Reaction mass of rel-{(1R,2S)-1-methyl-2-[(2R)-5-methylhex-4-en-2-yl]cyclopropyl}methanol and rel-{(1S,2R)-1-methyl-2-[(2R)-5-methylhex-4-en-2-yl]cyclopropyl}methanol

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:

November, 2014 - April, 2015.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SEPA (GB/T 21801-2008)

Version / remarks:

2008

Deviations:

no

Principles of method if other than guideline:

Analysis of COD :
The COD was determined to substitute the ThOD which cannot be calculated as the purity of test substance is 87.8%, and the structure of the impurity was unknown.
Stock solution I of the test substance (104 mg/L) was prepared by weighing 0.0052 g of test substance and dissolving it into 50 mL acetone.
Prepare 30 mg/L samples by diluting 0.90 60 mL stock solution (104 mg/L) of test substance to total volume of 2 mL with deionized water after dried. Then 2 mL of each concentration was added into COD reagents, each sample with 5 parallel.

Analytical Result of COD :
COD of the test substance was 2.55 mg O2/mg based on the COD analysis.


Heat all samples at 150ºC for 120 minutes, and then determine the COD of samples after cooling them to room temperature.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

GR-50-1408 is the Givaudan identification code which was employed for ROSYFOLIA during the early, developmental and testing period.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Inoculum preparation :
During the test the temperature was kept at 21.5°C~22.1°C, pH was kept from 7.31 to 7.74. The total oxygen uptake in the inoculum blank was 29 mg O2/L at the end of the test, not exceeding 60 mg O2/L.

Activated sludge was obtained from a sewage plant treating predominantly domestic sewage. On return to the laboratory the sludge was washed by Basal Salts Medium and centrifuged at 4°C, 2500 rpm for 10 minutes. The washing procedure was repeated three times. Thereafter, a small amount of the washed sludge was weighed and then dried at 100°C until the weight of the dried sludge did not change significantly anymore. Then the dry weight percentage of suspended solids of the activated sludge was calculated to be 10%. Finally, 40g wet sludge centrifuged was mixed with 1L of the Basal Salts Medium (BSM) to obtain an activated sludge with a mixed liquor suspended solids level of 4g/L.

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:

Four groups: inoculum control, test suspension, procedure control and inhibition control were set up simultaneously. Inoculum control only contained inoculated mineral salts medium. Test suspension contain mineral salts medium and a measured amount of test substance in order to determine whether there was any change in the test chemical during the testing period. Procedure control contained inoculated mineral salts medium and a measured amount of a reference substance for validating the test results. The inhibition control contained inoculated mineral salts medium and a measured amount of a reference substance and test substance to check the inhibitory effect of the test substance to inoculum.
The amount of oxygen taken up by the microbio population during biodegradation of the test substance (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of COD. For a test substance to be considered readily biodegradable, biodegradation based on oxygen consumption must reach 60% in 28 days and this pass level must be reached within 10 days after 10% biodegradation occurs (I.e 10-day window).

Test conditions
Duration: 28 days
Vessels: Glass bottles of 500ml
Water: Deionised water
Measuring apparatus: Automatic respirometer (OxiTop® Control 6, WTW, Germany)
Test medium: Basal salts medium
Controls: Inoculated same without test substance
pH: 7.31~7.74
Temperature: (22±1)°C

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

8.44

Sampling time:

28 d

Remarks on result:

other: Average of two incubates (13.1% BOD ad 3.80% BOD) on Day 28.

Details on results:

During the test, the temperature was kept at 21.5°C to 22.1°C. The pH value of the content in the test container was maintained between 7.31 and 7.74. The total oxygen uptake in the inoculum blank was 29 mg O2/L at the end of the test, not exceeding 60 mg O2/L. Biodegradation of the reference substance, sodium benzoate, reached 65.9% in 14 days, exceeding the validation criteria of > 60%. The difference of replicate values for inoculum blank and test, during the test, was both less than 20%. Biodegradation of inhibition control was 50.6% at 14 days exceeding the validation create criteria of > 25%, indicating that there was no inhibition effect to the inoculum. Thus, the test was considered to be valid.

