Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jun.22,2016 to Jul. 20, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))

Version / remarks:

Guidelines for Testing of Chemicals, 302C, Inherent Biodegradability: Modified MITI Test (II). Paris: OECD, 1981.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: HJ/T 153-2004

Version / remarks:

HJ/T 153-2004, The guidelines for the testing of chemicals [S]. Beijing: SEPA, 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: GB/T 21801-2008

Version / remarks:

GB/T 21801-2008, Chemical Inherent Biodegradation-Modified MITI Test (II) Beijing: SAC, 2008.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Guidelines for the Testing of Chemicals, Degradation and Accumulation

Version / remarks:

CRC-MEP. The Guidelines for the Testing of Chemicals, Degradation and Accumulation. 2nd edition. Beijing: China Environment Press. 2013: 74-81.

Deviations:

not applicable

GLP compliance:

yes

Specific details on test material used for the study:

Batch: RC-1045Study specific test item informationPurity/composition correction factor: No correction factor requiredChemical name (IUPAC), synonym or trade name: Amides, tallow, N,N-bis(2-hydroxypropyl)CAS Number: 1454803-04-3Test item handling: No specific handling conditions required

Oxygen conditions:

not specified

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

Preparation of the InoculumsActivated sludge, surface soil and surface water were sampled from ten sites distributed in four districts throughout Nanjing city, such as Chengdong, Chengbei, Jiangxinzhou and Jiangning. 1 L of the sludge, soil and water were collected and mixed thoroughly together.After removing floating matter, the mixture was allowed to stand and then the supernatant was filtrated through filter paper. After that the filtrate was adjusted to pH 7.0 with sodium hydroxide or phosphoric acid. Finally an appropriate volume of the filtrate was transferred to a fill-and-draw activated sludge vessel and aerated for about 23.5 h (Batch No.: IN201605231).Thirty minutes after stopping the aeration, about one third of the whole volume of supernatant was discarded. Then an equal volume of synthetic sludge (a solution at pH 7.0 containing 0.1% each of glucose, peptone and potassium orthophosphate) was added into the settled material which was then aerated again. This procedure was repeated once per day during one month.Before use the mixture was allowed to stand, and the supernatant was removed. A small quantity of sludge was taken to be centrifuged (2500 r/minx 10 min) and then weighed.Then the sludge was dried in the oven and weighed again in order to calculate the content of dry sludge was 12.6%. At last 15.88 g of centrifuged sludge was diluted 0.5 L with basal culture medium (BSM) to get an activated sludge suspension with a concentration of 4000 mg/L (dry basis).

Duration of test (contact time):

28 d

Initial conc.:

