 21 days (under anaerobic conditions). In the presence of sediments the 14C-labeled test substance migrated to the non-extractable fraction of the sediment. The parent compound disappeared in the process of an OECD 309 study completely while several metabolites were detected. The mineralization of the test substance was determined to be low under the conditions of both studies with maximum values of 5.7% in an OECD 308 study and 25.5% in an OECD 309 study (both values based on the production of 14CO2).

Key value for chemical safety assessment

Half-life in freshwater:

11.1 d

at the temperature of:

20 °C

Half-life in freshwater sediment:

21 d

at the temperature of:

20 °C

Additional information

The biodegradability and the transformation potential of the 14C-labeled test substance was investigated in a GLP guideline study according to OECD 309 (Aerobic Mineralization in Surface Water-Simulation Biodegradation Test). The test was conducted using surface waters collected from two locations: Love Point Lake (Kent Island, Maryland, USA) and Choptank River (Denton, Maryland, USA). After a test duration of 92 days a half-life of 7.3 days was recorded for samples from the river system whereas the DT50 for samples from the lake system was 11.1 days. The degree of production of 14CO2 was 25.5% in the river system samples and 18.1% in the lake system samples after 92 days of exposure. Radio chromatography analysis of ethyl acetate extracts resulted in a similar disappearance pattern of the parent test substance from both tested surface waters. The disappearance of the parent test material was associated with the appearance of several metabolites throughout the study.

Furthermore, the distribution and transformation of the 14C-labeled test substance was studied in a GLP OECD 308 test using aerobic and anaerobic sediments obtained from the same locations as in the OECD 309 study. In both systems a radioactivity shift from the water layer to the non-extractable fraction of the sediments was demonstrated. After a test duration of 100 days 69.3 and 90.2% of the applied radioactivity was detected in the non-extractable fraction of the aerobic samples taken from both systems. In the water layer only 6.1 and 11.7% were detected. The half-lives of the test substance under aerobic conditions ranged from 3.1 to 5.8 days and from 5 to 28.9 days under anaerobic conditions, respectively. The total amount of applied radioactivity transformed into 14CO2 in aerobic test systems after 100 days of incubation was 2.8% in the aerobic Love Point Lake test systems and 5.7% in the aerobic Choptank River system. Sterile aerobic samples of the lake and river systems revealed a similar distribution. In the anaerobic samples the production of CO2 and volatile organic gases was < 1% for both lake and river systems.

Both tests demonstrate that the test substance rapidly disappears from surface waters with half-lives of maximum 11.1 days whereas the maximum half-life in sediments was determined to be 21 days (under anaerobic conditions). In the presence of sediments the radioactivity migrates to the non-extractable fraction of the sediment. The parent compound disappeared in the process of the OECD 309 study completely while several metabolites were detected.The mineralization of the test substance was determined to be low under the conditions of both studies.