(3aα,4β,7β,7aα)-octahydro-4,7-methano-1H-indene

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:

key study

Study period:

08 November 2017 - 07 january 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

GLP compliance:

yes (incl. QA statement)

Remarks:

The test facility was operating in compliance with the OECD principles of Good Laboratory Practice. Date: 28/11/2017

Specific details on test material used for the study:

- Physical appearance: clear liquid
- Storage conditions: at room temperature
- Stable under storage conditions until: 30 March 2019 (retest date)

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic (adaptation not specified)

Details on inoculum:

A mixed population of sewage treatment micro-organisms was obtained on 8 November 2017 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and the filtrate maintained on continuous aeration in a temperature controlled room at approximately 21 °C prior to use.

Duration of test (contact time):

60 d

Initial conc.:

1 mg/L

Based on:

test mat.

Initial conc.:

3.29 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Mineral medium
The mineral medium used in this study was that recommended in the OECD Guidelines.
The deionized reverse osmosis water used for the preparation of the mineral medium and the mineral medium used for the test contained less than 1 mg/L Total Organic Carbon (TOC).

Preliminary work
The average volume of a completely filled 250-300 mL Biological Oxygen Demand (BOD) bottle was found to be 280 mL. This was calculated by measuring the volume of reverse osmosis water required to completely fill fifteen separate BOD bottles.

Test item preparation:
Following the results of the preliminary solubility work conducted and recommendations of the International Standards Organisation (ISO 10634, (1995)), the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in inoculated culture media. Using this method allows extremely small amounts of test item to be added to each bottle, the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.
A nominal amount of test item (100 mg) was dissolved in 10 mL of acetone to give a 100 mg/10 mL solvent stock solution. Aliquots (28 μL) of this solvent stock solution were dispensed onto 20 separate filter papers* and the solvent allowed to evaporate to dryness for approximately 15 minutes. A test item coated filter paper was then placed into 20 separate Biological Oxygen Demand (BOD) bottles prior to the addition of inoculated mineral medium to give a final concentration of 1.0 mg/L. The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution.
A test concentration of 1.0 mg/L was employed in the study as the Theoretical Oxygen Demand (ThOD) of the test item was calculated to be 3.29 mg O2/mg (see Annex 3). Hence, if complete degradation of the test item occurred, the oxygen depletion in the test vessels would be 3.29 mg O2/L and as such de-oxygenation of the test media would not occur.
As it was not a requirement of the Test Guidelines, no analysis was carried out 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 and is considered not to affect the purpose or integrity of the study.

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 and an aliquot (18 mL) dispersed in a final volume of 6 liters of inoculated mineral medium to give a test concentration of 3.0 mg/L. A filter paper* with 28 μL of acetone added and evaporated to dryness for approximately 15 minutes was added into 20 separate BOD bottles in order to maintain consistency between the test and reference item vessels. Each bottle was then filled with the 3.0 mg/L reference item solution.
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 treatment micro-organisms used in the study.
Aliquots (28 μL) of the solvent test item stock solution were dispensed onto eight separate filter papers* and the solvent allowed to evaporate to dryness for approximately 15 minutes. A test item coated filter paper was then placed into eight separate Biological Oxygen Demand (BOD) bottles. An aliquot (3.0 mL) of the sodium benzoate stock solution (see Section 3.3.3) was dispersed in a final volume of 3 litres of mineral medium. The BOD bottles containing the test item coated filter papers were then filled with the inoculated mineral medium containing sodium benzoate to give a final test concentration of 1.0 mg test item/L and 1.0 mg sodium benzoate/L.