30 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Preparation of the Test MediumThe Based Salt Medium (BSM) was prepared by adding 3 mL of each of stock solutions prepared in pre-aerated deionized water to 1 litre of deionized water. 10 L BSM was prepared (Batch No.: BSM201606221).Preparation of Solutions of the Test SubstanceTest Substance was added to the test bottles directly.A stock solution of the reference substance (sodium benzoate) at 1002 mg/L: 1.002 g of reference substance was dissolved in 1 L volumetric flask with BSM.Test Conditions(1) Average concentration of test chemicals: 30 mg/L.(2) Concentration of reference chemicals: 100 mg/L (W/V)(3) Concentration of activated sludge in "test" and ''blank inoculum control": 100 mg/L (W/V)(4) Concentration of activated sludge in "reference": 30 mg/L (W/V)(5) Test temperature: (25±2) °C(6) Period: 28 days(7) pH: 7.29-7.52(8) Stir vigorously with mechanical stirrer.Test ProcedureBottle 1 designated as "abiotic control" was then filled to a final volume of 300 mL with deionised water. Concentration of test chemical was 50 mg/L.For bottles 2-4 designated as "test", 7.5 mL of activated sludge suspension were added to each of these after which they were filled to a final volume of 300 mL with BSM. The concentration of activated sludge was 100 mg/L and concentration of test chemical was 50 mg/L.For bottle 5 designated as "procedure control", 30 mL stock solution of reference substance and 2.25 mL of activated sludge suspension were added. Bottle was then filled to a final volume of 300 mL with BSM. The concentration of activated sludge was 30 mg/L and concentration of the reference chemical was 100 mg/L.For bottle 6 designated as "blank inoculum control", 7.5 mL of activated sludge suspension was added and bottle was then filled to a final volume of 300 mL with BSM. The concentration of activated sludge was 1 00 mg/L.The equipment was then assembled, checked for air-tightness, stirrers were started and measurement of oxygen uptake under conditions of darkness began.The temperature, the operation of the stirrer and recorder was checked daily. Any changes in colour of the contents of the vessels were recorded. The BOD for the six bottles were determined and recorded.After the 28 days of testing, concentration of MLA-3202 in the testing bottles was analysed.Analysis of CODThe COD was determined to substitute the ThOD which cannot be calculated as the test substance is mixture.Stock solution of test substance (1150 mg/L): 0.0575 g test substance was weighted and made up to 50 mL with acetone.30.0 mg/L and 50.0 mg/L samples were prepared by adding 52.2 μL and 87.0 μL of stock solution of the test substance (1150 mg/L) into test tubes, with solvent was dried, Then 2 mL COD reagents were added, and 3 parallel for each concentration. Blank control was prepared by above method expect for stock solution of the test substance substituted by solvent.Heat all samples at 150 °C for 120 minutes, and then determine the COD of samples after cool them to room temperature.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

Not specified

Test performance:

The test was valid because the level of biodegradation of the reference substance, sodium benzoate, was 78.6% after 7 days(> 40%), and 84.9% after 14 days(> 65%), and recovery rate of residual amount of the test compound in the "abiotic control" test was found to be more than 1 0% after 28 days.

Parameter:

% degradation (O2 consumption)

Value:

77.9

Sampling time:

28 d

Details on results:

Analytical Result of CODCOD of the test substance was 2.64 mg 02/mg based on the COD analysis.Analytical Method of the Test Substance(1) SpecificityUsing analysis conditions mentioned, the chromatographic peak of test substance were emerged at retention time tR=3.1 min, tR=3.4 min, tR=3.6 min, tR=4.5 min and 5.1 min whereas this chromatographic peak did not emerge in the "blank inoculum control". Therefore, the GC-FID method was specific for test substance.(2) Calibration curveA series of standard solutions of test substance with concentration at 5.00, 10.0, 20.0, 50.0 and 100 mg/L were measured under the GC-FID using analysis conditions mentioned. Concentrations of the test substance were quantified by GC-FID using five of the principal peaks which were considered representative of the different physic-chemical characteristics of the individual components making up the complex nature. Based on the test result, linear regression equations was obtained with the peak areas vs. standard solution concentration, A= 3978c + 515, r2= 0.9988, where A represents peak area, and cis the concentration of the test item (mg/L). The results show that linearity for the concentration range of 5.00 mg/L to 100 mg/L is good.(3) PrecisionUnder the above conditions, 50.0 mg/L solution of the test substance was analysed for 6 times. The relative standard deviation was 1.80 %.(4) Recovery testThe recovery samples with concentrations at 5.00 mg/L were prepared in triplicate by adding 86.8 μL of the standard stock solution I (1152 mg/L) to 0.50 mL of activated sludge suspension (4000 mg/L) and making it up to 20 mL with BSM. 20 mL of the recovery samples were then extracted with 20 mL dichloromethane I 5 min. Then, the dichloromethane was removed with the helping of the rotary evaporation apparatus. Finally, the samples were diluted with n-hexane to 2 mL, and then filtered using 0.22 μm Millipore filters. The concentration of samples was determined with the analysis method mentioned above.The recovery samples with concentrations at 30.0 mg/L were prepared in triplicate by adding 521 μL of the standard stock solution I (l 152 mg/L) to 0.50 mL of activated sludge suspension (4000 mg/L) and making it up to 20 mL with BSM. 20 mL of the recovery samples were then extracted with 20 mL dichloromethane for 15 min. Then, the samples were filtered using 0.22 μm millipore filters. First 5 mL was discarded and the concentration of samples was determined with the analysis method mentioned.Measurements obtained from the recovery test were shown in Table 6. The results showed that mean recovery rate for 5.00 mg/L and 30.0 mg/L was 88.8% ~ 96.6% and 97.7%~105%, the relative standard deviation was 4.22 % and 3.81%, respectively. (5) Limit of detection (LOD) and limit of quantitation (LOQ)The LOD was 2.00 mg/L (S/N≥3), and the LOQ was 5.00 mg/L (S/N≥10).Inherent Biodegradation of Test SubstanceTest solutions were prepared in an inorganic salts medium, inoculated with a number of micro-organisms collected from 10 places in Nanjing city. During the test, the temperature was kept at (25±2) °C. The BOD results showed that inherent biodegradation of the test substance (MLA-3202) was 77.9 % after 28 days based on ThODNH3·Based on the residue analysis, biodegradation of the test substance (MLA-3202) was 100 % during the testing period.

