3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile

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:

The study was conducted between 30 March 2016 and 28 April 2016.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 102 using the headspace test and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Version / remarks:

2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: ES421 Pinyl Nitrile

SOURCE OF TEST MATERIAL
- Appearance/physical state: white solid
- Source and lot/batch No.of test material: SM15077102
- Expiration date of the lot/batch: 22 July 2016
- Purity: 98.08%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: approximately 4 °C, in the dark

REFERENCE ITEM

Information as provided by the Supplier (Sigma-Aldrich).
Identification: Sodium benzoate
Physical state/Appearance: white granular solid
Batch: SLBM0895V
Purity: >99.5%
Expiry Date: 05 June 2016
Storage Conditions: room temperature over silica gel

Oxygen conditions:

aerobic

Inoculum or test system:

other: A mixed population of sewage treatment micro-organisms was obtained on 30 March 2016 from the secondary treatment stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Details on inoculum:

Preparation of Inoculum
Upon receipt in the laboratory, the sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded). In order to reduce the inorganic carbon (IC) content of the inoculum, the filtrate was sparged with CO2-free air* for approximately 1 hour whilst maintaining its pH at 6.5 using concentrated orthophosphoric acid. After sparging, the pH was restored to its original value of 7.1 using 7 M sodium hydroxide and the inoculum allowed to settle for approximately 1 hour prior to removal of an aliquot (2 liters) of the supernatant for use in the test. The supernatant was maintained on aeration using CO2-free air until use.

* CO2-free air produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

other: mg carbon/L

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test System and Supporting Information

Mineral Medium
The mineral medium used in this study was that recommended in the OECD and ISO Guidelines. For details refer to 'Any other information on materials and methods incl. tables'.

Experimental Design and Study Conduct

Preliminary Solubility Work
Information provided by the Sponsor indicated that the water solubility of the test item was 13 mg/L. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation.

Test Item Preparation
Following preliminary solubility work and the recommendations of the International Standards Organisation (ISO, 1995) and in the published literature (Handley et al, 2002) the test item was dissolved in an auxiliary solvent prior to adsorption onto filter paper (Whatman GF/A, 21 mm diameter). The test item was heated to 45 °C prior to weighing. A nominal amount of test item (1036 mg) was dissolved in 10 mL of acetone to give a 1036 mg/10 mL solvent stock solution. An aliquot (25 µL) of this solvent stock solution was dispensed onto a filter paper and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was added to inoculated mineral medium (107 mL) to give a final concentration of 24.2 mg/L, equivalent to 20 mg carbon/L. The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution. The test vessels were then sealed using Teflon lined silicon septa and aluminum crimp caps.
A filter paper (Whatman GF/A, 21 mm diameter) was added to each control vessel in order to maintain consistency between the test and procedure control vessels. Acetone (25 µL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 minutes. The filter paper was added to each vessel. The test vessels were then sealed using Teflon lined silicon septa and aluminum crimp caps.

A test concentration of 20 mg carbon/L was employed in the study following the recommendations of the test guidelines.
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Reference Item Preparation
A reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium with the aid of ultrasonication for approximately 10 minutes. An aliquot (171.5 mL) of this stock solution was dispersed with inoculum (500 mL) and mineral medium to a final volume 5 liters, to give a test concentration of 34.3 mg/L, equivalent to 20 mg carbon/L. Aliquots (107 mL) of the 34.3 mg/L test concentration were dispensed to each of 33 replicate test vessels and the vessels sealed using Teflon lined silicon septa and aluminum crimp caps. A filter paper (Whatman GF/A, 21 mm diameter, with acetone evaporated off) was added to the procedure control vessels in order to maintain consistency between the test and procedure control vessels. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Toxicity Control
A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the study. Aliquots (107 mL) of the 34.3 mg/L reference item concentration were dispensed to 9 replicate test vessels. An aliquot (25 µL) of the 1036 mg/10 mL test item solvent stock solution was dispensed separately on to 9 filter papers and the solvent allowed to evaporate to dryness for approximately 15 minutes. The final concentration in the toxicity control vessels was 24.2 mg test item/L plus 34.3 mg reference item/L, equivalent to 40 mg carbon/L.