Preparation of Test system:
The following test preparations were prepared and inoculated in 250 mL Biological Oxygen Demand (BOD) bottles (darkened glass) with ground glass stoppers:
a) An inoculum control, twenty vessels, consisting of inoculated mineral medium plus a filter paper*.
b) The procedure control containing the reference item (sodium benzoate), twenty vessels, in inoculated mineral medium plus a filter paper to give a concentration of 3.0 mg/L.
c) The test item on a filter paper*, twenty vessels, in inoculated mineral medium to give a concentration of 1.0 mg/L.
d) The test item (1.0 mg/L) on a filter paper* plus the reference item (1.0 mg/L), eight vessels, in inoculated mineral medium to act as a toxicity control.
The reference item concentration was reduced to 1.0 mg/L in the toxicity control to prevent complete deoxygenation of the test media occurring.
A filter paper with 28 μL of acetone added was allowed to evaporate to dryness and 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 sewage treatment micro-organisms at a rate of 1 drop of inoculum per liter.
The pH of all of the above test preparations were measured using a Hach HQ40d Flexi handheld meter prior to being transferred by siphon to BOD bottles, which were firmly stoppered to exclude all air bubbles. Sufficient bottles were prepared to allow a single oxygen determination per bottle with duplicate bottles for each test preparation at each sampling occasion.
The BOD bottles were incubated in darkness in a temperature controlled water bath at temperatures of between 20 and 21 °C.

Assessment:
Dissolved oxygen concentrations in duplicate samples from the control, reference and test item series were determined, on Days 0, 2, 5, 7, 9, 14, 21, 28, 42 and 60 by means of a YSI 54A dissolved oxygen meter and BOD Probe. Dissolved oxygen concentrations in duplicate samples from the toxicity control series were determined on Days 0, 14, 28 and 60 only.
The dissolved oxygen depletion for each replicate flask for the test item, reference item and toxicity control for each time period is calculated as follows:
Oxygen depletion = (Mto - Mtx) - (Mbo - Mbx) mg O2/L
Where:
Mto = oxygen concentration in test flask at time 0
Mtx = oxygen concentration in test flask at time x
Mbo = mean oxygen concentration of inoculated control flasks at time 0
Mbx = mean oxygen concentration of inoculated control flasks at time x
The oxygen depletion for the inoculated control (blank) flasks is calculated as follows:
Blank oxygen depletion = Mbo - Mbx
The percentage biodegradation for each replicate test item, reference item and toxicity control flask was calculated as follows:
% biodegradation = dissolved oxygen depletion/(concentration of test substance x ThOD (or COD)) x100
Mean % biodegradation = (% biodegradation of replicate 1 + % biodegradation of replicate 2) / 2

Major Computerized Systems:
The following computerized systems were used in the study:
Shimadzu TOC TOC measurement
Delta control system Building management

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Value:

8

Sampling time:

14 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

9

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

9

Sampling time:

60 d

Details on results:

The oxygen depletion of the inoculated control did not exceed 1.5 mg O2/L after 28 days, the residual oxygen concentration in the test bottles remained at 7.55 mg O2/L or greater after 28 days in all test vessels and the difference between the extremes of replicate oxygen depletion values at the end of the test was less than 20% in all vessels thereby satisfying the validation criteria.
The test item attained 9% biodegradation after 28 days and, therefore, cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301D.
Extension of the test from 28 days to 60 days did not result in any increase in biodegration.
Variation in biodegradation rates on different sampling days was considered to be due to normal biological variation in respiration rates between the inoculum control and the test item vessels.
The toxicity control attained 27% biodegradation after 14 days, 31% biodegradation after 28 days and 30% biodegradation after 60 days therefore confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test. The slight decrease in biodegradation between Days 28 and 60 was considered to be due to normal biological variation in respiration rates between the inoculum control and the toxicity control vessels.