Key result

Parameter:

COD

Value:

2.64 mg O2/g test mat.

Results with reference substance:

The level of biodegradation of the reference substance, sodium benzoate, was 78.6% after 7 days(> 40%), and 84.9% after 14 days(> 65%),

Results of O₂Uptake during 28 Days Period

Time (d)	O2uptake (mg/L)
	“abiotic control”	“test”			“procedure control”	“blank inoculum control”
	Bottle 1	Bottle 2	Bottle 3	Bottle 4	Bottle 5	Bottle 6
0	0	0	0	0	0	0
1	0	8.64	8.52	6.83	0	0
2	0	13.6	13.7	12.7	85.6	2.19
3	0	32.4	33.8	26.5	112	4.38
4	0	28.3	39.5	34.1	118	5.11
5	0	43.0	44.4	41.5	124	5.65
6	0	46.5	48.1	43.4	130	6.09
7	0	49.2	51.9	46.3	134	7.24
8	0	54.1	57.0	47.7	138	8.34
9	0	57.2	60.7	51.8	141	8.79
10	0	62.0	68.5	58.6	141	9.26
11	0	66.6	72.8	63.4	142	10.1
12	0	69.9	76.2	66.6	144	10.7
13	0	71.4	78.6	67.8	145	11.4
14	0	73.3	80.8	68.8	145	12.3
15	0	74.8	82.4	70.7	147	12.4
16	0	78.9	86.8	74.0	150	15.0
17	0	82.8	91.2	77.5	153	18.0
18	0	84.3	93.2	80.7	155	20.1
19	0	84.3	93.2	81.2	155	20.1
20	0	84.3	93.2	81.2	155	20.1
21	0	84.3	93.2	81.2	155	20.1
22	0	84.3	93.5	81.4	155	20.1
23	0	84.3	94.9	81.4	155	20.1
24	0	85.1	96.9	81.4	155	20.1
25	0	85.9	97.9	81.4	155	20.1
26	0	86.3	99.0	81.4	155	20.1
27	0	87.9	100.6	81.9	155	21.0
28	0	88.2	101.7	81.9	155	21.2

 

Cumulative Biodegradation (%)

