Reaction mass of 3-(3,3-dimethyl-2,3-dihydro-1H-inden-5-yl)propanal and 3-(1,1-dimethyl-2,3-dihydro-1H-inden-5-yl)propanal and 3-(1,1-dimethyl-2,3-dihydro-1H-inden-4-yl)propanal

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study was performed according to OECD Guideline 301 D but was not performed according to GLP. However, this study is well documented and is considered reliable with restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Specific details on test material used for the study:

No additional information

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Activated sludge was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant treats predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted to 2 mg DW/L and distributed over the appropriate biological oxygen demand (BOD) bottles (van Ginkel and Stroo, 1992).
River water was sampled from the Rhine near Heveadorp, The Netherlands. The river water was aerated for 7 days to reduce the endogenous respiration. Particles in the river water were removed by sedimentation.

Duration of test (contact time):

112 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: The nutrient medium of the Closed Bottle test contained per liter of deionized water: 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4•2H2O, 22.5 mg MgSO4•7H2O, 27.5 mg CaCl2, and 0.25 mg FeCl3•6H2O. Ammonium chloride was omitted from the medium to prevent nitrification.
- Solubilising agent (type and concentration if used): None
- Test temperature: 22-24 °C
- Suspended solids concentration: 2 mg DW/L
- Continuous darkness: Yes
- Deionized water containing no more than 0.01 mg/L copper was prepared in a water purification system.
- Other: None

TEST SYSTEM
- Culturing apparatus: 0.3 L Biological oxygen demand (BOD) bottles with glass stoppers
- Number of culture flasks/concentration: 3 bottles containing only inoculum (control) and 3 bottles for test substance with the inoculum
- Method used to create aerobic conditions: No data
- Method used to create anaerobic conditions: Not applicable
- Measuring equipment: The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode (WTW Trioxmatic EO 200) and meter (WTW OXI 530) (Retsch, Ochten, The Netherlands). The pH was measured using a EUTECH instruments pH meter. The temperature was measured and recorded with a thermo couple connected to a data logger.
- Test performed in closed vessels: Yes
- Test performed in open system: No

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes

OTHERS:
- The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a pre-weighed 8 µm cellulose nitrate filter. This filter was dried for 1.5 hours at 104 °C and weighed after cooling. The dry weight was calculated by subtracting the weighed filters and by dividing this difference by the filtered volume.
The biological oxygen demand (BOD) mg/mg of the test compound was calculated by dividing the oxygen consumption by the concentration of the test substance in the closed bottle. The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD). The dissolved oxygen (DO) in the BOD bottles was measured over time for a maximum period of 84 to 112 days. A test was terminated when biodegradation exceeded 60 %.

Reference substance:

not specified

Preliminary study:

None

Test performance:

No data

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Remarks on result:

other: River

Parameter:

% degradation (O2 consumption)

Value:

18

Sampling time:

112 d

Remarks on result:

other: River

Parameter:

% degradation (O2 consumption)

Value:

5

Sampling time:

84 d

Remarks on result:

other: Sludge

Details on results:

The test substance was toxic to activated sludge up to 28 days.

Test conditions: The validity of the tests is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item showed toxic to sludge up to 28 days and undergoes 5% degradation after 84 days. In river water, the test item undergoes 0 and 18 % degradation after 28 and 112 days, respectively in the test conditions. Thus, the test item should be regarded as not readily biodegradable according to this test.

Executive summary:

This study was performed according to the OECD Guideline 301 D, to determine the biodegradability of the test substance by the Closed Bottle Test.

The test material was exposed to activated sewage sludge micro-organisms at a concentration of 2 mg/L with culture medium in closed bottles in the dark at 22-24 °C for 7, 14, 21, 28, 42, 56, 84 and 112 days. The degradation of the test material was assessed by the measurement of oxygen consumption.

 

Test item is partially degradable. The validity of the test is demonstrated by oxygen concentrations > 0.5 mg/L in all bottles during the test period.

 

Test substance	Inoculum	Biodegradation at day
		7	14	21	28	42	56	84	112
	Sludge	Toxic	Toxic	Toxic	Toxic	2	5	5
	River	Toxic	1	1	0	1	10	16	18

The test item showed toxic to sludge up to 28 days and undergoes 5% degradation after 84 days. In river water, the test item undergoes 0 and 18 % degradation after 28 and 112 days, respectively in the test conditions. Thus, the test item should be regarded as not readily biodegradable according to this test.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-07-09 to 2016-10-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Japenese test guideline followed, similar to OECD 301C : Biodegradability test of Chemical substances by Microorganisms, Japanese notification, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kanpokihatsu No. 110331009, March 31, 2011, the latest revision, April 2, 2012.