Results with reference substance:

Biodegradation of the reference substance, sodium benzoate, reached 65.9% at 14 days (> 60%).

 

 

The below table shows the data of biodegradation during the 28-day study. The results show that under the experimental conditions, biodegradation of the registration substance was 8.44% at the end of the test.

Time (d)	Biodegradation
	Test suspension			Procedure control	Inhibition control
	#1	#2	Mean	#5	#6
0	0	0	0	0	0
1	-1.31	2.53	0.61	47.3	30.6
2	-1.96	1.90	-0.03	51.2	33.6
3	0	2.53	1.27	53.3	35.9
4	0	3.80	1.90	56.3	37.1
5	0	3.80	1.90	58.1	39.1
6	1.31	5.06	3.18	59.9	41.6
7	0.65	5.69	3.17	61.4	44.2
8	1.96	5.69	3.83	62.6	46.7
9	1.96	5.69	3.83	63.2	47.9
10	1.31	5.06	3.18	63.5	48.5
11	1.96	4.43	3.20	64.4	49.1
12	3.92	5.06	4.49	64.7	49.7
13	3.92	5.06	4.49	65.3	50.1
14	3.92	5.06	4.49	65.9	50.6
15	5.88	4.43	5.16	66.2	51.2
16	5.88	4.43	5.16	66.8	51.6
17	5.88	4.43	5.16	66.8	51.6
18	6.54	5.06	5.80	67.7	52.2
19	7.19	4.43	5.81	67.4	52.4
20	7.84	5.06	6.45	68.3	53.0
21	8.50	4.43	6.46	68.6	53.2
22	9.80	4.43	7.12	68.6	54.0
23	9.80	4.43	7.12	69.2	54.0
24	10.5	3.80	7.13	68.9	54.6
25	10.5	3.80	7.13	69.5	54.6
26	11.8	3.80	7.78	69.5	55.0
27	12.4	4.43	8.42	69.8	55.6
28	13.1	3.80	8.44	70.1	55.4

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the valid conditions, biodegradatioon of the test substance (GR-50-1408) was 8.44% at the end of the test.

Executive summary:

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 2^nd edition), Beijing: China Environmental Press. 2013, “Chemicals-Ready biodegradability-Manometric respirometry test” (GB/T 21801-2008), and with reference to Procedure 301F of the “Guidelines for Testing of Chemicals” of the OECD: “Manometric Respirometry Test” (1992), the ready biodegradability of GR-50-1408 was determined in a 28-day oxygen depletion test (i.e., OECD 301F) using activated sludge from a domestic waste water treatment plant as the source of the microbial inoculum.

During the test, the temperature was kept at 21.5°C ~ 22.1°C, pH was kept from 7.31 to 7.74. The total oxygen uptake in the inoculum blank was 29 mg O₂/L at the end of the test, not exceeding 60 mg O₂/L. Biodegradation of the reference substance, sodium benzoate, reached 65.9% at 14 days (> 60%). The difference of replicate values for inoculum blank and test, during the test, was both less than 20%. Biodegradation of inhibition control was 50.6% at 14 days (> 25%), indicating that there was no inhibition effect to inoculum. Thus, the test was considered valid.

Under the valid conditions, biodegradation of the test substance (GR-50-1408) was 8.44% at the end of the test.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

December, 2019 - March, 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

GR-50-1408 is the Givaudan identification code which was employed for ROSYFOLIA during the early, developmental and testing period.

Oxygen conditions:

aerobic

Inoculum or test system:

mixture of sewage, soil and natural water

Details on inoculum:

On-site sludge sampling was carried out at 10 locations in Japan (samples were from surface water and surface soil of rivers, lake, and inland sea, and return sludge from sewage plants).

Activated sludge, which was prepared and was controlled in this test facility according to the test methods described, was used in this study (sampling period: November, 2019, initiation date of use: December 20, 2019).