Time (d)	Cumulative Biodegradation (%)
	“test”				“procedure control”
	Bottle 2	Bottle 3	Bottle 4	Mean	Bottle 5
0	0	0	0	0	0
1	9.44	8.96	8.53	8.97	0
2	12.4	12.1	13.1	12.5	50.8
3	30.6	30.9	27.6	29.7	66.1
4	36.3	36.2	36.2	36.2	69.5
5	40.8	40.8	44.7	42.1	73.0
6	44.1	44.2	46.5	44.9	76.7
7	45.9	47.0	48.8	47.2	78.6
8	50.0	51.2	49.1	50.1	81.2
9	52.9	54.6	53.7	53.7	82.9
10	57.7	62.3	61.6	60.5	82.9
11	61.8	66.0	66.6	64.8	83.5
12	64.7	68.9	69.9	67.8	84.3
13	65.6	70.7	70.4	68.9	85.0
14	66.6	72.0	70.6	69.7	84.9
15	68.2	73.7	72.9	71.6	85.7
16	69.9	75.5	73.7	73.0	87.1
17	70.8	77.1	74.3	74.1	88.4
18	70.1	76.9	75.7	74.3	89.0
19	70.1	76.9	76.4	74.5	89.0
20	70.1	76.9	76.4	74.5	89.0
21	70.1	76.9	76.4	74.5	89.0
22	70.1	77.2	76.6	74.7	89.0
23	70.1	78.8	76.6	75.2	89.0
24	71.1	80.8	76.6	76.2	89.0
25	71.9	81.9	76.6	76.8	89.0
26	72.4	83.0	76.6	77.3	89.0
27	73.1	83.8	76.1	77.7	88.9
28	73.2	84.7	75.8	77.9	88.8

 

pH of the Test Solutions

Sample		pH
		0 d	28 d
“abiotic control”	Bottle 1	7.35	7.48
“test”	Bottle 2	7.31	7.46
	Bottle 3	7.32	7.39
	Bottle 4	7.35	7.41
“procedure control”	Bottle 5	7.32	7.52
“blank inoculum control”	Bottle 6	7.29	7.35

 

COD of the Test Substance

Concentration (mg/L)	Total COD (mg/L)	Test substance COD (mg O2/mg)
30.0	79	2.63
30.0	81	2.70
30.0	84	2.80
50.0	131	2.62
50.0	127	2.54
50.0	128	2.56
Average		2.64

 

Precision of the GC-FID method for the test substance

Nominal Concentration (mg/L)	Peak Areas (Hz.s)	RSD (%)
50.0	204475	2.44
	202986
	202034
	210083
	207479
	195671

 

Recovery of the Test Substance in Recovery Samples (GC-FID)

Nominal Concentration (mg/L)	Measured Concentration (mg/L)	Recovery Rate (%)	Mean Recovery Rate (%)	RSD (%)
5.00	4.83	96.6	92.5	4.22
	4.61	92.3
	4.44	88.8
30.0	31.5	105	102	3.81
	31.1	104
	29.3	97.7

 

Residual Results and Degradation of the Test Substance

Sample	Time (d)	Bottle	Concentration (mg/L)	Degradation (%)
“abiotic control”	28	1	30.6	----
“blank inoculum control”	28	6	ND*	----
“test”	28	2	ND*	100
		3	ND*	100
		4	ND*	100
		Average	ND*	100

ND*: Not detected, since below the detection limit of 2 mg/L of instrument.

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

Substances are considered to be "inherently biodegradable" if the inherent degradation rate is equal to or greater than 20% during the 28-day test period. MLA-3202 can therefore be considered inherently biodegradable.

Executive summary:

The inherent biodegradation test on the test substance (MLA-3202) was performed according to "The guidelines for the testing of chemicals" SEPA(HJ/T 153-2004), "The guidelines for the testing of chemicals, Degradation and Accumulation" (the 2nd edition) (Beijing: China Environment Press. 2013), and Procedure 302C of the 'Guidelines for Testing of Chemicals' of the OECD: "Inherent Biodegradability: Modified MITI Test (II)" (1981).

 

Test solutions were prepared in an inorganic salts medium, inoculated with a number of micro-organisms collected from 10 places in Nanjing city. During the test, the temperature was kept at (25±2) °C. The test was valid because the level of biodegradation of the reference substance sodium benzoate was 78.6% after 7 days (> 40%), and 84.9% after 14 days (> 65%), and the recovery rate of residual amount of the test compound in the "abiotic control" is found to be more than 10% after 28 days.

 

The BOD results showed that inherent biodegradation of the test substance (MLA-3202) was 77.9% after 28 days based on the calculation of ThOD_NH3.