Qualifier:

according to guideline

Guideline:

other: Biodegradability test of Chemical substances by Microorganisms, Japanese notification, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kanpokihatsu No. 110331009, March 31, 2011, the latest revision, April 2, 2012.

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not applicable

Principles of method if other than guideline:

Not appicable

GLP compliance:

yes

Remarks:

GLP statement (October 18, 2016)

Specific details on test material used for the study:

No additional information

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

Inoculum from activated sludge provided by Chemicals Evaluation and Research Institute, Japan.

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:

TEST CONDITIONS
- Composition of medium: Basal medium was prepared by adding 0.3% of each liquid A, B, C and D (as specified in JIS K 0102-2008, 21) into purified water (which was adapted to quality of Japanese pharmacopeia) and adjusted the pH to 7,0 +/- 0,04.
- Test temperature: 25 ± 1 °C
- pH: 7.0 +/- 0,04
- pH adjusted: Yes
- Suspended solids concentration: 2500 mg/L (BOD measurement), 2900 mg/L (recovery test)

TEST SYSTEM
- Culturing apparatus: glass bottles
- Test substance or reference substance or activated sludge were added to the media in test vessels. At the end of BOD measurement, the pH values were 7.3 for the [water + test substance] and all 6.5 for [sludge + test substance]-1,2 and 3 respectively.
- Amount of the test solution: 30 mg
- Number of culture flasks/concentration: 3 bottles (test suspensions); 1 bottle (inoculum blank); 1 bottle (activity control); 1 bottle (abiotic control)
- Test suspensions: Test substance (Bottle No.2 : 30 mg, No.3 : 30 mg; No.4 : 30 mg) + 300 mL (296,4 mL of basal medium + 3.6 mL of activated sludge).
- Measuring equipment: The BOD was measured continuously for 28 days using a closed system oxygen consumption measuring apparatus (OM-3100A, Ohkura Electric Co.).
- Test performed in closed vessels: Yes
- Test performed in open system: No
- Other: In the apparatus, the temperature, stirring recording meter and air-tightness of the closed system were checked once a day except holidays during BOD measurement period. Observation was made on color of the contents in test bottles using [inoculum blank] as a comparative reference. After the end of BOD measurement, observations on dissolution state of the test substance and growth of the activated sludge were also made.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes; Mineral medium 296,4 mL + activated sludge 3,6 mL
- Abiotic sterile control: Yes; Test substance (30 mg) + purified water 300 mL
- Toxicity control: Yes; Aniline 29,5 µL (30 mg) + basal medium 296,4 mL + 3,6 mL of activated sludge

Reference substance:

aniline

Preliminary study:

None

Test performance:

No data

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Remarks on result:

other: The BOD of the test suspensions (test substance + sludge, n=3) calculated from oxygen consumption were -1.6, -1.9 and -2.0 mg, respectively.The degradabilities based on the BOD after 28 days were calculated to be all 0% (calculated values were all -2%).

Details on results:

Degradability based on the BOD
The BOD of the test suspensions (test substance + sludge, n=3) calculated from oxygen consumption were -1.6, -1.9 and -2.0 mg, respectively.
The degradabilities based on the BOD after 28 days were calculated to be all 0% (calculated values were all -2%).
(Table 1 a), b), c))

Degradability based on the DOC
The DOC (theoretical value 25,0 mg) in the [water + test substance] after 28 days was 12,9 mg, and the DOC in the [sludge + test substance]-1,2 and 3 after 28 days (as corrected with the value in the [inoculum blank]) were 25,8, 24,1 and 23,8 mg respectively.
The degradability based on the DOC after 28 days was not calculated since the DOC in the [water+ test substance] was less than 90% of the theoretical value.
(Tables 1 and 2)

Degradability based on the residual test substance amount
The residual amounts of the test substance in the [water + test substance] after 28 days was 21,2 mg and the residual amounts of the test substance in the [sludge + test substance]-1,2 and 3 were 0,3, 0,4 and 0,6 mg respectively.
As the residual amount of the test substance in the [water + test substance] was less than 90% of the initial amount, the disappearance rate was calculated instead of degradability. the disappearance rates of the residual amount of the test substance werfe calculated to be 29 % for the [water + test substance] and 99, 99 and 98 % for the [sludge + test substance]-1,2 and 3 respectively.
The disappearance in the [water + test substance] was considered because of volatilization. As results of LC/MS analysis for the confirmation of existence of transformation products, only the test substance ion was detected, and no transformation product ion was detected.
(Tables 1 and 4)

Results with reference substance:

The degradability of reference substance based on the BOD exceeds 40% after 7 days and 65% after 14 days, respectively.