The activated sludge, which was cultivated for 22 hours after feeding with the synthetic sewage, was used. The synthetic sewage was prepared according to the following method: glucose, peptone, and potassium dihydrogenphosphate were dissolved in purified water, and the pH of the solution was adjusted to 7.0±1.0.

Decision of additive amount of activated sludge :
Additive amount of the activated sludge into the test vessel was 2.51 mL on the basis of the concentration of suspended solids in the activated sludge which was determined by the following method:
Method In accordance with JIS K 0102:2019
Date December 23, 2019
Result 3580 mg/L

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Remarks:

Substance specific analytics performed on Day 28 for Parent and metabolites performed on sample incubates.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Preparation of test solutions :
The following test solutions were prepared.
Measurement of pH of the test solutions was not performed at the preparation of them because the test item is not dissolved at 100 mg/L in water.
a) Addition of test item or aniline
1) Test solution (water + test item) (n=1, Vessel No. 1)
In one test vessel, 30 mg of the test sample was accurately weighed with an electronic analytical balance and added to 300 mL of purified water, so that the concentration of the test item reached 100 mg/L.
2) Test solution (sludge + test item) (n=3, Vessel Nos. 2, 3 and 4)
In each test vessel, 30 mg of the test sample was accurately weighed with an electronic analytical balance and added to the basal culture medium [the volume subtracting the volume (2.51 mL) of activated sludge from 300 mL], so that the concentration of the test item reached 100 mg/L.
3) Test solution (sludge + aniline) (n=1, Vessel No. 6)
In one test vessel, 29.5 µL (30 mg) of aniline was taken out with a microsyringe and added to the basal culture medium [the volume subtracting the volume (2.51 mL) of activated sludge from 300 mL], so that the concentration of aniline reached 100 mg/L.
4) Test solution (control blank) (n=1, Vessel No. 5)
In one test vessel, the basal culture medium [the volume subtracting the volume (2.51 mL) of activated sludge from 300 mL] was added.
b) Inoculation of activated sludge
The activated sludge was added to each test vessel, so that the concentration of the suspended solid reached 30 mg/L.

Conditions of incubation :
Incubation temperature 25 +/- 1ºC (measured value: 25.0ºC)
Incubation duration 28 days (under dark conditions)
Stirring method Each test solution was stirred with a stirrer

Observations and measurements :
a) Observation of test solutions
At the start and the end of the incubation, appearances of the test solution (water + test item), the test solutions (sludge + test item), and the test solution (control blank) were observed and recorded.
During the incubation period, appearances of the test solutions were observed once a day.
b) Measurement of biochemical oxygen demand (BOD)
During the incubation period, BOD of the test solutions was measured continuously with a closed system oxygen consumption measuring apparatus. The incubation temperature was measured and recorded once a day.

Reference substance:

aniline

Parameter:

% degradation (test mat. analysis)

Value:

19

Sampling time:

28 d

Remarks on result:

other: Average biodegradation of test item on Day 28 determined by HPLC, based on triplicate incubates of Sludge + Test Item (26%, 14%, 16% dissipation of Parent molecule).

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Remarks on result:

other: Average of 3 individual incubates (0%, -1%, -1 % BOD) in Sludge + Test Item samples.

 

		Sludge + test item
		Vessel No. 2	Vessel No. 3	Vessel No. 4	Average
Percentage biodegradation by BOD	%	0	-1	-1	-1
Percentage biodegradation of test item (by HPLC)	%	26	14	16	19

 

Analytical results of test solutions :

Analytical results of the test solutions on Day 28 were as follows.

		Water + test item	Sludge + test item			Average	Theoretical amount	Table	Fig.
		Vessel No. 1	Vessel No. 2	Vessel No. 3	Vessel No. 4
BOD^*2	mg	0.0	0.4	-0.8	-0.5	-0.3	89.4	1	1
Residual amount and percentage residue of test item (by HPLC)	mg	30.0	22.1	25.8	25.1	24.4	30.0	5	5
	% (i)	100	74	86	84	81	-
*3 Produced amount and percentage production of GR-89-1938-1^*4 (by HPLC)	mg	0	8.0	5.4	5.8	6.4	32.3	6
	% (ii)	0	25	17	18	20	-
*3 Produced amount and percentage production of GR-50-2063-3^*5 (by HPLC)	mg	0	0.52	0	0.29	0.27	29.7	7
	% (iii)	0	1.76	0	0.97	0.91	-
Mass balance (i) + (ii) + (iii)	%	100	101	103	103	102

           *2     The value of the test solution (control blank) was subtracted from the values of the test solutions (sludge + test item).