 

Based on the residue analysis, inherent biodegradation of the test substance (MLA-3202) was 1 00 % during the testing period.

 

Substances are considered to be "inherently biodegradable" if the inherent degradation rate is equal to or greater than 20% during the 28-day test period. MLA-3202 can therefore be considered inherently biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 July 2016 to 04 August 2016

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:

Organization for Economic Co-operation and Development (OECD), OECD guidelines for Testing of Chemicals, Section 3, Degradation and Accumulation, guideline No. 301 B: "Ready Biodegradability: CO2 Evolution Test" adopted July 17, 1992.

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Version / remarks:

Council Regulation (EC) No. 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C.4. "Biodegradation: determination of the 'ready' biodegradability, C.4-C: Carbon dioxide (CO2) evolution test (Modified Sturm Test).

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 9439

Version / remarks:

ISO International Standard 9439 “Water Quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - carbon dioxide evolution test (1999).

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 10634

Version / remarks:

ISO International Standard 10634 "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium" (1995).

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

Test item 207258/AIdentification MLA-3202Appearance Clear amber-red liquidBatch RC-1045Purity/Composition UVCBTest item storage At room temperatureStable under storage conditions until 17 February 2019 (expiry date)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Source: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', Heeswijk-Dinther, The Netherlands, receiving predominantly domestic sewage.Treatment: The freshly obtained sludge was preconditioned to experimental conditions by continuous aeration until further treatment. The concentration of suspended solids was determined to be 3.4 g/L in the concentrated sludge. Before use, the sludge was allowed to settle (44 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.Reason for selection: The test has been accepted internationally for determining the 'ready' biodegradability of test items under aerobic conditions.

Duration of test (contact time):

28 d

Initial conc.:

17 mg/L

Based on:

test mat.

Initial conc.:

12 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Reference item concentration and preparation of test solutionsA solution of sodium acetate was prepared by dissolving 1003.9 mg in Milli-RO water and making this up to a total volume of 250 mL. Volumes of 20 mL from this stock solution were added to 2 litres of the test medium of the positive control bottle and the toxicity control bottle, resulting in a final concentration of 40 mg sodium acetate per litre (12 mg TOC/L).Test concentration and preparation of test solutionsMLA-3202 was a clear amber-red liquid UVCB. The test item was tested in duplicate at a concentration of 17 mg/L, corresponding to 12 mg TOC/L. For calculation of the organic carbon content the ratio of the components was taken into account.In a pre-test MLA-3202 was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. Therefore, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components (test item bottle A: 33.1 mg; test item bottle B: 33.1 mg and toxicity control bottle: 33.3 mg). To this end, 10 mL of Milli-RO water was added to each weighing bottle containing the test item.After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms.Test procedure and conditionsTest duration: 28 days (last CO2 measurement on day 29).During the test period, the test media were aerated and stirred continuously.Test vessels: 2 litre glass brown coloured bottles.Milli-RO water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.Stock solutions of mineral components: A) 8.50 g KH2PO4; 21.75 g K2HPO4; 67.20 g Na2HPO4.12H2O; 0.50 g NH4Cl dissolved in Milli-RO water and made up to 1 litre, pH 7.4 ± 0.2B) 22.50 g MgSO4.7H2O dissolved in Milli-RO water and made up to 1 litre.C) 36.40 g CaCl2.2H2O dissolved in Milli-RO water and made up to 1 litre.D) 0.25 g FeCl3.6H2O dissolved in Milli-RO water and made up to 1 litre.Mineral medium: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli-RO water.Barium hydroxide: 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.Synthetic air (CO2 < 1 ppm): A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).Illumination: The test media were excluded from light.Preparation of bottlesPre-incubation medium: The day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle.This mixture was aerated with synthetic air overnight to purge the system of CO2.Type and number of bottles: Test suspension: containing test item and inoculum (2 bottles).Inoculum blank: containing only inoculum (2 bottles)Positive control: containing reference item and inoculum (1 bottle).Toxicity control: containing test item, reference item and inoculum (1 bottle).Preparation: At the start of the test (day 0), test and reference item were added to the bottles containing the microbial organisms and mineral components.The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before.Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.Determination of CO2Experimental CO2 production: The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).Measurements: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made over a period of at least 14 days.Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator.On day 28, the pH of all test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.Theoretical CO2 production: The theoretical CO2 production was calculated from the molecular formula.Measurements and recordingpH: At the start of the test (day 0) and on day 28, before addition of concentrated HCl.Temperature of medium: Continuously in a vessel with Milli-RO water in the same room.