Table 1 a) BOD measurement, pH

Bottle No.	Sample description	BOD (mg)				pH
		day 7	day 14	day 21	day 28	day 0	day 28
1	Water + test substance (Abiotic control)	0.2	0.5	0.9	1.4	7.8	7.3
2	Sludge + test substance - 1 (Test suspension – 1)	7.0	10.9	13.2	14.5	-	6.5
3	Sludge + test substance - 2 (Test suspension – 2)	6.9	10.8	13.0	14.2	-	6.5
4	Sludge + test substance - 3 (Test suspension – 3)	5.5	10.3	12.8	14.1	-	6.5
5	Sludge + aniline (Activity control)	71.1	93.8	101.5	103.6	-	8.0
6	Inoculum blank (Control blank)	4.9	9.4	13.3	16.1	7.0	7.3

Table 1 b) Measured values (day 28)

		Sludge + test substance			Water + test substance	Theoretical value
		No.1	No.2	No.3
		Bottle 2	Bottle 3	Bottle 4	Bottle 1
BOD*^1	mg	-1.6	-1.9	-2	1.4	85.1
DOC*^1	mg	25.8	24.1	23.8	12.9	25.0
Test substance (GC)	mg	0.3	0.4	0.6	21.2	30.0
	% (1)	1	1	2	71	-
Transformation product (Hivernal neo acid) (HPLC)	mg	33.8	33.8	33.4	<0.1	32.4
	% (2)	105	104	103	<1	-
Transformation product (Molecular weight 190.10)	mg	1.1	1.1	1.8	<0.2	28.2
	% (3)	4	4	6	<1	-
Transformation product (Molecular weight 190.14)	mg	1.0	1.1	0.9	<0.9	28.2
	% (4)	4	4	3	<3	-
Total ( (1) + (2) + (3) + (4) )	%	114	113	114	71	-

*¹ : Value of [sludge + test substance] is corrected with BOD or DOC value of [Inoculum blank]

Table 1 c) Degradability (%)

		Sludge + test substance			Average	Water + test substance
		No.1	No.2	No.3
		Bottle 2	Bottle 3	Bottle 4		Bottle 1
BOD	%	0 (-2) *^2	0 (-2) *^2	0 (-2) *^2	0	-
DOC *^3	%	NA	NA	NA	-	-
Test substance *^4	%	99	99	99	99	99

*² : Where % degradability is calculated to be negative, this value is shown into brackets.
*³ : Degradability is not calculated because the DOC in [water + test substance] is less than 90 % of theoretical value
*⁴: Disappearance rate was calculated because the test substance in [water + test substance] was less than 90 % of the initial amount

Table 2 Results of DOC measurement

Bottle No.	Sample description	Added amount (mg)	Theoretical value (mgC)	DOC in bottle (mgC)	DOCs-DOCb (mgC)	Ratio to theoretical value (%)	Degradability (%)
1	Water + test substance (Abiotic control)	30.0	25.0	12.9	-	52	-
2	Sludge + test substance - 1 (Test suspension – 1)	30.0	25.0	26.3	25.8	103	NA*
3	Sludge + test substance - 2 (Test suspension – 2)	30.0	25.0	24.6	24.1	96	NA*
4	Sludge + test substance – 3 (Test suspension – 3)	30.0	25.0	24.3	23.8	95	NA*
5	Sludge + aniline (Activity control)	-	-	-	-	-	-
6	Inoculum blank (Control blank)	-	-	0.5	-	-	-
	Average (Bottle 2,3,4)					98

*Not available

Equations : Theoretical value : added amount (mg) x Carbon ratio
Carbon ratio in the test substance 0.832

Ratio to theoretical value : [sludge + test substance] = (DOCs-DOCb) / theoretical value x 100
[water + test substance] = (DOCc) / Theoretical value x 100

Degradability (%) = 1 – (DOCs – DOCb) / DOCc] x 100

DOCs : DOCs (mg) in [sludge + test substance]

DOCb : DOC (mg) in [inoculum blank]

DOCc : DOC (mg) in [water + test substance]

