Reaction mass of 1,2,3,4,5,6,7,8-octahydro-5,5-dimethylnaphthalene-2-carbaldehyde and 1,2,3,4,5,6,7,8-octahydro-8,8-dimethyl-2-naphthaldehyde

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

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

Specific details on test material used for the study:

- Physical Description: Liquid
- Expiration Date: January 2012
- Storage Conditions: Room Temperature
- Carbon Content: 80.83% (as determined by elemental analysis)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Test Inoculum: Activated sludge was collected from the Cambridge Wastewater Treatment Facility, Cambridge, Maryland on August 19, 2011. The Cambridge facility treats predominantly residential wastes. The sludge was sieved using a 2-mm screen, the total suspended solids (TSS) were measured and then it was aerated at test temperature until its adjustment.
- Preconditioning of Inoculum: The activated sludge was diluted in test medium to approximately 30 mg total suspended solids/L and than aerated with CO2-free air for approximately one week until its final adjustment on Day 0 of the initial test.
- Inoculated Medium: The inoculated medium used in the initial test was prepared by diluting the preconditioned activated sludge in test medium to approximately 4 mg total suspended solids/L. The pH was measured and aliquots were removed and processed before dissolved organic (DOC) and dissolved inorganic (DIC) carbon measurements were conducted.

Duration of test (contact time):

60 d

Initial conc.:

20 mg/L

Based on:

IC (inorganic carbon)

Parameter followed for biodegradation estimation:

inorg. C analysis

Details on study design:

EXPERIMENTAL DESIGN
Two tests of biodegradability were conducted. The initial test was a standard ready biodegradability test conducted according to OECD 310 Guidelines. The second test was an enhanced biodegradation screening test, also based upon OECD 310 Guidelines. The main enhancement of the second test was its vessel preparation using inoculum initially prepared for the initial test. In preparation for its use in the second test, the inoculum was dosed with test substance and preconditioned for the length of the initial test. Each test contained one blank control, two reference, and one treatment group. The blank control group was used to measure the background IC production of the inoculated medium and was not dosed with a carbon source. One reference group was dosed with rapeseed oil, a substance known to be biodegradable, at a concentration of 20 mg carbon/L. The second reference group was dosed with 1-octanol, also known to be biodegradable, at a concentration of 20 mg C/L. The treatment group was dosed with the test substance at a concentration of 20 mg carbon/L. The initial test was conducted for 28 days. The second test was conducted for 60 days as a further enhancement. Measurements of biodegradability for both studies were conducted once a week.

MATERIALS AND METHODS
- Test Medium: The test medium was a mineral salts media and was prepared using high quality water (NANOpure®). All chemicals and reagents used in the preparation of the test medium were reagent grade or better. The constituents of the test medium are
not known to contain any contaminants that are reasonably expected to be present or known to be capable of interfering with the study.
- Test Apparatus and Conditions: The test chambers were glass serum bottles with a nominal volume of 160 mL. The chambers were sealed with Teflon septa and crimp caps. The test was conducted at 20±3ºC and the test temperature was measured every working day using a min/max thermometer. The pre-exposure vessel used to store inoculum for the second test was a 4 L Erlenmeyer flask sealed with an aluminum foil lined stopper and Parafilm® wrap and was identified by project number, test substance ID and test concentration. Based on the volume of inoculum needed for the second test, the 4L flask was selected to provide a headspace to liquid ratio equivalent to that used in the biodegradation test vessels. The test chambers for the initial and second tests were mixed at a rate sufficient to keep the bottle contents mixed and in suspension throughout the conduct of the tests using a shaker table. Test chambers were identified by project number, test substance ID, test concentration and replicate.
- Initial Biodegradation Test: Aliquots of inoculated test medium were dispensed into 160 mL replicate bottles to give a headspace to liquid ratio of approximately 1:2 (107 mL in a 160 mL bottle). The blank controls contained only inoculated medium and were not dosed with a carbon source. Reference group test chambers were dosed with sufficient amounts of the appropriate reference substance necessary to deliver 20 mg C/L. Treatment group test chambers were dosed with sufficient test substance necessary to deliver 20 mg C/L. A volume of inoculated test medium sufficient to conduct the second test and to give a headspace to liquid ratio of approximately 1:2 (e.g. 2.67 L in a 4 L vessel) was dispensed into a pre-exposure vessel. An amount of test substance necessary to deliver 20 mg C/L was added to the vessel. The pre-exposure vessel was sealed with an aluminum foil lined stopper and Parafilm® and a magnetic stir bar was employed to mix the contents for the duration of the initial test. The stirrer was cycled on and off approximately every 15 minutes to prevent heating of the stirrer motor.
- Second Biodegradation Test: At the end of the initial biodegradation test, the inoculated test medium for the second test was prepared by combining 200 milliliters of the pre-exposed inoculated test medium, prepared at the start of the initial test and incubated for its duration, per 800 milliliters of freshly prepared test medium and aerated with CO2-free air for approximately 30 minutes. The approximate concentration of total suspended solids in the final mixture was approximately 0.8 mg/L. Following aeration, the pH, dissolved organic (DOC) and inorganic carbon (DIC) levels of the mixture were measured. Aliquots of inoculated test medium were dispensed into 160 mL replicate bottles to give a headspace to liquid ratio of approximately 1:2 (107 mL in a 160 mL bottle). The blank controls contained only inoculated medium and were not dosed with a carbon source. Reference group test chambers were dosed with appropriate sufficient reference substance necessary to deliver 20 mg C/L. Treatment group test chambers were dosed with sufficient test substance necessary to deliver 20 mg C/L.

Reference substance:

other: Rapeseed Oil

Reference substance:

other: 1-Octanol

Key result

Parameter:

% degradation (inorg. C analysis)

Value:

-1.8

Sampling time:

28 d

Remarks on result:

other: Initial test

Parameter:

% degradation (inorg. C analysis)

Value:

1.4

Sampling time:

60 d

Remarks on result:

other: Second test

Details on results:

- The temperature range recorded during the study was 19.2 to 21.4ºC. The temperature range recorded during the storage of the inoculum before the study was 20.6 to 21.2ºC. The Initial TSS of the collected activated sludge was 5,313 mg/L. The inoculum used to prepare test chambers in the initial test had a pH of 7.5, a DOC concentration of 0.21 mg/L and a DIC concentration of 0.29 mg/L (both values extrapolated). The inoculum used to prepare test chambers in the second test had a pH of 7.5, a DOC concentration of 2.7 mg/L and a DIC concentration of 0.85 mg/L (extrapolated value).
- The blank control chambers evolved an average of 1.01 mg IC/L for Day 28 in the initial test. The blank control chambers evolved an average of 0.84 mg IC/L for Day 28 and 0.46 mg IC/L for Day 60 in the second test.The average cumulative percent biodegradation for the test substance at the end of the 28-day initial test was –1.8%. The average cumulative percent biodegradation for the test substane at the end of the 60-day second test was 1.4%. The test substance may not be considered readily biodegradable under aerobic conditions since the pass level of 60% ThIC was not achieved in the initial test.

Results with reference substance:

The viability of the inoculum and validity of the test were supported by the results of the reference substances, rapeseed oil and 1-octanol, from which an average of 75.4% and 67.0% of theoretical IC were evolved respectively after 28 days in the initial test. After 28 days in the second test, averages of 63.7% and 64.1% of theoretical IC were evolved respectively, and averages of 90.2% and 67.2% were evolved after 60 days.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

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)

Qualifier:

according to guideline

Guideline:

ISO 14593:1999 (Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test))

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Storage: at ambient temperature on the dark
- Exipration date: 03/2010

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

Secondary activated sludge was obtained from the WWTP Nieuwgraaf in Duiven, The Netherlands (08-06-2009). The WWTP Nieuwgraaf is an activated sludge plant treating predominantly domestic waste water. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated overnight with moist air. The sludge was diluted to a concentration of 4 mg DW/L in the test vessels.

Duration of test (contact time):

56 d

Initial conc.:

30 mg/L

Based on:

ThIC

Parameter followed for biodegradation estimation:

inorg. C analysis

Details on study design:

- Test bottles: The test was performed in 120 mL serum flasks with butyl septa and crimp-on aluminum seals (control and reference) or Mininert valves (test substance) seals. The volume of the liquid phase was 80 mL.
- Nutrient solution and stock solutions: The nutrient medium of the carbon dioxide headspace test contained per liter of deionized water: 85 mg KH2PO4, 217.5 mg K2HPO4, 334 mg Na2HPO4 2H2O, 5 mg NH4Cl, 22.5 mg MgSO4 7H20, 27.5 mg CaCl2, 0.25 mg FeCl3 6H20. 1-Octanol was added to the vessels using a stock solution in dichloromethane of 1.0 g/L. The test substance is a poorly soluble substance in water. The test substance was directly added to the vessels.
- Test procedures: The carbon dioxide headspace test was performed according to the study plan. The study plan was developed from ISO 14593 Test Guidelines (2005) and OECD 310 Test Guidelines (2006). Use was made of 36 vessels containing only inoculum, 36 vessels containing test substance and inoculum, and 9 vessels containing 1-octanol and inoculum. One additional bottle of each series was included to allow determination of the pH at day 28. Volumes of 3 µL of the test substance were added to the vessels. The amounts of test substance added were checked by weighing 10 times, 3 µL of test substance. 1-Octanol in dichloromethane was added directly to bottles. The solvent was subsequently allowed to evaporate in a ventilated hood for 24 hours (Nyholm and Seiero, 1989). The concentrations of the test substance and 1-octanol in the vessels were 37.0 and 21.9 mg/L, respectively. The inoculum was diluted to 4 mg DW/L in the test vessels. 80 mL of each of the prepared solutions was dispensed into the respective group of test vessels. The time zero vessels were analyzed for total inorganic carbon using the TOC apparatus upon adding 1 mL of a freshly prepared 7 M NaOH solution with 1 mL plastic syringes. The remaining vessels were incubated in the dark. Triplicate vessels of all series were withdrawn for analyses of the carbon dioxide formed at day 3, 7, 10, 14, 17, 21, 24, 28, and 56. The biological reactions in the test vessels were stopped by injecting 1 mL of a 7 M NaOH solution through the septum or Mininert valves. Before analysis of the inorganic carbon, the test vessels were shaken for at least one hour. Next, the particles in the vessels were allowed to settle and a suitable sample was withdrawn from the vessel with a syringe for analysis. The conversion of carbon dioxide to carbonate with sodium hydroxide should result in a negligible carbon dioxide concentration in the headspace. This was checked by adding 10 and 20 mg/L of carbonate-carbon to the mineral salts medium in the test vessels (in triplicate). A control without addition of carbonate was included. These test vessels were incubated at 20°C on a rotary shaker at 180 rpm for 24 hours. Next, sodium hydroxide (1 mL, 7 M) was added and the vessels were shaken for one hour. The inorganic carbon was measured in the alkaline medium using the TOC analyzer.

Reference substance:

other: 1-Octanol

Key result

Parameter:

% degradation (inorg. C analysis)

Value:

0

Sampling time:

28 d

Parameter:

% degradation (inorg. C analysis)

Value:

41

Sampling time:

56 d

Details on results:

- Theoretical organic carbon and chemical oxygen demand: The calculated organic carbon contents of the test substance and 1-octanol are 0.81 and 0.74 mg/mg, respectively. The mean weight of 3.0 µL test substance (weighed 10 times) was 2.96 mg with a standard deviation of 0.11 mg. The mean weight was used to calculate a density of 0.99 g/mL. The purity of the test substance was 98.8%. It was assumed that the other compounds in the sample have organic carbon contents comparable the test substance. Using the 0.99 mg/mL, the organic carbon content of the test substance and the working volume of 80 mL in the test vessels, an inorganic carbon concentration of 30.0 mg/L was calculated for the test substance. This concentration was used to calculate the biodegradation percentages of the test substance. The organic carbon concentration in the test vessels originating from 1-octanol was 16.2 mg/L.
- Toxicity: Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate, by the test compound in the carbon dioxide headspace test was not determined because inhibition of the endogenous respiration of the microorganisms is more sensitive. Inhibition of the endogenous respiration of the inoculum by the test substance was detected. Therefore, inhibition of the biodegradation due to the "high" initial concentration of the test substance cannot be excluded.
- Test conditions: The pH of the media was 7.4 at the start of the test. The pH of the medium at day 28 was 7.1 (test) and 7.2 (control). The temperature range recorded during the test was 19 to 21 0 C.
- Validity of the test: The mean carbonate-carbon concentrations and standard deviations in vessels containing the nutrient solution, nutrient solution and 10 mg/L carbonate-carbon and nutrient solution and 20 mg/L of inorganic carbon were 2.1±0.7, 14.0±0.3 and 24.1±1.1 mg/L, respectively. The carbon added was therefore recovered for 119% and 110% at 10 and 20 mg/L carbonate-carbon, respectively. This demonstrates that carbon dioxide was removed from the headspace adequately. The validity of the test is demonstrated by an increase of the inorganic carbon content in the blank controls of < 15% of the inorganic carbon added initially as test substance at day 28. The viability of the inoculum and validity of the test were supported by the results of the reference substance, 1-octanol from which 95% of the theoretical carbon dioxide was evolved at day 14.
- The carbon dioxide headspace test: The test substance was not biodegraded in the carbon dioxide headspace test after 28 days and should therefore not be classified as readily biodegradable. After 56 days 41% biodegradation was achieved demonstrating partial degradation of the test substance.

Results with reference substance:

77% degradation after 7 days and 95% after 14 days.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Specific details on test material used for the study:

- Description: Almost colourless to very pale yellow liquid
- Expiry date: 31/12/1996

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Sewage plant Hochdahl, predominantly municipal sewage serving for a population equivalent to approximately 27000

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

- Dilution water and stabilisation of inoculated medium: Deionised water was used for the preparation of the test medium. A sufficient amount of mineral test medium (according to OECD 301 tests) was inoculated with a mixed bacterial inoculum (30 mg suspended solids of activated sludge per litre) and distributed to the test bottles ca. 200 mL of liquid in bottles of ca. 300 ml volume). After addition of a magnetic stirrer the bottles were stoppered and stabilised under laboratory conditions for one week. After stabilisation, the bottles were aerated with compressed air by means of a sintered glass tube until O2 saturation was reached and then spiked with a predetermined amount of test chemical. The bottles were stoppered again and incubated on a shaker at a temperature of 20-25°C. Since the oxygen measurements must carried out at 20 ± 0.5°C, the temperature was adjusted accordingly half an hour before each measurement.
- Addition of test substance: Because of the adequate solubility a stock solution of Sodium acetate Trihydrate was prepared and 0.5 mL (=20 mg ThOD) were added to the stabilised inoculated medium (final concentration of reference substance : 100 mg ThOD/L). Due to the insufficient water solubility of the test substance, the test substance was weighed on a piece of glass to an amount of about 20 mg ThOD and put directly into the test flask (final concentration of test substance: 100 mg/L ThOD). The weighted amount of test substance added to the test flask was registered for each individual flask number. The homogeneous distribution of the test substance was supported by continuous shaking of the test flasks during the whole incubation period.
- Validity and evaluation criteria: The results are valid if: the percentage degradation of the reference substance has reached the level of 60 % ThOD within 14 days; the difference between the extreme degradation values found in the triplicates is less than 20 % at the end of the test; the total oxygen uptake in the blank bottles after the first week of the test is lower than 3 mg O2/L and in the following weeks lower than 1 mg O2/L per week. It may be assumed that a chemical yielding a positive result (>60% ThOD or BOD/COD within 28-days) in a BODIS test will be rapidly biodegraded in the environment. Such test substances can be classified as „readily degradable " according to the criteria to be taken into account for the classification of substances as „dangerous for the environment". A test substance is classified as „ moderately biodegradable" if a degradation result in the range of ≥10 - < 60% ThOD or BOD/COD is reached within the 28-day period of test. A test substance is classified as „poorly biodegradable under pertaining test conditions " if a degradation result < 10% ThOD or BOD/COD is obtained.

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

5

Sampling time:

28 d

Parameter:

COD

Value:

2 768 mg O2/g test mat.

Results with reference substance:

91% biodegradation in 28 days

Test substance	Test conc. (mg/L)	Calc. Basis	Day 7	Day 14	Day 21	Day 28
Sodium Acetate	100	ThOD	71	84	88	91
Test substance	100	COD	0	4	4	5

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Description of key information

The test substance is considered to be not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

Three ready biodegradability tests were performed.

In the first test, the ready biodegradability of the test substance was determined by the Headspace Test. In the Headspace Test, inoculated test medium was dosed with a known amount of test substance as the nominal sole source of organic carbon and sealed. CO2 evolution from the ultimate aerobic biodegradation of the test substance is determined by measuring the inorganic carbon (IC) produced in the test bottles over that produced in the blank control bottles. The amount of IC produced by the test substance (corrected for that evolved by the controls and the amount added by basification, where applicable) is expressed as a percentage of the theoretical amount of IC (ThIC) that could have been produced if complete biodegradation of the test substance occurred. The study contained two tests, both following GLP. The initial test was a standard ready biodegradability test conducted according to OECD 310 Guidelines. The second test was an enhanced biodegradation screening test, also based upon OECD 310 Guidelines. The enhancements included low-level pre-adaptation, by utilization of inoculum pre-exposed to the test substance concurrently with the conduct of the first test and by extending the test duration to 60 days. Each test contained a blank control group, two reference groups and a treatment group. The blank controls were used to measure the background IC production of the inoculum medium and were not dosed with a carbon source. The reference chambers were dosed with either rapeseed oil or 1-octanol, substances known to be biodegradable, at a nominal concentration of 20 mg C/L. The treatment group test chambers were used to evaluate the test substance at a nominal concentration of 20 mg C/L. The results indicated that the activated sludge inoculum was active, degrading the reference substances rapeseed oil and 1-octanol respectively; mean of 75.4% and 67.0% in the initial test with a 28-day duration and 63.7% and 64.1% by Day 28 in the second test with a 60-day duration. The average cumulative percent biodegradation for the test substance was –1.8% in the initial test and 1.4% at the end of the second test. Based upon the initial test, the test substance may not be considered readily biodegradable.

In the second test, ready biodegradability was determined in a carbon dioxide sealed vessels (headspace) test performed according to OECD 310, and ISO 14593 Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice. The test substance did cause a reduction in the endogenous respiration. The test substance is therefore considered to be inhibitory to the inoculum. The test substance was biodegraded 0% at day 28 and 41% at day 56 in the carbon dioxide headspace test. The biodegradation reached at the last day of the test demonstrates that the test substance is partially degraded in the carbon dioxide headspace test. The validity of the test is demonstrated by an inorganic carbon content in the blank controls of 15% of the inorganic carbon added initially as test substance. Octanol was degraded 95% after 14 days.

In the third test, the biodegradability of the test substance was tested in a Two Phase Closed Bottle Test / BODIS-Test (BOD Test for insoluble substances). The method represents a modification of the Closed Bottle Test (OECD 301D) especially suited for poorly soluble compounds. Under the chosen conditions, 5% of the test substance was degraded within the 28-day test period calculated on the basis of the COD (Chemical Oxygen Demand). Based on the obtained data the test substance was classified as poorly biodegradable under pertaining test conditions.