Reference substance:

acetic acid, sodium salt

Preliminary study:

The ThCO2 of MLA-3202 was calculated to be 2.65 mg CO2/mg

Test performance:

Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

>= 46 - <= 50

Sampling time:

28 d

Details on results:

The relative biodegradation values calculated from the measurements performed during the test period revealed 50% and 46% biodegradation of MLA-3202 (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.In the toxicity control, more than 25% biodegradation occurred within 14 days (50%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.The temperature recorded in a vessel with water in the same room varied between 21.4 and 22.9 °C.

ThCO₂calculation of MLA-3202

Percent Composition		Formula				MW	%C:	ThCO2:
33.1 22.9 13.6 11 6 3.2 2.0 1.5 1.1 5.6	99.4	C24 C22 C24 C24 C22 C26 C24 C18 C26	H47 H45 H45 H49 H43 H45 H43 H34 H47	N1 N1 N1 N1 N1 N1 N1   N1	O3 O3 O3 O3 O3 O3 O3 O3 O3	397.6 371.6 395.6 399.6 369.6 419.6 393.6 282.4 421.6	0.72 0.71 0.73 0.72 0.71 0.74 0.73 0.77 0.74	2.66 2.61 2.67 2.65 2.62 2.73 2.68 2.81 2.71
	5.6
100.0							0.72	2.65

 

pH values of different test media

Test medium:	At the start of the test:	On day 28:
Blank control (A)	7.5	7.6
Blank control (B)	7.5	7.6
Positive control	7.5	8.0
MLA-3202 (A)	7.7→7.6^1	7.4
MLA-3203 (B)	7.6	7.6
Toxicity control	7.6	7.9

¹: Adjusted using 1 HCl

 

Notes: Except for the percentages biodegradation, all calculations are performed without rounding off.

Produced CO₂: negative values are expressed as 0.00 mL HCl

 

HCl titrated in duplicate blank bottles

Day	HCl (0.05 M) titrated (mL)
	Blank A	Blank B	Mean Value
2 5 7 9 14 19 23 27 29 29 29	47.40 45.70 45.28 45.84 44.41 43.57 42.70 43.60 43.16 46.45 48.38	45.97 45.43 44.30 45.50 44.20 43.66 42.46 43.31 42.41 45.82 48.21	46.69 45.57 44.79 45.67 44.31 43.62 42.58 43.46 42.79 46.14 48.30

 

HCl titrated in Ba(OH)₂solution (background bottles)

Day	HCl (0.05 M) titrated (mL)
	Blank A	Blank B	Mean Value
2 5 7 9 14 19 23 27 29 29 29	49.48 49.97 48.92 48.83 49.21 49.24 49.01 50.00 49.54 49.43 49.75	49.54 49.32 48.55 48.71 48.99 49.33 49.51 50.00 48.89 49.80 49.46	49.51 49.65 48.74 48.77 49.10 49.29 49.26 50.00 49.22 49.62 49.61

 

CO₂production in the blank

Day	HCl (0.05 M) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)
	Ba(OH)2^1)	Blank (mean)
2 5 7 9 14 19 23 27 29 29 29	49.51 49.65 48.74 48.77 49.10 49.29 29.26 50.00 49.22 49.62 49.61	46.69 45.57 44.79 45.67 44.31 43.62 42.58 43.46 42.79 46.14 48.30	2.83 4.08 3.95 3.10 4.80 5.67 6.68 6.55 6.43 3.48 1.31	3.1 4.5 4.3 3.4 5.3 6.2 7.3 7.2 7.1 3.8 1.4	3.1 7.6 11.9 15.3 20.6 26.9 34.2 41.4 48.5 52.3 53.7

