[1R-(1α,2β,5α)]-2-isopropyl-5-methylcyclohexyl 5-oxo-L-prolinate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-12 to 2018-02-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline No. 301 D. There was one deviation from the study plan (concerning temperature values). This deviation did not affect the integrity of the study. Indeed, the temperature values recorded between days 21 and 22 were not taken into account since there was a problem related to the temperature sensor at this time, and some temperature values were aberrant or unavailable. This minor deviation was considered not to affect the results of the test as no impact was observed on the inoculum blank respiration throughout the duration of the test. Except this, all validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

10 January 2017

Specific details on test material used for the study:

None

Oxygen conditions:

aerobic

Inoculum or test system:

other: river water

Details on inoculum:

- Origin: River water was sampled from “La Mourachonne”, situated downstream of a plant treating domestic wastewater (Pegomas, France).
- Pre-conditioning: The river water was aerated for about 5 days before use as inoculum at the test temperature to reduce endogenous respiration.

Duration of test (contact time):

62 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Test water: Mineral medium (reconstituted water), as prescribed by the OECD Guideline No. 301D
- Composition of medium: The mineral medium was prepared by adding one mL of each of the following stock solutions (a to d) to each litre of ultrapure water: a) 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.40 mg Na2HPO4•2H2O, 0.50 mg NH4Cl. The pH of this solution was approximately 7.4 ± 0.1; b) 36.40 mg CaCl2•2H2O, c) 22.50 mg MgSO4•7H2O and d) 0.25 mg FeCl3•6H2O.). This reconstituted water was aerated for at least 20 min and thereafter set aside at the test temperature for about 20 h before use.
- Test temperature: Temperatures recorded continuously and situated between 20.9 and 21.4°C throughout the test (average value: 21.1°C), and complied with the requirements as laid down in the study plan (22°C ± 2°C, constant within 1°C).
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: All-glass sealed with ground glass stoppers BOD (Biological Oxygen Demand) bottles of approximately 300 mL capacity. Each test vessel was uniquely identified with study code, replicate number, date of the start of the biodegradability test (=J0) and type of series.
- Inoculum: A concentration of inoculum of approximately 10^4 cells.L-1 was used.
- Number of culture flasks/concentration: 20 bottles containing test item, inoculum and mineral medium (test suspension)

SAMPLING
- Sampling frequency: Determinations of the concentration of dissolved oxygen using an oxygen electrode in duplicate vessels were made at day 0, 7, 14, 21, 28, 35, 42, 49, 56 and 62 for the “test suspension” and “inoculum blank” series, and only at the start of the test and after 7 and 14 days of incubation for the “procedure control” and the “toxicity control” series.
- Temperature: Measured continuously in the growth chamber.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 20 bottles containing inoculum and mineral medium
- Test suspension: 18 bottles containing test item, inoculum and mineral medium
- Procedure control: 6 bottles containing reference substance, inoculum and mineral medium
- Toxicity control: 6 bottles containing test item, reference substance, inoculum and mineral medium

Reference substance:

benzoic acid, sodium salt

Remarks:

2 mg/L

Preliminary study:

None

Key result

Parameter:

% degradation (O2 consumption)

Value:

8

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

61

Sampling time:

62 d

Details on results:

Test item QUESTICE L was biodegraded by 8% at day 28 in the Closed Bottle test. The test period was therefore extended to 62 days in order to demonstrate a potential inherent biodegradability or a non persistence of the test item. The biodegradation percentage at day 62 was 61% (most conservative value, based on only one replicate, since differences of the replicate values at day 28 were 25%). It should be noted that the test item had no inhibitory effect on the activity of the microbial inoculum, as demonstrated by the test results of the toxicity control, in agreement with OECD 301 (oxygen consumption in the toxicity control must be greater than 25% of the ThOD value within 14 days).

Calculated ThOD of the test item: 2.277 mg O2/mg, based on a MW of 267 g/mol (C15 H25 NO3)

Results with reference substance:

Calculated ThOD of the reference substance: 1.67 mg O2/mg, based on a MW of 144.1 g/mol (C7 H5 O2 Na). The biodegradation percentage of the reference substance, sodium benzoate, was 89 % at day 14.