           *3     Calculated from the total peak area of two peaks on the chromatograms.

           *4     1-Methyl-2-(5-methylhex-4-en-2-yl)cyclopropanecarboxylic acid

            *5        1-Methyl-2-(5-methylhex-4-en-2-yl)cyclopropanecarbaldehyde.

 

The average of the percentage biodegradation by BOD was -1% on Day 28 and it demonstrated that the test item did not undergo ultimate biodegradation under the test conditions of this study.

The percentage residue of the test item by HPLC was 100% in the test solution (water + test item) but 74–86% (average 81.3%) in the test solutions (sludge + test item) on Day 28.  The results showed that some of the test item underwent primary degradation by microorganisms.  Thus, the carboxylic acid form (GR-89-1938-1) and the aldehyde form (GR-50-2063-3) which were estimated as degradation products oxidized from the test item were determined: the percentage production of GR-89-1938-1 was 17–25% (average 20%) and that of GR-50-2063-3 was 0–1.76% (average 0.91%) in the test solutions (sludge + test item).  In general, an alcohol form is oxidized to an aldehyde form and the aldehyde form is oxidized to a carboxylic form by microorganisms under aerobic conditions.  Since GR-89-1938-1 was produced more than GR-50-2063-3, GR-50-2063-3 can be considered a minor and transitory intermediate in the degradation pathway of the test item.  Sums of the percentage residue of the test item and the percentage production of GR-89-1938-1 and GR-50-2063-3 were 100% to 103% (mass balance) and it showed that no degradation products except GR-89-1938-1 and GR-50-2063-3 were produced under the test conditions.

 

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The percentage biodegradation by BOD was -1% on Day 28 and it demonstrated that the test item did not undergo ultimate biodegradation under the test conditions of this study. The percentage residue of the test item by HPLC was 100% in the test solution (water + test item) but 74–86% (average 81.3%) in the test solutions (sludge + test item) on Day 28. Some of the test item was oxidized to 1-methyl-2-(5-methylhex-4-en-2-yl)cyclopropanecarboxylic acid (GR-89-1938-1, 17–25% production (average 20%)) and 1-methyl-2-(5-methylhex-4-en-2-yl)cyclopropanecarbaldehyde (GR-50-2063-3, 0–1.762% production (average 0.91%)) by microorganisms.

Reference 3

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August, 2013 - January, 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

GR-50-1408 is the Givaudan identification code which was employed for ROSYFOLIA during the early, developmental and testing period.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (Bois-de-Bay, Satigny, Switzerland) was used.

The sludge is collected in the morning, washed three times in the mineral medium (by centrifuging at 1000g for 10 minutes, discarding the supernatant and resuspending in mineral medium) and kept aerobic until being used on the same day.

The dry weight of suspended solids is determined by taking two 50 ml samples of the homogenised sludge, evaporating water on a steam bath, drying in an oven at 105 - 110 °C for two hours and weighing the residue.

Dry weight of suspended solids: 6.06 g/l, diluted to 2.62 g/L.

Duration of test (contact time):

63 d

Initial conc.:

30 mg/L

Based on:

test mat.

Details on study design:

The method used is basically the one described under No. 302C in the OECD guidelines for Testing of Chemicals. However, fresh activated sludge was sued instead of sludge cultivated as described in the guideline and no quantitative analysis of the test substance was performed.