¹⁾: “Strength” of untreated 0.0125 M Ma(OH)₂solution

 

CO₂production and percentage biodegradation of the positive control item

Day	HCl (0.05 M) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
	Blank (mean)	Positive control
2 5 7 9 14	46.69 45.57 44.79 45.67 44.31	45.56 23.80 30.22 36.61 33.63	1.13 21.77 14.57 9.06 10.68	1.2 23.9 16.0 10.0 11.7	1.2 25.2 41.2 51.2 62.9	1 29 48 60 73

¹⁾: Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of sodium acetate: 85.9 mg CO₂/2L

 

CO₂production and percentage biodegradation of the test item (bottle A)

Day	HCl (0.05 M) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
	Blank (mean)	Bottle A
2 5 7 9 14 19 23 27 29 29 29	46.69 45.57 44.79 45.67 44.31 43.62 42.58 43.46 42.79 46.14 48.30	45.66 40.43 36.62 39.97 37.71 40.17 37.21 40.30 41.44 46.00 48.35	1.03 5.14 8.17 5.70 6.60 3.44 5.37 3.16 1.35 0.14 0.00	1.1 5.6 9.0 6.3 7.3 3.8 5.9 3.5 1.5 0.1 0.0	1.1 6.8 15.8 22.0 29.3 33.1 39.0 42.5 43.9 44.1 44.1	1 8 18 25 33 38 44 48 50 50 50

¹⁾: Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of the test item: 87.7 mg CO₂/2L

 

CO₂production and percentage biodegradation of the test item (bottle B)

Day	HCl (0.05 M) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
	Blank (mean)	Bottle B
2 5 7 9 14 19 23 27 29 29 29	46.69 45.57 44.79 45.67 44.31 43.62 42.58 43.46 42.79 46.14 48.30	45.34 38.84 39.38 40.70 34.11 39.98 40.89 41.85 43.20 46.90 48.56	1.35 7.72 5.41 4.97 10.20 3.64 1.69 1.61 0.00 0.00 0.00	1.5 8.5 6.0 5.5 11.2 4.0 1.9 1.8 0.0 0.0 0.0	1.5 10.0 15.9 21.4 32.6 36.6 38.5 40.2 40.2 40.2 40.2	2 11 18 24 37 42 44 46 46 46 46

¹⁾: Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of the test item: 87.7 mg CO₂/2L

 

CO₂production and percentage biodegradation of the toxicity control

Day	HCl (0.05 M) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
	Blank (mean)	Toxicity control
2 5 7 9 14	46.69 45.57 44.79 45.67 4.31	39.94 20.65 30.71 34.16 22.98	6.75 24.92 14.08 11.51 21.33	7.4 27.4 15.5 12.7 23.5	7.4 34.8 50.3 63.0 86.4	4 20 29 36 50

¹⁾: Calculated as the ratio between CO₂produced (cumulative) and the sum of the ThCO₂of the test item and positive control: 174.2 mg CO₂/2L (ThCO₂test item: 88.2 mg CO₂/2L + ThCO₂sodium acetate: 85.9 mg CO₂/2L)

 

Comparison of biodegradation of the test item in bottles A and B

Day	Biodegradation (%)
	Bottle A	Bottle B	Mean A and B	∆A-B^1)
2 5 7 9 14 19 23 27 29 29 29	1 8 18 25 33 38 44 48 50 50 50	2 11 18 2 37 42 44 46 46 46 46	2 10 18 25 35 40 44 47 48 48 48	1 3 0 1 4 4 0 2 4 4 4

¹⁾: Absolute difference in biodegradation between bottles A and B

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

MLA-3202 was biodegraded significantly (50% and 46%) during the test period. However, since at least 60% biodegradation was not reached within 10 days immediately following the attainment of 10% biodegradation (10-day window), the criterion for ready biodegradability was not met. Thus, under the conditions of this test MLA-3202 was not readily biodegradable.