Preparation of Test System
The following test preparations were prepared and incubated in 125 mL glass Wheaton bottles (total volume when full 160 mL) each containing 107 mL of solution:
a) An inoculated control consisting of inoculated mineral medium, plus a filter paper (Whatman GF/A, 21 mm diameter, with acetone evaporated off), 33 replicate vessels.
b) The procedure control containing the reference item (sodium benzoate) in inoculated mineral medium, plus a filter paper (Whatman GF/A, 21 mm diameter, with acetone evaporated off), to give a final concentration of 20 mg carbon/L, 33 replicate vessels.
c) The test item in inoculated mineral medium, plus a filter paper (Whatman GF/A, 21 mm diameter, with acetone evaporated off), to give a final concentration of 20 mg carbon/L, 29 replicate vessels.
d) The test item plus the reference item in inoculated mineral medium, plus a filter paper (Whatman GF/A, 21 mm diameter, with acetone evaporated off), to give a final concentration of 40 mg carbon/L to act as a toxicity control, 9 replicate vessels.
A filter paper was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels.
Test media a) to d) were inoculated with the prepared inoculum at a final concentration of 100 mL/L. Aliquots (107 mL) of the test media were dispensed into replicate vessels to give a headspace to liquid ratio of 1:2. Sufficient vessels were prepared to allow a single inorganic carbon determination per vessel with triplicate vessels for the inoculum control, procedure control, test item and toxicity control at each sampling occasion (five replicates for analysis on Day 28). Additional inoculum control and procedure control were prepared to provide samples for Dissolved Organic Carbon (DOC) analyses on Days 0 and 28 (duplicate vessels per sampling occasion). All vessels were sealed using Teflon lined silicon septa and aluminum crimp caps and incubated in darkness at approximately 20 °C with constant shaking at approximately 125 rpm (INFORS Version 2 Multitron® Incubator).

Data Evaluation

Calculation of Mean Total Inorganic Carbon (TIC)
The mean TIC (mg/L) in the inoculum control vessels (replicates R1-R5) on Day 28 was calculated using the following equation:
Mean TIC (mg/L) = Mean TIC (mg) in inoculum control vessels on day 28/V x 1000
Where:
V = volume of liquid in each vessel (107 mL)

Calculation of the Percentage Inorganic Carbon Produced in the Inoculum Control Bottles in Relation to the Amount of TOC Added Initially as Test Item
The amount of inorganic carbon (%) produced from the inoculum control vessels on Day 28 in relation to the amount of organic carbon (20 mg C/L) added as test item initially was calculated using the following equation:

% = Mean TIC in the control vessels on day 28 (mg C/L)/Nominal test item TOC concentration in test vessels (mg C/L) x 100
Where:
% = percentage of organic carbon added as test substance

Validation Criteria
Test items giving a result of ≥60% yield of ThIC within 28 days should be regarded as readily biodegradable. This level must be reached within 10 days of the biodegradation exceeding 10%. The test is considered valid if the reference item biodegradation rate is ≥60% by day 14. The toxicity control should attain ≥25% biodegradation by day 14 for the test item to be considered as non-inhibitory. The TIC produced from the control bottles at the end of the test should be ≤15% of the TOC added initially as test item.














References

Handley et al. (2002) The Use of Inert Carriers in Regulatory Biodegradation Tests of Low Density Poorly Water-soluble Substances, Chemosphere 48 pp. 529-34.
International Standards Organisation (ISO), ISO 10634 (1995) 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.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (inorg. C analysis)

Value:

0

Sampling time:

28 d

Remarks on result:

not readily biodegradable based on QSAR/QSPR prediction

Details on results:

Definitive Test
Total inorganic carbon values for the test item, procedure control, toxicity control and inoculum control vessels at each analysis occasion are given under 'Any other information on results incl. tables'. Percentage biodegradation values for the test item and procedure control and the toxicity control are given under 'Any other information on results incl. tables'. The results of the Dissolved Organic Carbon analyses performed on Days 0 and 28 are given under 'Any other information on results incl. tables'.

Validation Criteria
The mean TIC in the inoculum control vessels on Day 28 was 1.05 mg/L, equivalent to 5% of the organic carbon added initially as test item to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.

Biodegradation
The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 310. Statistical analysis of the Day 29 IC values for the control and test item vessels showed there were no statistically significant differences (P≥ 0.05) between the control and the test item. The test item was therefore considered not to have a toxic effect on the sewage sludge micro-organisms used in the study and this was confirmed by the toxicity control results. The toxicity control attained 39% biodegradation after 14 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test. Sodium benzoate attained 70% biodegradation after 14 days and 84% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Variation in the biodegradation rates obtained on different sampling days (see 'Any other information on results incl. tables') was considered to be the result of normal biological variation between the respiration rates of replicate vessels. Due to the sacrificial nature of the study design, the biodegradation rates obtained on each sampling occasion were for individual replicate vessels and not the result of cumulative biodegradation values determined from a single vessel sampled on numerous occasions and as such variation in biodegradation rates on different sampling days was to be expected. DOC analyses conducted on samples taken from the reference item vessels on Days 0 and 28 (see 'Any other information on results incl. tables') showed that the replicate reference item vessels attained 97% biodegradation for each replicate vessel. The biodegradation rates for the reference item were higher than those determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to biodegradation and hence CO2 evolution occurring.