A measured volume of inoculated mineral medium, containing a known concentration of test substance as the nominal sole source of organic carbon, is stirred in a closed flask at a constant temperature (± 1°C) for up to 28 days or more. Evolved carbon dioxide is absorbed in sodium hydroxide pellets. The consumption of oxygen is determined by measuring the pressure drop in the respirometer flask. The BOD, amount of oxygen taken up by the microbial population during biodegradation of the test chemical (corrected for uptake by blank inoculum, measured simultaneously) is expressed as a percentage of ThOD (Theoretical Oxygen Demand, calculated from the elemental composition, assuming that carbon is oxidised to carbon dioxide; hydrogen to water and nitrogen metabolised to ammonium, nitrite or nitrate).

Preparation of the flasks :
Test substance samples (~13.47 mg, corresponding to 30 mg/l in 449 ml of test volume) are weighed in small aluminium boats and added directly to the test flasks of the Oxitop that were previously charged with 432 ml of mineral medium. Then, 17.1 ml of suspended sludge, diluted to a concentration of 2.62 g/l dry matter, corresponding to 44.8 mg dry weight is added. Flasks containing sludge only (100 mg/l) are charged with 432 ml of mineral medium. Then, 17.1 ml of suspended sludge, diluted to a concentration of 2.62 g/l dry matter, are added.

Reference substance samples (~43.70 mg, corresponding to 100 mg/l in 437 ml of test volume) are weighed in small aluminium boats and added directly to the test flasks of the Oxitop that were previously charged with 432 ml of mineral medium. Then, 5.0 ml of suspended sludge, diluted to a concentration of 2.62 g/l dry matter, corresponding to 13.1 mg dry weight is added. Flasks containing sludge only (30 mg/l) are charged with 432 ml of mineral medium. Then, 5.0 ml of suspended sludge, diluted to a concentration of 2.62 g/l dry matter, are added.

Except when the test substance has an acid or alkaline character, the pH of each flask is not measured but assumed to be the same as the mineral medium, in order not to remove any floating undissolved test substance from the test medium by dipping a glass electrode in it. Neutral test substances, even sodium benzoate, were shown not to affect the pH of the medium by more than 0.1 pH unit. Two sodium hydroxide pellets are placed in the quivers on top of the bottle, and the flasks are closed tightly with the measuring heads. The flasks are allowed to equilibrate to the test temperature. The measurement is started by programming the measuring unit of the Oxitop test flasks, and the test flasks are placed in the temperature controlled cupboard of the Oxitop system. After temperature equilibration, the controller of the instrument starts the data acquisition (time zero of the experiment).

Performance of the test :
Everyday the oxygen consumption of each flask is recorded and correct temperature and stirring are checked.

At the end of the test period, the pH of each flask is measured again.

Test and reference substances :
Nominal concentrations
- test substance : 30 mg/l
- reference substance : 100 mg/l

Test temperature and duration :
Test temperature : 21.1°C – 21.9°C
Test duration : 63 days

Reference substance:

benzoic acid, sodium salt

Remarks:

The activity of the inoculum is checked by performing a simultaneous ready biodegradability test according to OECD Guideline No. 301F with sodium benzoate (Fluka, Buchs, Switzerland, Art. No. 71300, purity: min. 99.0 %). The test conditions are the same.

Key result

Parameter:

% degradation (O2 consumption)

Value:

29

Sampling time:

28 d

Remarks on result:

other: Average of Two Incubates (28% BOD and 30% BOD) on Day 28.

Parameter:

% degradation (O2 consumption)

Value:

33

Sampling time:

63 d

Remarks on result:

other: Average of Two Incubates (34% BOD and 33% BOD) on Day 63.

Details on results:

At the test concentration GR-50-1408 did not significantly inhibit the intrinsic respiration of the inoculum.

Results with reference substance:

Degradation of sodium benzoate exceeded 40 % after 7 days (actual = 73%) and 65 % after 14 days (actual = 88%): the activity of the inoculum was thus verified (validity criterion).

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

Thus, GR-50-1408 cannot be regarded as inherently and ultimately biodegradable according to this test. Howvever, GR-50-1408 should be regarded as inherently primarily biodegradable according to this test method.