Executive summary:

Determination of ‘ready’ biodegradability: carbon dioxide (CO₂) evolution test (modified Sturm test) of MLA-3202.

 

The study procedures described in this report were in compliance with the OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the Council Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-C and ISO 9439, 1999 and ISO 10634, 1995.

 

MLA-3202 was a clear amber-red liquid UVCB. The test item was tested in duplicate at a concentration of 17 mg/L, corresponding to 12 mg TOC/L. For calculation of the organic carbon content the ratio of the components was taken into account. The Theoretical CO₂production (ThCO₂) of MLA-3202 was calculated to be 2.65 mg CO₂/mg.

 

The study consisted of six bottles:

-2 inoculum blanks (no test item),

-2 test bottles (MLA-3202),

-1 positive control (sodium acetate) and

-1 toxicity control (MLA-3202 plus sodium acetate).

 

Since MLA-3202 was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, 10 mL of Milli-RO water was added to each weighing bottle containing the test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test medium.

 

The relative biodegradation values calculated from the measurements performed during the test period revealed 50% and 46% biodegradation of MLA-3202 (based on ThCO₂), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

 

In the toxicity control, more than 25% biodegradation occurred within 14 days (50%, based on ThCO₂). Therefore, the test item was assumed not to inhibit microbial activity.

 

Since all criteria for acceptability of the test were met, this study was considered to be valid.

 

MLA-3202 was designated as not readily biodegradable.

 

However, it is expected that MLA-3202 will be inherently biodegradable, as an inherent biodegradability test has more optimal conditions than a ready test.

Description of key information

Key value determined in a GLP accredited laboratory using Modified MITI Test (II) in accordance with OCED Guideline 302C, "The guidelines for the testing of chemicals" SEPA(HJ/T 153-2004) and "The guidelines for the testing of chemicals, Degradation and Accumulation" (the 2nd edition) (Beijing: China Environment Press. 2013).

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Additional information

Ready Biodegradability

Determination of ‘ready’ biodegradability: carbon dioxide (CO₂) evolution test (modified Sturm test) of MLA-3202.

The test item was tested in duplicate at a concentration of 17 mg/L, corresponding to 12 mg TOC/L. For calculation of the organic carbon content the ratio of the components was taken into account. The Theoretical CO₂production (ThCO₂) of MLA-3202 was calculated to be 2.65 mg CO₂/mg.

 

Since MLA-3202 was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, 10 mL of Milli-RO water was added to each weighing bottle containing the test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test medium.

 

The relative biodegradation values calculated from the measurements performed during the test period revealed 50% and 46% biodegradation of MLA-3202 (based on ThCO₂), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

In the toxicity control, more than 25% biodegradation occurred within 14 days (50%, based on ThCO₂). Therefore, the test item was assumed not to inhibit microbial activity.

 

MLA-3202 was designated as not readily biodegradable.

However, it is expected that MLA-3202 will be inherently biodegradable, as an inherent biodegradability test has more optimal conditions than a ready test.

Inherent Biodegradability

The inherent biodegradation test on the test substance (MLA-3202) was performed using the "Inherent Biodegradability: Modified MITI Test (II)".

 

Test solutions were prepared in an inorganic salts medium, inoculated with a number of micro-organisms collected from 10 places in Nanjing city. During the test, the temperature was kept at (25±2) °C.

 

The BOD results showed that inherent biodegradation of the test substance (MLA-3202) was 77.9% after 28 days based on the calculation of ThOD_NH3.

Based on the residue analysis, inherent biodegradation of the test substance (MLA-3202) was 100 % during the testing period.

Substances are considered to be "inherently biodegradable" if the inherent degradation rate is equal to or greater than 20% during the 28-day test period. MLA-3202 can therefore be considered inherently biodegradable.