Results with reference substance:

Sodium benzoate attained 70% biodegradation after 14 days and 84% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions 'Any other information on results incl. tables'.

Inorganic Carbon Values on Each Analysis Occasion

Day	Inorganic Carbon (mg IC)
	Inoculum Control					Procedure Control					Test Item					Toxicity Control
	R1	R2	R3	R4	R5	R1	R2	R3	R4	R5	R1	R2	R3	R4	R5	R1	R2	R3	R4	R5
0	0.09	0.08	0.08	-	-	0.08	0.08	0.07	-	-	0.07	0.08	0.07	-	-	0.08	0.09	0.09	-	-
2	0.09	0.08	0.10	-	-	1.21	1.28	1.29	-	-	0.08	0.08	0.07	-	-	-	-	-	-	-
5	0.09	0.10	0.09	-	-	1.74	1.65	1.61	-	-	0.09	0.09	0.09	-	-	-	-	-	-	-
7	0.09	0.08	0.08	-	-	1.53	1.55	1.48	-	-	0.08	0.09	0.08	-	-	1.58	1.51	1.65	-	-
9	0.09	0.10	0.10	-	-	1.47	1.43	1.61	-	-	0.08	0.09	0.09	-	-	-	-	-	-	-
14	0.09	0.08	0.09	-	-	1.61	1.56	1.59	-	-	0.08	0.08	0.09	-	-	1.97	1.64	1.61	-	-
16	0.10	0.11	0.11	-	-	1.61	1.66	1.67	-	-	0.09	0.09	0.09	-	-	-	-	-	-	-
21	0.11	0.11	0.11	-	-	1.61	1.70	1.74	-	-	0.11	0.10	0.12	-	-	-	-	-	-	-
28	0.12	0.11	0.11	0.11	0.11	1.79	1.87	2.07	1.87	1.82	0.12	0.12	0.10	0.11	0.10	-	-	-	-	-

R₁– R₅= Replicates 1 to 5

Percentage Biodegradation Values

Day	% Biodegradation
	Procedure Control	Test Item	Toxicity Control
0	0	0	0
2	55	0	-
5	74	0	-
7	67	0	35
9	65	0	-
14	70	0	39
16	72	-1	-
21	73	0	-
28	84	0	-

R₁– R₂= Replicates 1 and 2

LOQ= Limit of Quantification (determined down to 1.0 mg C/L).

Dissolved Organic Carbon (DOC) Values on Days 0 and Day 28

Test Vessel		DOC Concentration
		Day 0			Day 28
		mg C/L	mg C/L Corrected for Mean Control Value	% of Nominal Carbon Content	mg C/L	mg C/L Corrected for Mean Control Value	% Biodegradation
Inoculum Control	R1	1.19	-	-	0.97	-	-
	R2	1.10	-	-	0.88	-	-
Procedure Control	R1	21.75	20.60	103	1.28	0.35	97
	R2	21.76	20.61	103	1.50	0.57	97

R₁– R₂= Replicates 1 and 2

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item, ES421 Pinyl Nitrile, attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 310.

Executive summary:

Introduction

A study was performed to assess the ready biodegradability of the test item, ES421 Pinyl Nitrile, in an aerobic aqueous medium. The method followed was designed to be compatible with OECD Guidelines for Testing of Chemicals (2014) No. 310 and the ISO Guideline No 14593 “Water quality – Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium – Method by analysis of inorganic carbon in sealed vessels (CO₂Headspace Test)”.

Methods

The test item, at a concentration of 20 mg C/L, was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in the dark at a temperature of approximately 20°C for 28 days.

In view of the difficulties associated with the evaluation of the biodegradability of organic compounds with low water solubility, a modification to the standard method of preparation of the test concentration was performed. An approach endorsed by the International Standards Organisation (ISO, 1995) is to dissolve the test item in an auxiliary solvent prior to adsorption onto filter paper. 

The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

Results

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 310.

References

International Standards Organisation (ISO), ISO 10634 (1995) 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.

Description of key information

A study was performed to assess the ready biodegradability of the test item,ES421 Pinyl Nitrile, in an aerobic aqueous medium. The method followed was designed to be compatible with OECD Guidelines for Testing of Chemicals (2014) No. 310 and the ISO Guideline No 14593 “Water quality – Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium – Method by analysis of inorganic carbon in sealed vessels (CO₂Headspace Test)”.

Methods

The test item, at a concentration of 20 mg C/L, was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in the dark at a temperature of approximately 20°C for 28 days.

In view of the difficulties associated with the evaluation of the biodegradability of organic compounds with low water solubility, a modification to the standard method of preparation of the test concentration was performed. An approach endorsed by the International Standards Organisation (ISO, 1995) is to dissolve the test item in an auxiliary solvent prior to adsorption onto filter paper. 

The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

Results

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 310.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed