(2Z)-2-phenylhex-2-enenitrile

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:

supporting study

Study period:

07-11-2008 to 19-01-2009

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Well documented guideline study (although not under GLP) with some acceptable deviations. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

other: China GB/T 21856-2008. Chemicals, Ready Biodegradation, CO2 Evolution Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

No toxicity control and no abiotic-sterile control performed

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

yes

Remarks:

See above.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

yes

Remarks:

See above

GLP compliance:

no

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): Activated sludge microorganisms were obtained from Nanjing suojincun waste water treatment plant (China). The municipal waste water treatment plant treats predominantly domestic sewage and waste waters
- Laboratory culture: See below.
- Method of cultivation: See below.
- Storage conditions: Room temperature (at ca. 21°C).
- Storage length: Used on day of collection (< 1 week).
- Preparation of inoculum for exposure: The received sludge is washed, centrifuged at 10000 rpm for 10 minutes. This procedure is repeated three times. A small amount of the washed sludge is weighed and dried for suspended solids determination. The wet sludge is suspended in water to obtain activated sludge of ca. 4 g SS/L concentration.
- Concentration of sludge: 4 g/L solids concentration determined prior to use (and typically to 30 mg SS/L final concentration in test vessels per guideline).
- Initial cell/biomass concentration: Not reported.
- Water filtered: yes – Deionized water used to prepare Basal Salts Medium and Stock Solutions.
- Type and size of filter used, if any: Stock solutions A-C are poured through sterilization filter units with 0.45 micron pore cellulose membrane, sterile filtrates are refrigerated before use for a period of up to 6 months. Stock solution D is made up immediately before use. Test medium is prepared before use in deionized water. 10 mL solution A, 800 mL DI water, 1 mL solutions B,C and D the dilution to 1 L DI water.

Initial conc.:

18.9 mg/L

Based on:

TOC

Remarks:

Test substance as mg TOC/L added directly.

Parameter followed for biodegradation estimation:

CO2 evolution

Parameter followed for biodegradation estimation:

inorg. C analysis

Parameter followed for biodegradation estimation:

DOC removal

Remarks:

DOC theoretical removal was determined from CO2 evolution (not directly measured)

Details on study design:

TEST CONDITIONS
- Composition of medium: Solution a: KH2PO4 8.50 g/L; K2HPO4 21.75 g/L; Na2HPO4.2H2O 33.40 g/L ; NH4Cl 0.50 g/L ; pH = 7.4 ; Solution b: CaCl2 27.50 g/L ; Solution c: MgSO4.7H2O 22.50 g/L ; Solution d: FeCl3.6H2O 0.25 g/L. Stock solutions A-C are poured through sterilization filter units with 0.45 micron pore cellulose membrane, sterile filtrates are refrigerated before use for a period of up to 6 months. Stock solution D is made up immediately before use. Test medium is prepared before use in deionized water. 10 mL solution A, 800 mL DI water, 1 mL solutions B,C and D the dilution to 1 L DI water.
The test vessels (carboys) were filled with 1280 mL of BSM (Basal Salts Medium). 220 mL of the prepared inoculum suspensions was added to each vessel. The mixture was aerated with CO2-free air for approximately 24 hours to purge the system of carbon dioxide. After the aeration period, the test item at concentration 18.9 mg TOC/L was directly added to two of the carboys with 500 mL purged BSM to begin the test period. Purged BSM (500 mL) was added to each of the carboys used as Inoculum Blank controls (containing no test item). The final volume in each carboy was 2 L.
Test item concentration: ca. 18.9 mg TOC/L, equivalent
Procedure control: Reference substance aniline, concentration 1 gram in 1 L BSM
Toxicity control: Not applicable.
- Solubilising agent (type and concentration if used): None
- Test temperature: 22 +/- 2°C, maintained in temperature controlled room
- pH: ; pH of the test preparations was determined on Day 28. No unusual variation was reported from day 0 to day 28.
- pH adjusted: No
- Aeration of dilution water: Yes.
- Continuous darkness: Yes (use of ‘brown flasks’).

TEST SYSTEM
- Culturing apparatus: Preparations were prepared and inoculated in 2.5 litre test culture vessels each containing ca. 2 litres of solution
- Number of culture flasks/concentration: In duplicate
- Method used to create aerobic conditions: The CO2 scrubbing apparatus was set up to remove CO2 at a constant rate from the air supplied to the carboy solutions with 2.5 L brown flask. This was done by diverting the air through two absorber bottles with 200 mL of 0.5 mol/L NaOH at a rate of 30-100 mL/min.
- Measuring equipment: Samples were analyzed for CO2 by 702 SM Titrino and titration
- Test performed in closed vessels due to significant volatility of test substance: No.
- Test performed in open system: Yes.
- Details of trap for CO2 and volatile organics if used: Not applicable.

SAMPLING
- Sampling frequency: CO2: - CO2 evolution was determined periodically and quantitatively on days 0, 2, 5, 7, 11, 14, 18, 21, 25, and 28.
CO2 scrubbing apparatus was set up to remove CO2 at a constant rate from the air supplied to the carboy solutions with 2.5 L brown flasks. This was done by diverting the air through two absorber bottles with 200 mL of 0.5 mol/L NaOH at a rate of 30 to 100 mL/min. Six inoculated carboys were required for the testing of the test item, two for the Inoculum Blank, two for the Reference Control, and two for the test item itself. Periodically, the CO2 absorber bottle nearest the carboy was removed for titration. The remaining two absorber bottles were moved one place closer to the carboy, and a new absorber bottle containing 100 ml of fresh 0.0125 mol/L Ba(OH)2 was placed at the far end of the series. Final titrations were performed on day 28. - DOC (theoretical) was determined by calculation based on CO2 evolution during the relevant time period (not directly measured).- Sampling method: See measuring equipment above.
- Sterility check if applicable: Not reported.
- Sample storage before analysis: No.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes.
- Abiotic sterile control: No.
- Toxicity control: No.
- Other: Procedure control - using reference item (aniline, concentration 1 gram in 1 L BSM) only

Reference substance:

aniline

Test performance:

All validity criteria were considered to be met.
(1) The inorganic carbon content of the test item suspension in the BSM at the beginning of the test must be less than 5% of the total carbon content. The total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg COi/L of medium. (Actual: 27.9 mg CO/L)
2) Viability of the microorganisms was checked by means of a reference control. Biodegradation of the reference substance aniline reached > 60% on 14 day (actual: 71.2% and 66.8% respectively)
3) The difference of extremes of replicate values of the removal of the test item at the plateau, at the end of the test, is less than 20%. (Actual test item day 28: 20.7% and 23.4% and reference item: 88.1% and 82.2%)

Key result

Parameter:

% degradation (CO2 evolution)

Value:

>= 20.7 - <= 23.4

Sampling time:

28 d

Remarks on result:

other: mean (n=2) degradation 28-days = 22.1%

Details on results:

The pH of the BSM at test start was 7.4. No unusual variation in pH was noted from day 0 to day 28 in any of the test vessels.
The Inoculum Blanks released 27.9 mg CO/L over the test period.
The air temperature during the test period ranged from 22 ± 2°C.
All validity criteria were considered to be met.

Results with reference substance:

Sodium benzoate attained (n=2) 71.2% and 66.8% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item mean biodegradation in duplicate was 22.1 % at day 28.

Executive summary:

The ready biodegradability test was carried out according to China GB/T 21856-2008. Chemicals, Ready Biodegradation, CO2 Evolution Test, OECD 301B Ready Biodegradability: CO2 Evolution (Modified Sturm Test)a and EU Method C.4-C and US EPA OPPTS 835.3110 guidelines. The test item, at a concentration of 19.8 mg TOC/L, was exposed to activated domestic sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at approximately 22°C for 28 days. The degradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference substance: aniline were used for validation purposes. The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion. The total CO2 evolution in the inoculum control vessels on Day 28 was 27.9 mg/L and the difference between the values for CO2 production at the end of the test for the replicate vessels was < 20% and therefore satisfied the validation criteria. Aniline attained > 60% degradation, specifically 71.2% and 66.8% after 14 days and 82.2% to 88.1% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. All validity criteria were met. The final value for the test item day 28 indicated it attained 20.7% to 23.4% degradation after 28 days. This equates to mean (n=2) biodegradation of 22.1%. Under the conditions of this study, the test item is not readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24-05-2006 to 30-05-2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study to ISO Guideline or which is equivalent to recognised Guidelines performed under GLP. All relevant validity criteria were met with acceptable deviations.

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))

Deviations:

yes

Remarks:

Test terminated on day 27 not day 28. This was not considered a significant deviation given the study conclusion.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

See above

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: September 2005 ; signature: February 2006

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, 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 secondary treatment stage of the sewage treatment plant at Villette (STEP Villette, av. De Theonex 105, 1226 THONEX Geneva, Switzerland)), which treats predominantly domestic sewage.
- Storage conditions: See pretreatment field.
- Storage length: < 1 week
- Preparation of inoculum for exposure:
- Pretreatment: The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed three times by settlement (centrifuge) and suspension in culture medium. To remove any excessive amounts of dissolved organic carbon (DOC) the solution was stirred and maintained on pure oxygen at room temperature. The final inoculum concentration in test bottles was 4 mg/L dry weight activated sludge solids.
- Concentration of sludge: The sludge was diluted in the test bottles to 4 mg DW/L.

Duration of test (contact time):

28 d

Initial conc.:

23.76 mg/L

Based on:

test mat.

Remarks:

20.00 mg C/L

Parameter followed for biodegradation estimation:

inorg. C analysis

Details on study design:

TEST CONDITIONS
- Composition of medium: 10 mL of Solution A [KH2PO4: 8.50 g/L; K2HPO4: 21.75 g/L; Na2HPO4,2H2O: 33.40 g/L; NH4Cl: 0.50 g/L]; 1 mL of Solution B [CaCl2,2H2O: 36.40 g/L]; 1 mL of Solution C [MgSO4,7H2O: 22.50 g/L]; 1 mL of Solution D [FeCl3,6H2O: 0.25 g/L] and purified water diluted to 1 L.
- Solubilising agent (type and concentration if used): None.
- Test temperature: 23 ±2 °C
- pH: Not reported ; typically pH 7.2 – 7.4
- pH adjusted: No
- Aeration of dilution water: Not reported
- Suspended solids concentration: 4 mg/L dry weight
- Continuous darkness: No. The test was conducted in diffuse light.

TEST SYSTEM
- Culturing apparatus: 250 mL glass flasks with continuous stirring (fill volume ca. 75 mL)
- Number of culture flasks/concentration: In duplicate (test item); In duplicate (Inoculum blank); duplicate flasks (reference item) ; duplicate (Toxicity Control) ; N/A (Abiotic Sterile Control)
- Method used to create aerobic conditions: Gas tight syringe/teflon septum.
- Measuring equipment: Measuring the increase of IC (Inorganic Carbon) in the headspace and the liquid phase of each vessel by gas-tight syringe and needle through the teflon septum (0.5 ml) and direct injection on the TOC (Total Organic Carbon) analyser. The sampling of the liquid phase was done through the bottom of the vessel and the sample was filtered prior to analysis. Analysis conducted in duplicate.

SAMPLING
- Sampling frequency: Every three or four days to day 27.
- Sampling method: Measuring the increase of IC (Inorganic Carbon) in the headspace and the liquid phase of each vessel by gas-tight syringe and needle through the teflon septum (0.5 ml) and direct injection on the TOC (Total Organic Carbon) analyser. The sampling of the liquid phase was done through the bottom of the vessel and the sample was filtered prior to analysis. Analysis conducted in duplicate.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes.
- Abiotic sterile control: No.
- Toxicity control: Yes.
- Other: Positive reference control (Sodium Benzoate).

Reference substance:

benzoic acid, sodium salt

Remarks:

34.27 mg/L or 39.99 mg C/L

Parameter:

% degradation (inorg. C analysis)

Value:

0

Sampling time:

27 d

Remarks on result:

other: Day 27 can be considered equal to day 28 measurement.

Details on results:

Applicant assessment indicates: The test item was biodegraded by 0 % at day 28 in the CO2 evolution test. The multi-constituent test substance is therefore classified as not readily biodegradable.

References:
1. OECD (2006), Introduction to the section of the OECD Guidelines for Testing of Chemicals on Degradation and Accumulation
2. GHS revision 4 (2011), Globally harmonized system of classification and labelling of chemicals (GHS). Chapter 4.1. Hazardous to the Aquatic Environment. United Nations, 2011

Results with reference substance:

The reference item attained 90.26% degradation at 4 days and 96.23% at day 13 thereby confirming the suitability of the inoculum and test conditions.

All validity criteria were met.

1. The blank control TIC at day 27 was 1.18 mg C/L (< 3.0 mg C/L)

2. The Reference Item attained 96.23% degradation at day 13 (> 60% degradation at day 14)

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item mean biodegradation in duplicate was 0 % at day 28.

Executive summary:

The ready biodegradability test was carried out according to ISO 14593 equivalent or similar to OECD TG 310 guideline under GLP. The test item, at a concentration of 23.76 mg/L was exposed in duplicate to activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment which treats predominantly domestic sewage (Villette: Geneva, Switzerland) with culture medium in sealed culture vessels in diffused light at 23°C ± 2°C for 27 days. The sludge was diluted in the test bottles to 4 mg DW/L. The degradation of the test item was assessed by the regular measurement of inorganic carbon (IC) production on days 0 to day 27. This was achieved by measurement of the IC (Inorganic Carbon) in the headspace and the liquid phase of each vessel by gas-tight syringe and needle through the teflon septum (0.5 ml) and direct injection on the TOC (Total Organic Carbon) analyser. Analysis conducted in duplicate. The reference item attained 96.23% degradation after 13 days thereby confirming the suitability of the inoculum and test conditions. The mean biodegradation for duplicate test flasks at 28 days for the test item was 0%. Under the conditions of the study, test item is considered as not readily biodegradable.

Description of key information

Biodegradation: not readily biodegradable, mean biodegradation 0 % (28-days), eq. or similar to OECD TG 310, 2006

Supporting study: not readily biodegradable, mean biodegradation (n=2) 22.1% (28-days), OECD TG 301B, 2009

Note: the supporting study is non-GLP. There is evidence that substance biodegradation will occur however the OECD TG 301 criteria for 'ready biodegradability' has not been met at the current time.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

Key Study : eq. or similar to OECD TG 310, 2006 : The ready biodegradability test was carried out according to ISO 14593 equivalent or similar to OECD TG 310 guideline under GLP. The test item, at a concentration of 23.76 mg/L was exposed in duplicate to activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment which treats predominantly domestic sewage (Villette: Geneva, Switzerland) with culture medium in sealed culture vessels in diffused light at 23°C ± 2°C for 27 days. The sludge was diluted in the test bottles to 4 mg DW/L. The degradation of the test item was assessed by the regular measurement of inorganic carbon (IC) production on days 0 to day 27. This was achieved by measurement of the IC (Inorganic Carbon) in the headspace and the liquid phase of each vessel by gas-tight syringe and needle through the teflon septum (0.5 ml) and direct injection on the TOC (Total Organic Carbon) analyser. Analysis conducted in duplicate. The reference item attained 96.23% degradation after 13 days thereby confirming the suitability of the inoculum and test conditions. The mean biodegradation for duplicate test flasks at 28 days for the test item was 0%. Under the conditions of the study, test item is considered as not readily biodegradable.

 

Supporting study : OECD TG 301B, 2009 : The ready biodegradability test was carried out according to China GB/T 21856-2008. Chemicals, Ready Biodegradation, CO2 Evolution Test, OECD 301B Ready Biodegradability: CO2 Evolution (Modified Sturm Test)a and EU Method C.4-C and US EPA OPPTS 835.3110 guidelines. The test item, at a concentration of 19.8 mg TOC/L, was exposed to activated domestic sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at approximately 22°C for 28 days. The degradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference substance: aniline were used for validation purposes. The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion. The total CO2 evolution in the inoculum control vessels on Day 28 was 27.9 mg/L and the difference between the values for CO2 production at the end of the test for the replicate vessels was < 20% and therefore satisfied the validation criteria. Aniline attained > 60% degradation, specifically 71.2% and 66.8% after 14 days and 82.2% to 88.1% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. All validity criteria were met. The final value for the test item day 28 indicated it attained 20.7% to 23.4% degradation after 28 days. This equates to mean (n=2) biodegradation of 22.1%. Under the conditions of this study, the test item is not readily biodegradable.

Note: the supporting study is non-GLP. There is evidence that substance biodegradation will occur however the OECD TG 301 criteria for 'ready biodegradability' has not been met at the current time.