Table 5.2.1/1: Percentage biodegradation of the test item, the reference substance and of the toxicity control throughout the test

		Time (days)
		0	7	14	21	28	35	42	49	56	62
Test Suspension (2 mg test item.L^-1)	Replicate 1	0.00	1.43	2.64	2.09	7.25	29.31	25.47	58.41	55.78	79.49
	Replicate 2	0.00	0.33	4.17	3.18	8.78	10.21	17.35	52.04	22.40	61.05
	Mean	0	1	3	3	8	20	21	55	39	70
Procedure Control (2 mg reference substance.L^-1)	Replicate 1	0.00	82.19	87.72	-	-	-	-	-		-
	Replicate 2	0.00	78.89	89.82	-	-	-	-	-		-
	Mean	0	81	89	-	-	-	-	-		-
Toxicity Control* (2 mg reference substance.L^-1 + 2 mg test item.L^-1)	Mean	0	83	92	-	-	-	-	-		-

* % biodegradation_{tox. Control}=  (BOD tox. cont / ThODref. subst.) * 100

with BOD tox. cont = (mTox(o) - mTox(n)) - (ma(o) - ma(n)) / mg test ref susbst.L^-1in vessel

and where:

 n = number of days after the start of the test

mTox(0) = mean toxicity control value at day 0

 mTox (n) = mean toxicity control value at day n

 ma(0) = mean test suspension value at day 0

 ma(n) = mean test suspension value at day n.

Table 5.2.1/2: Dissolved oxygen concentrations (mg.L^-1) throughout the test

	Time (days)
	0	7	14	21	28	35	42	49	56	62
Inoculum Blank	8.53	8.11	7.92	7.96	7.52	7.29	7.09	7.06	7.03	6.99
	8.52	7.99	7.89	7.58	7.39	7.37	6.72	6.87	6.96	7.04
Mean	8.53	8.05	7.91	7.77	7.46	7.33	6.91	6.97	7.00	7.02
Test Suspension (2 mg test item.L^-1)	8.50	7.96	7.76	7.65	7.10	5.97	5.72	4.28	4.43	3.37
	8.51	8.02	7.70	7.61	7.04	6.85	6.10	4.58	5.96	4.22
Mean	8.51	7.99	7.73	7.63	7.07	6.41	5.91	4.43	5.20	3.80
Procedure Control(2 mg reference substance.L^-1)	8.50	5.28	4.95	-	-	-	-	-	-
	8.47	5.36	4.85	-	-	-	-	-	-
Mean	8.49	5.32	4.90	-	-	-	-	-	-
Toxicity Control (2 mg reference substance.L^-1 + 2 mg test item.L^-1)	8.48	5.19	4.83	-	-	-	-	-	-
	8.50	5.25	4.46	-	-	-	-	-	-
Mean	8.49	5.22	4.65	-	-	-	-	-	-

Table 5.2.1/3. Analytical monitoring of nitrate & nitrite concentrations in inoculum blanks and test suspensions throughout the test.

 

Time (days)

0

7

14

21

28

35

42

49

56

62

Inoculum Blank

Concentration of nitrate (mg N.L^-1)

Rep. 1

<0.100

<0.100

<0.100

<0.100

<0.100

<0.100

0.239

0.373

0.515

0.497

Rep. 2

<0.100

0.123

0.148

<0.100

<0.100

<0.100

0.154

0.434

0.510

0.491

Concentration of nitrite (mg N.L^-1)

Rep. 1

<0.020

<0.020

<0.020

<0.020

0.027

0.432

0.264

0.056

<0.020

<0.020

Rep. 2

<0.020

<0.020

<0.020

<0.020

<0.020

0.391

0.366

<0.020

<0.020

<0.020

Test Suspension (2 mg test item.L^-1)

Concentration of nitrate (mg N.L^-1)

Rep. 1

<0.100

<0.100

0.104

<0.100

<0.100

<0.100

<0.100

0.420

<0.100

0.625

Rep. 2

<0.100

<0.100

0.126

<0.100

<0.100

<0.100

<0.100

0.329

0.547

<0.100

Concentration of nitrite (mg N.L^-1)

Rep. 1

<0.020

<0.020

<0.020

<0.020

<0.020

<0.020

0.457

0.022

0.453

<0.020

Rep. 2

<0.020

<0.020

<0.020

<0.020

<0.020

0.060

0.420

0.141

<0.020

0.475

LOQ_Nitrate: 0.1 mg NO₃.L^-1; LOQ

Results of test measurements and conditions  

Analysis of nitrite and nitrate concentrations in inoculum blanks and test suspensions throughout the test (seeTable 4 of the Appendix II for details) revealed there was no nitrification process due to nitrogen from the test item. Indeed, the values obtained withinoculum blanks and test suspensions were in the same order of magnitude (approx. the same nitrogen quantity was oxidised in both case; this nitrogen was probably the one from the mineral medium (NH₄Cl)).