Results with reference substance:

The reference item, sodium benzoate, attained 81% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 79% biodegradation was attained with 72% biodegradation being attained after 60 days. The slight decrease in biodegradation between Days 14, 28 and 60 was considered to be due to normal biological variation in respiration rates between the inoculum control and the procedure control vessels. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

1. Dissolved oxygen demand

		Dissolved Oxygen (mg O2/L)
		Day
Test series		0	2	5	7	9	14	21	28	42	60
a) Inoculum control	R1	8.70	8.60	8.25	8.20	8.10	8.20	7.80	8.10	7.50	7.20
	R2	8.70	8.60	8.05	8.15	8.20	8.20	7.70	7.80	7.30	7.30
b) Procedure Control	R1	8.65	5.30	4.90	4.25	4.80	4.05	3.70	3.80	3.80	3.70
	R2	8.65	5.25	4.85	4.25	4.00	4.15	3.50	4.10	3.70	3.55
c) Test Item	R1	8.65	8.35	7.90	7.80	8.05	7.95	7.50	7.70	7.00	6.90
	R2	8.70	8.30	7.90	7.70	7.55	7.90	7.40	7.55	7.10	7.00
d) Toxicity control	R1	8.65	-	-	-	-	6.80	-	6.30	-	5.80
	R2	8.65	-	-	-	-	6.80	-	6.30	-	5.60

2. Oxygen Depletion and Mean Percentage Biodegradation Values

			days
Test series			2	5	7	9	14	21	28	42	60
a) Inoculum Control	Mean O2 depletion (mg O2/L)		0.100	0.550	0.525	0.550	0.500	0.950	0.750	1.300	1.450
b) Procedure Control	O2 depletion (mg O2/L)	R1	3.250	3.200	3.875	3.300	4.100	4.000	4.100	3.550	3.500
	O2 depletion (mg O2/L)	R2	3.300	3.250	3.875	4.100	4.000	4.200	3.800	3.650	3.650
	% Biodegradation (mean)		66	65	77	74	81	82	79	72	72
c) Test Item	O2 depletion (mg O2/L)	R1	0.200	0.200	0.325	0.050	0.200	0.200	0.200	0.350	0.300
	O2 depletion (mg O2/L)	R2	0.300	0.250	0.475	0.600	0.300	0.350	0.400	0.300	0.250
	% Biodegradation (mean)		8	7	12	10	8	9	9	10	9
d) Toxicity Control	O2 depletion (mg O2/L)	R1	-	-	-	-	1.350	-	1.600	-	1.400
	O2 depletion (mg O2/L)	R2	-	-	-	-	1.350	-	1.500	-	1.600
	% Biodegradation (mean)		-	-	-	-	27	-	31	-	30

3. pH values of the test Preparations on day 0

Test preparations	pH
Inoculum Control	7.2
Procedure Control	7.4
Test item	7.4
Toxicity Control	7.4

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The registered substance attained 9% biodegradation after 28 days and therefore cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301D.
Extension of the test from 28 days to 60 days did not result in any increase in biodegration.

Executive summary:

This study was performed to assess the ready biodegradability of the registered substance in an aerobic aqueous medium. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301D, "Ready Biodegradability; Closed Bottle Test” referenced as Method C.4-E of Commission Regulation (EC) No. 440/2008, and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 Paragraph (o).

The registered substance, at a concentration of 1.0 mg/L, was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 20 and 21 °C for 60 days. At the request of the Sponsor the study was extended from 28 to 60 days.

Following the recommendations of the International Standards Organisation (ISO 10634, (1995)), the registered susbtance was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in inoculated mineral media. Using this method allows extremely small amounts of substance to be added to each bottle, the registered substance is evenly distributed throughout the test medium and the surface area of substance exposed to the test organisms is increased thereby increasing the potential for biodegradation. The degradation of the registered substance was assessed by the determination of the amount of oxygen consumed. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

The registered substance attained 9% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301D.

Extension of the test from 28 days to 60 days did not result in any increase in biodegration.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

19 September 2018 - 20 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

The test facility was operating in compliance with the OECD principles of Good Laboratory Practice. Date: 10/01/2018

Specific details on test material used for the study:

Molecular Formula: C10H16
Molecular Weight: 136.24 g/mol
Relative Density: 0.935 g/cm3 at 20 C
Water Solubility: 2.87 mg/L
Vapour Pressure: 260 Pa at 20 °C
Log Kow: 4.8
Total Organic Carbon
(calculated by the study director): 88.16 %
Theoretical Oxygen Demand
(calculated by the study director): 3.29 mg O2/mg
Appearance: Colorless, clear liquid
Expiration Date: March 19, 2020
Storage Conditions: Keep in a cool, well-ventilated place away from moisture. Store in tightly closed containers.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

The study was performed with aerobic activated sewage sludge from the aeration stage of the local wastewater treatment plant, ARA Birs (Birsfelden / Switzerland), which treats predominantly domestic sewage.
No pre-adaptation of the inoculum to the test item was done.
Preparation of Inoculum
The aerobic activated sewage sludge was washed three times by centrifugation, decantation of the supernatant liquid phase and resuspension of the solid material in tap water and finally in mineral medium. Aliquots of the homogenized final sludge suspension were weighed, thereafter dried and the dry weight of the suspended solids was determined.
Based on this determination, calculated amounts of wet sludge were suspended in mineral medium to obtain a concentration equivalent to 4 g dry material per liter. During the holding period of one day prior to use, the sludge was aerated with CO2-free air at room temperature. Prior to use, the dry weight of the sludge was again determined and the sludge was diluted with mineral medium to a concentration of 1 g dry material per liter. Defined volumes of the diluted activated sludge were added to the mineral medium in the test vessels to obtain a final concentration of 4 mg dry material per liter.

Duration of test (contact time):

ca. 28 d

Initial conc.:

19.6 mg/L

Based on:

test mat.

Initial conc.:

17.3 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

inorg. C analysis

Details on study design:

MATERIALS AND METHODS
Test System
The test method and test system (inoculum) are consistent with the testing guidelines and should provide a rational basis to assess the ready biodegradability of the test item under aerobic conditions.

Mineral Medium
The mineral medium was prepared according to the testing guidelines and is considered not to contain any contaminant that would affect the integrity or outcome of the study.
The purified water used in the test was ultrapure water obtained from an ELGA PURELAB Option-Q water purification system.
Analytical grade salts were dissolved in ultrapure water to obtain the following stock solutions:
1) KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4 × 2H2O 33.40 g/L
NH4Cl 0.50 g/L
The pH of this solution was 7.4.
2) CaCl2 × 2H2O 36.40 g/L
3) MgSO4 × 7H2O 22.50 g/L
4) FeCl3 × 6H2O 0.25 g/L, stabilized with one drop of concentrated HCl per liter.
To obtain 1 liter of the final mineral medium, 10 mL of stock solution No. 1 and 1 mL each of
stock solutions Nos. 2, 3 and 4 were added to approximately 800 mL ultrapure water, mixed
and made up to 1000 mL with ultrapure water. The pH was adjusted from 7.6 to 7.4 with a
diluted hydrochloric acid solution.

Materials
Test vessels: The test was performed in “500 mL” Wheaton glass serum bottles, with a total volume of around 536 mL, sealed with aluminium crimp caps with Teflon lined chlorobutyl septa.
The test vessels were labeled with the study number and all necessary information to ensure unique identification.
Shaker The sealed bottles were placed on an orbital shaker, with a shaking rate of 150 rpm, sufficient to keep the bottle contents well mixed and in suspension. The shaker was placed in a temperature-controlled environment.
IC analyzer: IC analyses were performed using a TOC infrared gas analyzer equipped with an automatic sampler (i.e. vario TOC cube from Elementar Analysensysteme GmbH, Langenselbold, Germany)

Experimental Conditions
Test temperature: The test was conducted at 20 - 21°C, in a temperature-controlled environment. The temperature in an additional bottle, filled with water and incubated under the same conditions as the test bottles was continuosly recorded throughout the study. The incubator temperature was as well continuously recorded.
Shaking: The sealed bottles were placed on an orbital shaker, with a shaking rate of 150 rpm, sufficient to keep the bottle contents well mixed and in suspension. The shaker was placed in a temperature-controlled environment.
Light conditions: The bottles were incubated in the dark.
Test duration: 28 days.

Study Design
1. Number of Bottles
The following number of bottles was prepared for each series (including bottles on reserve):
a) 34 bottles for the test item biodegradation, containing the test item in inoculated mineral medium,
b) 34 bottles for the inoculum control, containing inoculated mineral medium alone (blank),
c) 20 bottles for the procedure control, containing the reference item in inoculated mineral medium,
d) 15 bottles for the toxicity control, containing both, the test item and the reference item in inoculated mineral medium.
The number of bottles prepared was sufficient for each series to allow at least triplicate bottles to be measured on each sampling occasion. For the last sampling, five bottles (instead of triplicate bottles) from sets a), b) and c) were analyzed to enable 95 % confidence intervals to be calculated for the mean percentage biodegradation value.
2. Preparation of the Bottles
Immediately before use, sufficient inoculated mineral medium was prepared by adding diluted activated sludge (see Section 5.1.2) to mineral medium. Aliquots of inoculated medium (well mixed) were dispensed into replicate bottles to give a headspace to liquid ratio of 1:4.1 (i.e.430 mL medium into 536mL-capacity bottles).
The test item (9 μL) was injected into sealed bottles containing the inoculated mineral medium using a high precision syringe.
The blank bottles were treated in a similar fashion, but excluding the test item.
The reference item 1-octanol (12 μL) was injected into sealed bottles containing the inoculated mineral medium using a high precision syringe.
In the toxicity control bottles both, test item (9 μL) and reference item (12 μL), were injected into sealed bottles containing the inoculated mineral medium using a high precision syringe.
All bottles were sealed with Teflon-lined chlorobutyl septa and aluminum caps and incubated in the dark at 20 - 21°C on a rotary shaker.

Test Concentrations
The following test item and reference item concentrations (rounded values) were applied:
Test item: 19.6 mg/L or 17.3 mg/L or 9 µL/bottle of test item
Procedure control:23.1 mg/L or 17 mg/L or 12 µL/bottle of reference item
Inoculum control:/
Toxicity control: 19.6 mg/L or 17.3 mg/L or 9 µg/L of test item and 23.1 mg/L or 17 mg/L or 12 µL/bottle of reference item
These target concentrations were calculated taking into consideration the following:
- The total organic carbon (TOC) content of the test item of 88.16 %, calculated based on the information (molecular formula and purity) provided by the Sponsor.
- The TOC content of the reference item of 73.8 %.
- The relative density of the test item (i.e. 0.935 g/cm3) and of the reference item (i.e.0.827 g/cm3), the volume of inoculated medium in the test bottle and the application method.

Sampling
Bottles were sacrificed for analysis on the following sampling days:
Test item and inoculum control: Exposure Day 0, 3, 5, 7, 10, 14, 21 and 28, Procedure control: Exposure Day 0, 3, 7, 14 and 28,
Toxicity control: Exposure Day 0, 7, 14 and 28.
Triplicate bottles were measured on each sampling occasion throughout the test. Five bottles from each series (except the toxicity control) were analyzed at the end of the test, on Day 28, to enable 95 % confidence intervals to be calculated for the mean percentage biodegradation value.
In addition, the pH was measured in a test item and an inoculum control bottle (which have not been made alkaline) at the start and end of incubation.

DATA EVALUATION
1. Percentage Biodegradation
The theoretical maximum IC production (ThIC) equals the amount of total organic carbon (TOC) from the test substance added to each bottle at the start of the test assuming 100 % mineralization of the test item into CO2.
The total mass [mg] of inorganic carbon (TIC) in each bottle is calculated from:
TIC = mg IC in the liquid + mg IC in the headspace
= (VL x CL) + (VH x CH)
where: VL = volume of liquid in the bottle [liter],
CL = concentration of IC in the liquid [mg C/L],
VH = volume of the headspace [liter],
CH = concentration of IC in the headspace [mg C/L].
By addition of alkali and equilibration, the concentration of IC in the headspace is negligible (VH x CH = 0). Hence only the concentration of IC in the liquid phase needs to be determined and:
TIC = (VL x CL)
The amount of IC produced must be corrected for endogenous IC production and is, therefore, determined by the difference in IC production between the test and inoculum control bottles.
The percentage biodegradation based on IC production is calculated from:
%biodegradation = ((TICt - TICb)/ TOC) × 100
where: TICt = mg TIC in test bottle at time t,
TICb = mean mg TIC in inoculum control (blank) bottles at time t,
TOC = mg TOC added to test bottle at the start of the test.
For the toxicity control the percent biodegradation is based on the theoretical IC yield anticipated from only the reference component of the mixture, according to the guidelines.
2. Ready Biodegradability
Test items giving a result of ≥ 60 % mean degradation based on CO2 evolution (within 28 days) are regarded as readily biodegradable. This level must be reached within 10 days of biodegradation exceeding 10 %.

Reference substance:

other: 1-octanol

Key result

Parameter:

% degradation (inorg. C analysis)

Value:

-4.1

St. dev.:

2.1

Sampling time:

28 d

Details on results:

The percent biodegradation of the test item was calculated based on a total organic carbon content (TOC) of 0.882 mg C/mg test item.
The inorganic carbon (IC) production in the test vessels containing the test item THDCPD was in the range of or slightly below the IC production of the inoculum control vessels during the whole test period. At the end of the test, on Exposure Day 28, the mean percentage biodegradation of the five test item bottles sampled was calculated to be -4.1 % with a 95 % confidence interval of ± 2.1 %.
In conclusion, THDCPD was found to be not biodegradable under the test conditions within 28 days.

Results with reference substance:

The percent biodegradation of the reference item 1-octanol was calculated based on a total organic carbon content (TOC) of 0.738 mg C/mg.
In the procedure control, the reference item was degraded by an average of 69 % and 72 % by Exposure Day 7 and 14, respectively, thus confirming suitability of the activated sludge (> 60 % degradation by Exposure Day 14). At the end of the 28-day exposure period, the mean percentage biodegradation of the five procedure control bottles sampled was 85 % with a 95 % confidence interval of ± 1.4 %.

Biodegradation in the Toxicity Control

The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the total organic carbon content (TOC) of the reference item in the mixture, only, as requested by the guidelines.

The mean percent biodegradation in the toxicity control was 72 % and 78 % by Exposure Day 14 and at the end of the 28-day exposure period, respectively.

To assess if the test item may had inhibitory effect on the activity of the inoculum the following calculation formula was applied for the Day 28:

(% degradation reference item − %degradation toxicity control)x100/ %degradation reference item

The resulting value was 8 %, which is lower than 25 %. Thus, according to the test guidelines, the test item THDCPD had no inhibitory effect on activated sludge microorganisms at the tested

concentration of 19.6 mg/L.

Total Inorganic Carbon in the Inoculum Control

The mean concentration of total inorganic carbon (TIC) in the inoculum control on Day 28 was 2.8 mg C/L. Therefore, the test is valid because the mean concentration of TIC in the inoculum

control at the end of the test was < 3 mg C/L.

pH Measurement

The pH was measured in a test item and an inoculum control bottle (which have not been made alkaline) at the start of the test and was found to be 7.4 for both. The measurements at the end of incubation on Day 28 resulted in a pH value of 7.3 for both.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item THDCPD was found to not biodegradable after 28 days of exposure to activated sludge under the conditions of the conducted CO2 in Sealed Vessels Test (Headspace Test).
This valid test was performed according to the OECD Guideline for Testing of Chemicals, No. 310 (2014) and the Method C.29 of Commission Regulation (EU) No 260/2014.

Executive summary:

The test item THDCPD was investigated for its ready biodegradability in a Headspace Test (CO2 in sealed vessels) over 28 days according to the OECD Guideline for Testing of Chemicals, No. 310 (2014) and the Method C.29 of Commission Regulation (EU) No 260/2014.

The inorganic carbon (IC) production in the test vessels containing the test item THDCPD was in the range of or slightly below the IC production of the inoculum control vessels during the whole test period. At the end of the test, on Exposure Day 28, the mean percentage biodegradation of the five test item bottles sampled was calculated to be -4.1 % with a 95 % confidence interval of ± 2.1 %.

In conclusion, THDCPD was found to be not biodegradable under the test conditions within 28 days.

In the procedure control, the reference item 1-octanol was degraded by an average of 69 % and 72 % by Exposure Day 7 and 14, respectively, thus confirming suitability of the activated sludge (> 60 % degradation by Exposure Day 14). At the end of the 28-day exposure period, the mean percentage biodegradation of the five procedure control bottles sampled was 85 % with a 95 % confidence interval of ± 1.4 %.

In the toxicity control, containing both the test item and the reference item, no inhibitory effect on the biodegradation of the reference item was determined. Thus, THDCPD had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 19.6 mg/L.

The validity criteria of the test were met.

Description of key information

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

9% biodegradation after 28 days and 60 days (inoculum: activated sludge)

Under the test conditions, the percentage biodegradation of the test item did not reach 60% in 14 day-window, so the test item can be considered as not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

Two reliable (K1) studies are available to assess the biodegradation of tetrahydrodicyclopentadiene (THDCPD):

- An OECD 301 D test (Key study) was performed to assess the ready biodegradability of the registered substance in an aerobic aqueous medium. The registered substance, at a concentration of 1.0 mg/L, was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 20 and 21 °C for 60 days. At the request of the Sponsor the study was extended from 28 to 60 days.

Following the recommendations of the International Standards Organisation (ISO 10634, (1995)), the registered susbtance was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in inoculated mineral media. Using this method allows extremely small amounts of substance to be added to each bottle, the registered substance is evenly distributed throughout the test medium and the surface area of substance exposed to the test organisms is increased thereby increasing the potential for biodegradation. The degradation of the registered substance was assessed by the determination of the amount of oxygen consumed. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

The registered substance attained 9% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301D.

Extension of the test from 28 days to 60 days did not result in any increase in biodegration.

- Headspace Test (CO2 in sealed vessels) over 28 days according to the OECD Guideline for Testing of Chemicals, No. 310 (2014) and the Method C.29 of Commission Regulation (EU) No 260/2014 was perfomed to assess the ready biodegradability of tetrahydrodicyclopentadiene (supporting study). The inorganic carbon (IC) production in the test vessels containing the test item THDCPD was in the range of or slightly below the IC production of the inoculum control vessels during the whole test period. At the end of the test, on Exposure Day 28, the mean percentage biodegradation of the five test item bottles sampled was calculated to be -4.1 % with a 95 % confidence interval of ± 2.1 %. In conclusion, THDCPD was found to be not biodegradable under the test conditions within 28 days.

In the procedure control, the reference item 1-octanol was degraded by an average of 69 % and 72 % by Exposure Day 7 and 14, respectively, thus confirming suitability of the activated sludge (> 60 % degradation by Exposure Day 14). At the end of the 28-day exposure period, the mean percentage biodegradation of the five procedure control bottles sampled was 85 % with a 95 % confidence interval of ± 1.4 %. In the toxicity control, containing both the test item and the reference item, no inhibitory effect on the biodegradation of the reference item was determined. Thus, THDCPD had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 19.6 mg/L. The validity criteria of the test were met.

As a conclusion, tetrahydrodicyclopentadiene is not considered as readily biodegradable based on results of both OECD tests (301 D and 310, GLP).