Table 4 Residual test substance amount in test bottles

Bottle No.	Sample description	A	B	C	D
		Peak area (pA.sec)	Test substance in bottle (mg)	Added amount (mg)	Residual test substance rate (%)	Disappearance rate (%)
1	Water + test substance (Abiotic control)	800.206	21.2	30.0	71	29
2	Sludge + test substance – 1 (test suspension – 1)	10.026	0.3	30.0	1	99
3	Sludge + test substance - 2 (Test suspension – 2)	16.821	0.4	30.0	1	99
4	Sludge + test substance – 3 (Test suspension – 3)	23.140	0.6	30.0	2	99
5	Sludge + aniline (Activity control)	-	-	-		98
6	Inoculum blank (Control blank)	<1	<0.1	0.0	-	-
	Detection limit	1	0.1	-	-	-
	Average (Bottle 2,3,4)				1	99

 

Standard solution	Concentration	E	600 mg/L
	Peak area	F	1184.484 pA.sec
Volume of solution for GC analysis		G	0.050 L
Volume of test solution for GC analysis		H	0.300 L
Volume of test solution in bottle		I	0.300 L
Average recovery		J	95.8 %

 

Equations : B = A / F x E x G x (I / H) / (J / 100)   

D = (A / F x E x G x (I / H) / (J / 100)) / C x 100

Disappearance rate (%) = (initial amount – residual amount) / initial amount x 100

Detection limit (mg) was derived by rounding B up to first decimal place.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The BOD of the test suspensions (test substance + sludge, n=3) calculated from oxygen consumption were -1.6, -1.9 and -2.0 mg, respectively. The degradabilities based on the BOD after 28 days were calculated to be all 0% (calculated values were all -2%). In conclusion, the test substance should be regarded as not readily biodegradable according to this test, but disappears totally (104%) to form Hivernal Neo Acid.

Executive summary:

The non ready biodegradability of the test substance was determined by a Japanese test similar to the 301C OECD guideline test : Japanese notification, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kanpokihatsu No. 110331009, March 31, 2011, the latest revision, April 2, 2012 and following GLP.

A nominal concentration (100 mg/L) of test substance was introduced into the system and activated sludge was used as inoculum.

The BOD of the test suspensions (test substance + sludge, n=3) calculated from oxygen consumption were -1.6, -1.9 and -2.0 mg, respectively. The degradabilities based on the BOD after 28 days were calculated to be all 0% (calculated values were all -2%).

The residual amount of the test substance was around 1 %, and some transformation products were generated under the conditions of the test :

-Hivernal neo ACID was around 104%,

-transformation product (molecular weight of 109.10 g/mol) was around 5%

-transformation product (molecular weight of 190.14 g/mol) was around 4%

-and 2 unknown transformation products were generated.

It was suggested that all these transformation products were equal or higher in polarity than the test substance.

Degradabilities of the reference substance (aniline) were 71,1% after 7 days and 93,8% after 14 days. The activity of the inoculum is thus verified and the test is considered as valid.

In conclusion, according to this test, the test substance should be regarded as not readily biodegradable but disappears totally (104%) fo form Hivernal Neo Acid.

Description of key information

OECD Guideline 301C, GLP, key study, validity 1:

0% biodegradation after 28 days

Not readily biodegradable but the substance disappear totally to form Hivernal Neo Acid (104 %).

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

To assess the biodegradation potential of the registered substance, two experimental studies are available.

The first study (LSI Medience Corporation, 2016), assessed as the key study, was performed on the registered substance according to a Japanese test guideline similar to the OECD Guideline 301 C and following GLP statement. In this study, a nominal concentration of test substance (100 mg/L) was introduced into the system and activated sludge was used as inoculum. The degradabilities based on the BOD after 28 days was calculated to be 0%. The residual amount of the test substance was around 1 % but some transformation products were generated under the conditions of the test, particularly Hivernal Neo Acid which was around 104%. In conclusion, according to this test, the registered substance should be regarded as not readily biodegradable but disappears totally (104%) fo form Hivernal Neo Acid.

The second study (AkzoNobel, 2012), assessed as a supporting study, was perfored on the registered substance. This screening non-GLP study was performed according to the OECD Guideline 301D using two inoculums: activated sewage sludge and river water. The registered substance showed toxic effect to sludge up to 28 days and undergoes 5% degradation after 84 days. In river water, the substance undergoes 0 and 18 % degradation after 28 and 112 days, respectively in the test conditions. Thus, the registered substance is not readily biodegradable according to this test. This result supports the key GLP study. However, the biodegradation reached at day 112 demonstrates that the substance (certainly the transformation product) is expected to degrade over time .