Thus, a correction for the oxygen consumed by nitrification was not relevant for the present study.

Validity criteria of the test:   

Inoculum blank: Oxygen depletion in the inoculum blank did not exceed 1.5 mg dissolved oxygen/L after 28 days.

Residual [O₂]: Oxygen concentrations were > 0.5 mg/L in all bottles during the test period.

Replicate values: The differences of the replicate (test suspension) values at day 28 were less than 20 %.

Reference substance: The biodegradation percentage of the reference substance, sodium benzoate, was 89% at day 14.

Thus all validity criteria of the test have been fulfilled in the present study.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item QUESTICE L was biodegraded by 8% at day 28. The test period was extended to 62 days in order to demonstrate a potential inherent biodegradability or a non-persistence of the test item. The biodegradation percentage at day 62 was 61% (most conservative value between the two replicates). Therefore, QUESTICE L cannot be considered as readily biodegradable under the experimental conditions, but is considered as not persistent.

Executive summary:

A ready biodegradability study was performed to assess the biotic degradation of the test item QUESTICE L according to the OECD Guideline 301 D (Closed bottle test) and in compliance with GLP.

Twenty BOD bottles were filled with mineral medium inoculated with river water and test item added at a nominal concentration of 2 mg.L-1. In the meantime, one series of blanks were filled with inoculated mineral medium. Furthermore, six bottles containing reference substance sodium benzoate (2 mg.L-1) were tested in order to check the procedure. A toxicity control, containing both the test item and the reference substance, was also performed in order to check the absence of test item effect on the microbial inoculum. The concentrations of dissolved oxygen in duplicate bottles from each group incubated at 22°C ± 2°C in darkness were measured at the start of the test and thereafter once a week until the end of the test.

The validity of the test was demonstrated by an endogenous respiration < 1.5 mg O2.L-1 after 28 days and by a biodegradation of the reference substance of 89% of its ThOD after 14 days of incubation. Moreover, the differences of the replicate values at day 28 were less than 20% and oxygen concentrations were > 0.5 mg.L-1 in all bottles during the test period. Furthermore, the toxicity control showed that the test item had no inhibitory effect on the activity of the microbial inoculum.

Substances are considered to be readily biodegradable in the Closed Bottle test if oxygen consumption is equal to or greater than 60% of the ThOD value in the ten-day window within the 28-day period of the test (the 10-day window begins when the degree of biodegradation has reached 10% of the ThOD).

In this study, test item QUESTICE L was biodegraded by 8% at day 28. The test period was therefore extended to 62 days in order to demonstrate a potential inherent biodegradability or a non persistence of the test item. The biodegradation percentage at day 62 was 61% (most conservative value). Therefore, QUESTICE L cannot be considered as readily biodegradable under the experimental conditions, but it is not classified persistent.

Results synopsys:

Test item: QUESTICE L

Test: Closed Bottle test (according to OECD 301 D) and under GLP conditions.

Result: Not readily biodegradable, but 61% reached after 62 days.

Description of key information

The test item QUESTICE L was biodegraded by 8% at day 28. The test period was extended to 62 days in order to demonstrate a potential inherent biodegradability or a non-persistence of the test item. The biodegradation percentage at day 62 was 61% (most conservative value). Therefore, QUESTICE L cannot be considered as readily biodegradable under the experimental conditions, but can be classified as not persistent.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

For that endpoint, an experimental ready biodegradability study was available in order to assess the biotic degradation of the test item according to the OECD Guideline 301 D and in compliance with GLP. The validity of the test was demonstrated by an endogenous respiration < 1.5mg O2/L and by a biodegradation of the reference substance of 89% of its ThOD after 14 days of incubation. Moreover, the differences of the replicate values at day 28 were less than 20% and oxygen concentrations were > 0.5 mg/L ^{in all bottles during the test period}. Furthermore, the toxicity control showed that the test item had no inhibitory effect on the activity of the microbial inoculum, because the degradation was > 25% after 14 days of incubation..

The validity criteria of the 301 D test were respected. The study satisfies guidelines requirements for ready biodegradability studies and was therefore considered as acceptable for that endpoint.