Executive summary:

In the test conditions GR-50-1408 undergoes 29% biodegradation after 28 days (33% after 63). At the test concentration GR-50-1408 did not significantly inhibit the intrinsic respiration of the inoculum.

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June - October, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

GR-50-1408 is the Givaudan identification code which was employed for ROSYFOLIA during the early, developmental and testing period.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (Bois-de-Bay, Satigny, Switzerland) was used.

The sludge is collected in the morning, washed three times in the mineral medium (by centrifuging at 1000g for 10 minutes, discarding the supernatant and resuspending in mineral medium) and kept aerobic until being used on the same day.

The dry weight of suspended solids is determined by taking two 50 ml samples of the homogenised sludge, evaporating water on a steam bath, drying in an oven at 105 - 110°C for two hours and weighing the residue.

Dry weight of suspended solids: 6.94 g/l, diluted to 2.62 g/l

To obtain a concentration of 30 mg/l (dry weight) in 437 ml total volume, 5.00 ml of sludge (inoculum) was added to 432 ml of mineral medium.

Duration of test (contact time):

55 d

Initial conc.:

30 mg/L

Based on:

test mat.

Details on study design:

The method used is basically the one described under No. 301F in the OECD guidelines for Testing of Chemicals.

A measured volume of inoculated mineral medium, containing a known concentration of test substance as the nominal sole source of organic carbon, is stirred in a closed flask at a constant temperature (± 1°C) for up to 28 days. Evolved carbon dioxide us absorbed in sodium hydroxide pellets. The consumption of oxygen is determined by measuring the pressure drop in the respirometer flask. The BOD, amount of oxygen taken up by the microbial population during biodegradation of the test chemical (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of ThOD (Theoretical Oxygen Demand, calculated from the elemental composition, assuming that carbon is oxidised to carbon dioxide; hydrogen to water and nitrogen to ammonium, nitrite or nitrate).


Stock solutions of mineral components :
The following stock solutions were prepared:

Solution A:

KH2PO4 8.5 g
K2HPO4 21.75 g
Na2HPO4 · 2 H2O 33.4 g
NH4Cl 0.5 g

dissolved in water and made up to 1 litre.

Solution B:

CaCl2 27.5 g

dissolved in water and made up to 1 litre.


Solution C:

MgSO4 · 7 H2O 22.5 g

dissolved in water and made up to 1 litre.


Solution D:

FeCl3 · 6 H2O 0.25 g
HCl Conc. one drop

dissolved in water and made up to 1 litre.

Mineral medium :
Prepared by mixing 50 ml of solution A and 2000 ml deionised water, adding 5 ml of each of the solutions B, C and D and making up to 5 litres with deionised water. The pH is measured and if necessary adjusted to 7.4 ± 0.2 with phosphoric acid or potassium hydroxide.

Test substance samples (13.10 mg, corresponding to 30.0 mg/l in 437 ml of test medium) are weighed in small aluminium boats and added directly to the test flasks of the Oxitop. For reference substance samples 43.70 mg (corresponding to 100.0 mg/l in 437 ml of test medium) are weighed in small aluminium boats and added directly to the test flasks of the Oxitop.

Flasks are filled with 432 ml of mineral medium. Samples of test or reference substance are added. Then 5.00 ml of suspended sludge diluted to a concentration of 2.62 g/l dry matter is added. Except when the test substance has an acid or alkaline character, the pH of each flask is not measured but assumed to be the same as the mineral medium, in order not to remove any floating undissolved test substance from the test medium by dipping a glass electrode in it. Neutral test substances, even sodium benzoate, were shown not to affect the pH of the medium by more than 0.1 pH unit. Two sodium hydroxide pellets are placed in the quivers on top of the bottle, and the flasks are closed tightly with the measuring heads. The flasks are allowed to equilibrate to the test temperature. The measurement is started by programming the measuring unit of the Oxitop test flasks, and the test flasks are placed in the temperature controlled cupboard of the Oxitop system. After temperature equilibration, the controller of the instrument starts the data acquisition (time zero of the experiment).

