3a,4,5,6,7,7a-hexahydro-5-methoxy-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:

11 March 1991 to 08 April 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

The inoculum used was 10 % by volume of activated sludge plant secondary effluent filtered through a Whatman filter paper (541) to remove coarse particulate matter. The level of dissolved inorganic carbon (DIC) was reduced by sparging the filtered effluent with nitrogen after prior adjustment of the pH to 6.5.

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

other: Nominal Carbon

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: mineral salts medium. The mineral salts medium was as recommended in the 1988 OECD Ring Test on the harmonisation or ready biodegradability tests with the following deviation: the ferric chloride stock solution contained 0.25 g not 0.2 g. The EDTA stock solution contained 0.4 g as recommended in the 1988 OECD Ring Test and not 0.5 g as specified in the draft Ecotoxicology SOP 158 01. The slight difference in composition of the mineral salts medium should have no effect on the results.
- The test material was added directly to the appropriate vessels with a high quality liquid delivery syringe. The volume added (1-2 μL) was negligible compared to the total volume of 100 mL in each vessel and was not taken into consideration when determining the results.
- Test temperature: 18 – 22°C. The sealed vessels were incubated at 20°C on a rotary shaker.

TEST SYSTEM
- Culturing apparatus: 160 mL vessels (hypovials) sealed with a butyl rubber septum and an aluminium crimp seal.
- The headspace in each vessel has a volume of 60 mL and, when filled with air, contains approximately 6 times the mass of oxygen required for the complete oxidation of the test material.
- Number of culture flasks/concentration: 4
- Measuring equipment: Ionics 555 Inorganic Carbon Analyser
- Test performed in closed vessels due to significant volatility of test substance: yes

SAMPLING
- Analysis of both the headspace gas and the liquid medium for CO2/DIC was performed on day numbers: 4, 8, 14, 22, 24, and 28 using an Ionics 555 Inorganic Carbon Analyser and the extent of biodegradation determined.

STATISTICAL METHODS:
Determination of Percentage Biodegradation
In determining the percentage biodegradation of a test (or reference) material it is necessary to account for any biodegradation of material in the control. Therefore the percentage biodegradation is given by:
[(TIC1 – TICc) / TC] x 100 %
where TICt = μg Total Inorganic Carbon in test bottle
TICc = μg Total Inorganic Carbon in control bottle
TC = μg organic carbon added to test vessel as test material
The Total Inorganic Carbon is the summation of the results of the analysis of the liquid and gas phases of each bottle after reference to the appropriate inorganic carbon standard curve.

On the final day of the study more than one vessel is analysed for both the test material and the control. In this case TICc is the arithmetic mean mass of inorganic carbon in the control vessels. The percentage biodegradation is calculated for each individual test vessel and the mean percentage biodegradation and standard deviation are determined and the results expressed as:
Mean % biodegradation ± (S.t / √n)
where
S is the standard deviation
t is the 't' value (two tailed test) at n-1 degrees of freedom at the 0.05 significance level
n is the number of results used to obtain the standard deviation

Reference substance:

not specified

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0.3

Sampling time:

28 d

Details on results:

- The air temperature during the 28 day test period was in the range of 18 – 22 °C.
- The test material failed the test and therefore cannot be classed as being readily and ultimately biodegradable.

Table 1: Percentage Biodegradation of the Test Material

Day No.	% Biodegradation
4	-2.7
8	-1.3
14	3.5
22	-4.0
24	-1.3
28	0.3
95 % confidence interval	-8.0 – 8.7

 

Validity criteria fulfilled:

not specified

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of this study, the test material failed the test and therefore cannot be classed as being readily and ultimately biodegradable.

Executive summary:

The ultimate biodegradability of the test material was investigated in a study conducted largely in accordance with the standardised guideline OECD 301B, under GLP conditions.

The test used was the sealed vessel test which is suitable for determining the ready and ultimate biodegradability of organic substances. The test was conducted in 160 mL vessels (hypovials) containing 100 mL mineral salts medium inoculated with secondary effluent and the respective test or reference substance. Multiple vessels were prepared sealed with a butyl rubber septum and an aluminium crimp seal. The headspace in each vessel had a volume of 60 mL and, when filled with air, contains approximately 6 times the mass of oxygen required for the complete oxidation of the substance. The sealed vessels were incubated at 20°C on a rotary shaker. Analysis of both the headspace gas and the liquid medium for CO2/DIC was performed on day numbers: 4, 8, 14, 22, 24, and 28 using an Ionics 555 Inorganic Carbon Analyser and the extent of biodegradation determined.

After 28 days 0.3 % biodegradation of the test material was recorded.

Under the conditions of this study, the test material failed the test and therefore cannot be classed as being readily and ultimately biodegradable.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 August 1997 to 18 November 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 A (Inherent Biodegradability: Modified SCAS Test)

Version / remarks:

1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.12 (Biodegradation: Modified SCAS Test)

Version / remarks:

1988

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source: Environment Centre Pilot Plant
- The inoculum used was acclimatised (unit 12) effluent from the SCAS test, filtered through a Whatman filter paper (541) to remove coarse particulate matter.
- The level of dissolved inorganic carbon (DIC) was reduced by sparging the filtered effluent with nitrogen after prior adjustment of the pH to 6.5.

Duration of test (contact time):

28 d

Initial conc.:

10.1 mg/L

Based on:

test mat.

Remarks:

SCAS Test

Initial conc.:

12.1 mg/L

Based on:

other: Carbon in Sealed Vessel Test

Initial conc.:

15.2 mg/L

Based on:

test mat.

Remarks:

Sealed Vessel Test

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST METHODS

SCAS TEST
The test method used to acclimatise microorganisms to the test material was the modified Semi-Continuous Activated Sludge (SCAS) test which is one of the OECD Test Guidelines for assessing the inherent biodegradability of organic chemicals under aerobic conditions. The test has been designed to determine inherent biodegradability and those materials that fail the test (< 70% removal) are unlikely to be efficiently biodegraded in conventional sewage treatment processes.
The test material was added to activated sludge in an SCAS aeration unit and aerated for approximately 23 hours; the aeration was then stopped, the mixture was settled and the supernatant liquor removed. Settled domestic sewage and test material were then added to the settled sludge and the aeration re-started. The cycle was repeated for up to 3 months during which time conditions favourable for the acclimatisation of the micro-organisms to the test material were ensured.

SEALED VESSEL TEST
The test used was based on the sealed vessel test methodology which was adapted for determining the inherent biodegradability of volatile organic substances. The sealed vessel test was conducted in 160 mL vessels (hypovials) containing 100 mL medium and up to 20 – 30 mg/L as organic carbon of test or reference material. Multiple vessels were prepared and were sealed with a butyl rubber septum and an aluminium crimp seal. The headspace in each vessel has a volume of 60 mL and, when filled with air, contains approximately 6 times the mass of oxygen required for the complete oxidation of the test material.
The sealed vessels were incubated at 20°C on a rotary shaker and on day 28 the vessels were removed and the concentration of carbon dioxide in the headspace gas determined. The seal was then broken and the concentration of inorganic carbon in the test medium was also determined. The total inorganic carbon in the vessel was calculated and corrected by subtracting the total inorganic carbon produced in the control vessels. The control vessels were identical to the test vessels except for the omission of the test material. From a knowledge of the initial organic carbon concentration added as test material, the extent of mineralisation could be determined.


EXPERIMENTAL DESIGN

SCAS TEST
The test material was tested in the SCAS test at 10.1 mg/L (assuming 100% active material in sample provided) in SCAS unit number 12. The test material was added directly (10 μL) to the test unit using a Gilson auto pipette. The SCAS units were located in a constant temperature room controlled at 20°C.

SEALED VESSEL TEST
The study was conducted in accordance to the specified Ecotoxicology SOP 158 05 with the following deviations to the method. The inoculum used was acclimatised (unit 12) effluent from the above SCAS test, filtered through a Whatman filter paper (541) to remove coarse particulate matter. The level of dissolved inorganic carbon (DIC) was reduced by sparging the filtered effluent with nitrogen after prior adjustment of the pH to 6.5. This was used to inoculate the mineral salts media. The volume of the inoculated media dispensed to each vessel was 100 mL. The volume of test/reference material was considered as negligible, 1.5 μL of the test and reference materials were introduced to the appropriate vessels using a high precision liquid delivery syringe.


CALCULATION OF RESULTS

SCAS TEST
- Not applicable since the purpose of this phase of the study was to acclimatise microorganisms to the test material.

SEALED VESSEL TEST
- In determining the percentage biodegradation of a test (or reference) material it is necessary to account for any biodegradation of material in the control. Therefore the percentage biodegradation is given by: [(TICt - TICc)/TC] x 100 %
where
TICt= μg Total Inorganic Carbon in test bottle
TICc = μg Total Inorganic Carbon in control bottle
TC = μg organic carbon added to test vessel as test material
- The Total Inorganic Carbon is the summation of the results of the analysis of the liquid and gas phases of each bottle after reference to the appropriate inorganic carbon standard curve. On the final day of the study all the vessels are analysed for both the test and control. In this case TICc is the arithmetic mean mass of inorganic carbon in the control vessels. The percentage biodegradation is calculated for each individual test vessel and the mean percentage biodegradation and standard deviation are determined and the results expressed as:
Mean % biodegradation ± (S.t / √n) where S
Where
S is the standard deviation
t is the t’value (two tailed test at n-1 degrees of freedom at the 0.05 significance level)
n is the number of results used to obtain the standard deviation

Reference substance:

other: Benzyl alcohol

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0.3

Sampling time:

28 d

Remarks on result:

other: 95 % confidence limits -1.8 - 2.4 %

Details on results:

SCAS TEST
- The ambient temperature over the test period remained in the range 19.5 - 22.0°C.

SEALED VESSEL TEST
- The air temperature over the 28 day test period was in the range of 20.0 - 22.0°C. This temperature range lies inside the recommended range 18 - 22°C as quoted in the Ecotoxicology SOP 158 and the OECD guideline.

CONCLUSIONS
- Micro-organisms were pre-exposed to the test material using the modified SCAS test system. Due to the volatile characteristics of the test material the biodegradability of the test material could not be determined by conventional analysis. The pre-exposed micro-organisms were used to inoculate a sealed vessel test.
- The average extent of mineralisation of the test material in a sealed vessel test using an acclimatised inoculum was 0.3% which is insufficient to classify the material as inherently ultimately biodegradable.

Results with reference substance:

Benzyl alcohol was selected as a reference substance because it has previously been shown to be readily biodegradable in the Sealed Vessel Test. Benzyl alcohol achieved 103.5 % biodegradation after 28 days. Consequently the inoculum can be considered to be active.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of this study, the average extent of mineralisation of the test material in a sealed vessel test using an acclimatised inoculum was 0.3% which is insufficient to classify the material as inherently ultimately biodegradable.

Executive summary:

The inherent biodegradability of the test material was investigated under GLP conditions and in accordance with the standardised guidelines OECD 302A and EU Method C.12, and largely in accordance with OECD 301B.

During the study micro-organisms were pre-exposed to the test material using the modified SCAS test system. Due to the volatile characteristics of the test material the biodegradability of the test material could not be determined by conventional analysis. The pre-exposed micro-organisms were used to inoculate a sealed vessel test.

The temperatures over the test period for both test methods were in the range 19.5 - 22.0°C and therefore are acceptable.

Benzyl alcohol was selected as a reference substance because it has previously been shown to be readily biodegradable in the Sealed Vessel Test. Benzyl alcohol achieved 103.5% biodegradation after 28 days. Consequently the inoculum can be considered to be active.

Under the conditions of this study, the average extent of mineralisation of the test material in a sealed vessel test using an acclimatised inoculum was 0.3% which is insufficient to classify the material as inherently ultimately biodegradable.

Reference 3

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 January 1992 to 25 March 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

yes

Remarks:

See principles of method if other than guideline

Principles of method if other than guideline:

The sealed vessel test is a CO2 production test based on OECD Guideline 301 B and recent studies conducted by the OECD aimed at harmonising the various tests of ready biodegradability. The ready test guidelines specify a maximum test duration of 28 days. If the test duration is extended to 56 days then the test is no longer an assessment of ready and ultimate biodegradability but is indicative of inherent and ultimate biodegradability, albeit under modified ready test conditions. This version of the sealed vessel test is not a recognised OECD/EEC test of inherent biodegradability. However, the test system is closed and it is therefore suitable for the examination of volatile materials.

GLP compliance:

yes

Oxygen conditions:

anaerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source: Secondary effluent from an un-acclimatised activated sludge plant at URL North.
- The inoculum used was 10% by volume of activated sludge plant secondary effluent, filtered through a Whatman filter paper (541) to remove coarse particulate matter. The level of dissolved inorganic carbon (DIC) was reduced by sparging the filtered effluent with nitrogen after prior adjustment of the pH to 6.5.

Duration of test (contact time):

56 d

Initial conc.:

10 mg/L

Based on:

other: Nominal organic carbon

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: mineral salts medium. The mineral salts medium was as recommended in the 1988 OECD Ring Test on the harmonisation or ready biodegradability tests with the following deviation: The stock solution concentrations of FeCl3.6H2O and EDTA disodium salt were 0.25 and 0.5 g respectively, and not 0.2 and 0.4 g. The slight difference in composition of the mineral salts medium should have no effect on the results. In the preparation of the phosphate buffer anhydrous disodium orthophosphate was used instead of the dihydrate salt and the quantity used was adjusted accordingly.
- The test material was added directly to the appropriate vessels with a high quality liquid delivery syringe. The volume added (1-2 μL) was negligible compared to the total volume of 100 mL in each vessel and was not taken into consideration when determining the results.
- Test temperature: 14 – 22°C. The sealed vessels were incubated at 20°C on a rotary shaker.

TEST SYSTEM
- Culturing apparatus: 160 mL vessels (hypovials) sealed with a butyl rubber septum and an aluminium crimp seal.
- The headspace in each vessel had a volume of 60 mL and, when filled with air, contained approximately 6 times the mass of oxygen required for the complete oxidation of the test material.
- Number of culture flasks/concentration: 4
- Measuring equipment: Ionics 555 Inorganic Carbon Analyser
- Test performed in closed vessels due to significant volatility of test substance: yes

SAMPLING
- A single determination of the extent of biodegradation was made after 56 days.

STATISTICAL METHODS:
Determination of Percentage Biodegradation
In determining the percentage biodegradation of a test (or reference) material it is necessary to account for any biodegradation of material in the control. Therefore the percentage biodegradation is given by:
[(TIC1 – TICc) / TC] x 100 %
where TICt = μg Total Inorganic Carbon in test bottle
TICc = μg Total Inorganic Carbon in control bottle
TC = μg organic carbon added to test vessel as test material
The Total Inorganic Carbon is the summation of the results of the analysis of the liquid and gas phases of each bottle after reference to the appropriate inorganic carbon standard curve.

On the final day of the study more than one vessel is analysed for both the test material and the control. In this case TICc is the arithmetic mean mass of inorganic carbon in the control vessels. The percentage biodegradation is calculated for each individual test vessel and the mean percentage biodegradation and standard deviation are determined and the results expressed as:
Mean % biodegradation ± (S.t / √n)
where
S is the standard deviation
t is the 't' value (two tailed test) at n-1 degrees of freedom at the 0.05 significance level
n is the number of results used to obtain the standard deviation

Reference substance:

not specified

Key result

Parameter:

% degradation (CO2 evolution)

Value:

-4.2

Sampling time:

56 d

Remarks on result:

other: 95 % confidence limits: -9.0 - 0.6 %

Details on results:

- The air temperature during the 56 day test period was in the range of 14 – 22°C.
- The test material exhibited little or no biodegradation under the modified ready test conditions.

Validity criteria fulfilled:

not specified

Interpretation of results:

not readily biodegradable

Conclusions:

The test material exhibited little or no biodegradation under the modified ready test conditions.

Executive summary:

The biodegradability of the test material was investigated largely in accordance with the standardised guideline OECD 301 B, under GLP conditions.

The test used was based on the sealed vessel test which is suitable for determining the ready and ultimate biodegradability of organic substances. However, by extending the test duration to 56 days from 28 days it is possible to obtain an indication of the inherent biodegradability of a substance. The test was conducted in 160 mL vessels (hypovials) containing 100 mL mineral salts medium inoculated with secondary effluent and the respective test or reference material. Multiple vessels were prepared sealed with a butyl rubber septum and an aluminium crimp seal. The headspace in each vessel has a volume of 60 mL and, when filled with air, contains approximately 6 times the mass of oxygen required for the complete oxidation of the substance. The sealed vessels were incubated at 20°C on a rotary shaker and a single determination of the extent of biodegradation was made after 56 days.

The air temperature during the 56 day test period was in the range of 14 – 22°C. The biodegradation of the test material after 56 days was -4.2%.

The test material exhibited little or no biodegradation under the modified ready test conditions.

Description of key information

King (1996)

Under the conditions of this study, the test material failed the test and therefore cannot be classed as being readily and ultimately biodegradable.

Gore (1998)

Under the conditions of this study, the average extent of mineralisation of the test material in a sealed vessel test using an acclimatised inoculum was 0.3% which is insufficient to classify the material as inherently ultimately biodegradable.

King (1993)

The test material exhibited little or no biodegradation under the modified ready test conditions.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

King (1996)

The ultimate biodegradability of the test material was investigated in a study conducted largely in accordance with the standardised guideline OECD 301B, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test used was the sealed vessel test which is suitable for determining the ready and ultimate biodegradability of organic substances. The test was conducted in 160 mL vessels (hypovials) containing 100 mL mineral salts medium inoculated with secondary effluent and the respective test or reference substance. Multiple vessels were prepared sealed with a butyl rubber septum and an aluminium crimp seal. The headspace in each vessel had a volume of 60 mL and, when filled with air, contains approximately 6 times the mass of oxygen required for the complete oxidation of the substance. The sealed vessels were incubated at 20°C on a rotary shaker. Analysis of both the headspace gas and the liquid medium for CO2/DIC was performed on day numbers: 4, 8, 14, 22, 24, and 28 using an Ionics 555 Inorganic Carbon Analyser and the extent of biodegradation determined.

After 28 days 0.3 % biodegradation of the test material was recorded.

Under the conditions of this study, the test material failed the test and therefore cannot be classed as being readily and ultimately biodegradable.

Gore (1998)

The inherent biodegradability of the test material was investigated under GLP conditions and in accordance with the standardised guidelines OECD 302A and EU Method C.12, and largely in accordance with OECD 301B. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study micro-organisms were pre-exposed to the test material using the modified SCAS test system. Due to the volatile characteristics of the test material the biodegradability of the test material could not be determined by conventional analysis. The pre-exposed micro-organisms were used to inoculate a sealed vessel test.

The temperatures over the test period for both test methods were in the range 19.5 - 22.0°C and therefore are acceptable.

Benzyl alcohol was selected as a reference substance because it has previously been shown to be readily biodegradable in the Sealed Vessel Test. Benzyl alcohol achieved 103.5% biodegradation after 28 days. Consequently the inoculum can be considered to be active.

Under the conditions of this study, the average extent of mineralisation of the test material in a sealed vessel test using an acclimatised inoculum was 0.3% which is insufficient to classify the material as inherently ultimately biodegradable.

King (1993)

The biodegradability of the test material was investigated largely in accordance with the standardised guideline OECD 301 B, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test used was based on the sealed vessel test which is suitable for determining the ready and ultimate biodegradability of organic substances. However, by extending the test duration to 56 days from 28 days it is possible to obtain an indication of the inherent biodegradability of a substance. The test was conducted in 160 mL vessels (hypovials) containing 100 mL mineral salts medium inoculated with secondary effluent and the respective test or reference material. Multiple vessels were prepared sealed with a butyl rubber septum and an aluminium crimp seal. The headspace in each vessel has a volume of 60 mL and, when filled with air, contains approximately 6 times the mass of oxygen required for the complete oxidation of the substance. The sealed vessels were incubated at 20°C on a rotary shaker and a single determination of the extent of biodegradation was made after 56 days.

The air temperature during the 56 day test period was in the range of 14 – 22°C. The biodegradation of the test material after 56 days was -4.2%.

The test material exhibited little or no biodegradation under the modified ready test conditions.