3-(4,4-dimethylcyclohex-1-en-1-yl)propanal

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:

24-01-2007 to 18-06-2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Guideline study performed not performed under GLP. Study well documented. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

Remarks:

The study was conducted prior to 01 June 2008. The study is well documented and/or the regulatory conclusion, thus does not require positive “GLP” status.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms (Test System) was obtained from the domestic waste sewage treatment plant at Rossdorf (Germany). The plant treats predominantly domestic sewage.
- Laboratory culture: See source of inoculum/activated sludge.
- Method of cultivation: The sample of activated sewage sludge was maintained on continuous aeration upon receipt.
- Storage conditions: See pretreatment field.
- Storage length: typically < 1 week
- Preparation of inoculum for exposure: See pre-treatment field.
- Pretreatment: The sample of activated sewage sludge was maintained on continuous aeration upon receipt. The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and again centrifuged. This procedure was repeated twice. An aliquot of the fmal sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 1.5 g dry material per litre were mixed with test water and then aerated over night until use. Determination of dry weight is made to inoculate final solution with 30mg/L dry weight activated sludge.
- Concentration of sludge: The sludge was diluted in the BOD bottles to 30 mg DW/L.
- Initial cell/biomass concentration: Not applicable.
- Water filtered: Not applicable. Culture medium was in accordance with OECD TG 301F.
- Type and size of filter used, if any: Not applicable.

Duration of test (contact time):

28 d

Initial conc.:

ca. 49.5 mg/L

Based on:

test mat.

Remarks:

Test item (test flasks 1, 2 and 3)

Initial conc.:

ca. 26.23 mg/L

Based on:

test mat.

Remarks:

Test item (test flasks 4 and 5)

Initial conc.:

ca. 10.66 mg/L

Based on:

test mat.

Remarks:

Test item (test flasks 6, 7 and 8)

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: See table 1. 1000 mL of Solution A [KH2PO4: 8.50 g/L; K2HPO4: 21.75g/L; Na2HPO4,2H2O: 33.40 g/L; NH4Cl: 0.50 g/L]; 1000 mL of Solution B [MgSO4,7H2O: 22.50 g/L ]; 1000 mL of Solution C [CaCl2,2H2O: 36.40 g/L]; 1000 mL of Solution D [FeCl3,6H2O: 0.25 g/L] each diluted in purified water to create stock solution. In order to prepare of the stock solution d) immediately before use, one drop of concentrated HCI per litre was added. 10 mL of stock solution a) and 1 mL of the stock solutions b) to d) were combined and filled to a final volume of 1000 mL with deionised water.
- Solubilising agent (type and concentration if used): None. Test item, reference item were directly weighed and dispersed into vessels.
- Test temperature: 22 ±1 °C
- pH: See table 1.
- pH adjusted: No. (final pH was in the range of 7.5 to 7.60 for test item vessels and 7.6 for inoculum control, 7.8 for procedure control, 7.6 for abiotic control and 7.7 for toxicity control)
- CEC (meq/100 g): Not reported.
- Aeration of dilution water: Not reported
- Suspended solids concentration: 30 mg/L dry weight
- Continuous darkness: Yes. The test was conducted in the dark.

TEST SYSTEM
- Culturing apparatus: 500mL glass flasks with continuous stirring (fill volume ca. 244 mL); The test item may adsorb at glass surfaces. To prevent any adsorption, the test flasks used in the study were silanized by applying “SyIon CT” to the test vessels. About 30 mL of Sylon CT were filled into the test vessel and vigorous shaken for about 15 seconds. The silanization agent was discarded and the vessels were rinsed twice with toluol. The remaining toluol was removed by rinsing the vessels twice with methanol. The methanol was discarded and the remaining amounts were allowed to evaporate for two days. Test vessel no. 3 was not silanized to function as control. Test vessel no. 8 was not silanized.
- Number of culture flasks/concentration: at least duplicate per concentration (test item); In duplicate (Inoculum blank control and Toxicity control); single flasks (Abiotic sterile control and Procedure control)
- Method used to create aerobic conditions: Sealed flasks with sensor head/CO2 trap (45% KOH solution)
- Method used to create anaerobic conditions: Not applicable.
- Measuring equipment: The respirometer/manometric system used during this study is an BSB/BOD Sensor System (Aqualytic, Germany). Evolved carbon dioxide is absorbed by the CO2 trap (45% KOH solution)
- Test performed in closed vessels due to significant volatility of test substance: Not reported.
- Test performed in open system: No.
- Details of trap for CO2 and volatile organics if used: See above.

SAMPLING
- Sampling frequency: Daily.
- Sampling method: The respirometer/manometric system used during this study is an BSB/BOD Sensor System (Aqualytic, Germany).
- Sterility check if applicable: Not reported.
- Sample storage before analysis: Not applicable.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes. See table 1.
- Abiotic sterile control: Yes.
- Toxicity control: Yes.
- Other: Positive reference control (Sodium Benzoate).

Reference substance:

benzoic acid, sodium salt

Remarks:

102.8 mg/L

Test performance:

1. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled for the flasks containing test item at end of 10-day window (as applicable) and on day 28. Therefore, the test is considered valid.
2. The BOD of the inoculated blank control was 30 mgO2/L and < 60 mgO2/L after 28 days
3. The pH at day 28 was in the range of 6.0 to 8.5 (actual 7.5 to 7.8 for test item vessels and controls)
4. The toxicity test attained 49% degradation at 14 days and 69% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment microorganisms used in the study.
5. Sodium Benzoate attained 96% degradation at 7 days thereby confirming the suitability of the inoculum and test conditions.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

mean (n=2) / test concentration ca. 50 mg/L test item

Value:

62.5

Sampling time:

28 d

Remarks on result:

other: test flasks 1 and 2 ; 10-day window not met ; mean biodegradation day 28 = 62.5%

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

test concentration ca. 50 mg/L test item

Value:

>= 62 - <= 63

Sampling time:

28 d

Remarks on result:

other: test flasks 1 and 2 ; 10-day window not met ; mean biodegradation day 28 = 62.5%

Parameter:

% degradation (O2 consumption)

Remarks:

mean (n=2) / test concentration ca. 11 mg/L test item

Value:

77

Sampling time:

28 d

Remarks on result:

other: test flasks 6 and 7 ; 10-day window not met ; mean biodegradation = 77%

Parameter:

% degradation (O2 consumption)

Remarks:

test concentration ca. 11 mg/L test item

Value:

>= 73 - <= 81

Sampling time:

28 d

Remarks on result:

other: test flasks 6 and 7 ; 10-day window not met ; mean biodegradation = 77%

Details on results:

(i) In the abiotic control the Oxygen Demand was 0 mgO2/L.
(ii) In the inoculum blank controls the oxygen demand was 30 mgO2/L and therefore less than 60 mgO2/L satisfying the requirements of the guideline.
(IIi) In the procedure control: sodium Benzoate attained 96% degradation after 7 days thereby confirming the suitability of the inoculum and test conditions.
(iv) In the two toxicity controls (both concentrations used ; including ca. 50 mg/L test item). Each control contained a different concentration of the test item and the same concentration of the reference item (sodium benzoate). At the highest test item concentration of ca. 52 mg/L, the degradation was 49% after 14 days and 60% after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge microorganisms because degradation was >25% within 14 days. Additionally, within the lower test item concentration of ca. 26 mg/L, the degradation was 69% after 14 days and 81% after 28 days of incubation.
(v) In all the test, reference item and controls the pH was in the range of 7.5 to 7.8 (thereby was in the range of 6.0 to 8.5 required by the guideline)
(vi) The difference of duplicate values for the degradation of the test item at the plateau, at the end of the test and at the end of the 10-day window should be less than 20 %. The validity criterion was met for the period of plateau phase.
(vi) In the test vessels; at the lowest test concentration of approx. 11 mg/L a degradation of 73% and 81 % after 28 days was found for the two replicates, respectively. The higher test concentrations of 26 mg/L and 50 mg/L reached a mean degradation of 43% and 63% after 28 days, respectively. Even if there was no clear dose response, the highest degradation rate was found at the lowest concentration.
(vii) The highest test item concentration was approx. 50 mg/L based on ThOD. This concentration corresponds to the lowest test concentration required by the OECD 301F test guideline. The maximum degradation at this concentration was 63% but the 10-day window criterion was not fulfilled.
(viii) There was no biodegradation found in the unsilanized vessels.
(ix) The mean test item biodegradation conducted in duplicate, was 62.5% at day 28. The 10-day window criteria was not met. The test item is readily biodegradable, but failing 10-day window.

Results with reference substance:

Sodium Benzoate attained 96% degradation after 7 days thereby confirming the suitability of the inoculum and test conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

The mean biodegradation was 62.5% at day 28 conducted in duplicate. The 10-day window criteria was not met. The test item is readily biodegradable, but failing 10-day window.

Executive summary:

The ready biodegradability test was carried out according to OECD TG 301F guideline under GLP. The test item, at a concentration of ca. 50 mg/L, ca. 26 mg/L and ca. 11 mg/L, respectively was exposed to activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment which treats predominantly domestic sewage (Rossdorf, Germany) with culture medium in sealed culture vessels in the dark at 22°C ± 1°C for 28 days. The sludge was diluted in the BOD bottles to 30 mg DW/L. The degradation of the test item was assessed by the measurement of daily oxygen consumption on days 0 and 28 using a respirometer/manometric system which utilised a BSB/BOD Sensor (Aqualytic, Germany). Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control and/or an abiotic control were used for validation purposes. In the test inoculum blank the oxygen uptake was ca. 30 mg O2/L. The pH value at the end of the test period 28 days did not exceed 7.6 in the test item systems and 7.8 in the reference item system. At the highest test item concentration of ca. 52 mg/L in the toxicity control, the degradation was 49% after 14 days and 60% after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge microorganisms because degradation was >25% within 14 days. The test system met the validation criteria of the guideline. Sodium Benzoate attained 96% degradation after 7 days thereby confirming the suitability of the inoculum and test conditions. In the test vessels; at the lowest test concentration of approx. 11 mg/L a degradation of 73% and 81 % after 28 days was found for the two replicates, respectively. The higher test concentrations of 26 mg/L and 50 mg/L reached a mean degradation of 43% and 63% after 28 days, respectively. Even if there was no clear dose response, the highest degradation rate was found at the lowest concentration. The highest test item concentration was approx. 50 mg/L based on ThOD. This concentration corresponds to the lowest test concentration required by the OECD 301F test guideline. The maximum degradation at this concentration was 63% but the 10-day window criterion was not fulfilled. There was no biodegradation found in the unsilanized vessels. The mean test item biodegradation conducted in duplicate, was 62.5% at day 28. The 10-day window criteria was not met. The test item is readily biodegradable, but failing 10-day window.

Description of key information

Biodegradation: ‘not readily biodegradable’ ; specifically: test item was found to be ‘readily biodegradable but failing 10-day window’ with mean biodegradation 62.5% (28-days; 10-day window not met), OECD TG 301F, 2007

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable but failing 10-day window

Type of water:

freshwater

Additional information

Key study : OECD TG 301F, 2007 : The ready biodegradability test was carried out according to OECD TG 301F guideline under GLP. The test item, at a concentration of ca. 50 mg/L, ca. 26 mg/L and ca. 11 mg/L, respectively was exposed to activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment which treats predominantly domestic sewage (Rossdorf, Germany) with culture medium in sealed culture vessels in the dark at 22°C ± 1°C for 28 days. The sludge was diluted in the BOD bottles to 30 mg DW/L. The degradation of the test item was assessed by the measurement of daily oxygen consumption on days 0 and 28 using a respirometer/manometric system which utilised a BSB/BOD Sensor (Aqualytic, Germany). Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control and/or an abiotic control were used for validation purposes. In the test inoculum blank the oxygen uptake was ca. 30 mg O2/L. The pH value at the end of the test period 28 days did not exceed 7.6 in the test item systems and 7.8 in the reference item system. At the highest test item concentration of ca. 52 mg/L in the toxicity control, the degradation was 49% after 14 days and 60% after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge microorganisms because degradation was >25% within 14 days. The test system met the validation criteria of the guideline. Sodium Benzoate attained 96% degradation after 7 days thereby confirming the suitability of the inoculum and test conditions. In the test vessels; at the lowest test concentration of approx. 11 mg/L a degradation of 73% and 81 % after 28 days was found for the two replicates, respectively. The higher test concentrations of 26 mg/L and 50 mg/L reached a mean degradation of 43% and 63% after 28 days, respectively. Even if there was no clear dose response, the highest degradation rate was found at the lowest concentration. The highest test item concentration was approx. 50 mg/L based on ThOD. This concentration corresponds to the lowest test concentration required by the OECD 301F test guideline. The maximum degradation at this concentration was 63% but the 10-day window criterion was not fulfilled. There was no biodegradation found in the unsilanized vessels. The mean test item biodegradation conducted in duplicate, was 62.5% at day 28. The 10-day window criteria was not met. The test item is readily biodegradable, but failing 10-day window.