Performance of the test :
Everyday the oxygen consumption of each flask is recorded and correct temperature and stirring are checked.

At the end of the test period (55 days), the pH of each flask is measured again.


Test and reference substances :
Nominal concentrations -
- test substance : 30 mg/l
- reference substance : 100 mg/l

Reference substance:

benzoic acid, sodium salt

Remarks:

Sodium benzoate (Fluka, Buchs, Switzerland, Art. No. 71300), purity : min. 99.0%, at a test concentration of 100 mg/L.

Key result

Parameter:

% degradation (O2 consumption)

Value:

22

Sampling time:

28 d

Remarks on result:

other: Average of two incubates (23% BOD, 20% BOD) on Day 28.

Parameter:

% degradation (O2 consumption)

Value:

31

Sampling time:

55 d

Remarks on result:

other: Average of two incubates (31% BOD, 30% BOD) on Day 55.

Details on results:

GR-50-1408 did not significantly inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered o be non toxic to the inoculum at the test concentration.

Results with reference substance:

Degradation of sodium benzoate exceeded 40% after 7 days (actual = 73%) and 65% after 14 days (avtual = 81%): the activity of the inoculum was thus verified (validity criterion)

	Days:		5	7	12	13	23	28	55
O2 uptake of sludge (inoculum blank)	3	B1	21.0	23.1	27.4	25.2	29.5	31.6	33.7
	4	B2	15.0	17.1	23.6	21.4	23.6	25.7	30.0
	mean	B	18.0	20.1	25.5	23.3	26.6	28.7	31.9
O2 uptake of Test Subst. + sludge	5	C1	16.5	20.6	35.5	35.0	45.2	49.4	59.7
	6	C2	16.9	21.1	31.6	33.7	42.1	46.4	59.0
O2 uptake of Test Subst.		C1-B	-1.5	0.5	9.5	11.7	18.7	20.8	27.8
		C2-B	-1.1	1.0	6.1	10.4	15.6	17.8	27.2
% biodegradation of test subst.		D1	-2	1	11	13	21	23	31
		D2	-1	1	7	12	17	20	30
	mean	D	-1	1	9	12	19	22	31

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item undergoes 22% biodegradation after 28 days (31% after 55 days) in the test conditions.

Executive summary:

GR-50-1408 did not significantly inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered to be non-toxic to the inoculum at the test concentration.

Thus GR-50-1408 should be regarded as not readily biodegradable according to this test.

Description of key information

Results from two key biodegradability screening studies (OECD 301F - Ready Biodegradability, OECD 302C - Inherent Biodegradability) and two supporting studies (OECD 301C, SEPA/OECD 301F) are presented.

KEY STUDIES :

OECD 301F (Givaudan Study No. 13-E146) : 22% BOD on Day 28, 31% BOD on Day 55 - Not Readily Biodegradable;

OECD 302C (Givaudan Study No. 13-E155) : 29% BOD on Day 28, 33% BOD on Day 63 - Not Inherently and Ultimately Biodegradable.

SUPPORTING STUDIES :

OECD 301C (CERI Study No. 16639) : 0% BOD on Day 28, 19% dissipation of Parent, test item was oxidized to 1-methyl-2-(5-methylhex-4-en-2-yl)cyclopropanecarboxylic acid (GR-89-1938-1, average production 20%)) and 1-methyl-2-(5-methylhex-4-en-2-yl)cyclopropanecarbaldehyde (GR-50-2063-3, , average production 0.91%) by microorganisms - Not Readily Biodegradable.

SEPA / OECD 301F (NIES Study No. S2014NC060-02) : 8.44% BOD on Day 28 - Not readily Biodegradable.

 

The registration substance was found to be neither readily nor inherently biodegradable under conservative biodegradation screening test conditions.

Both key study screening studies demonstrate a significant degree of BOD suggesting substantial primary degradation of the test item, presumably, in the first instance, to the Carboxylic Acid metabolite (GR-89-1938-1), as observed in the OECD 301C study, prior to undergoing further transformation.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable