4-hydroxy-3,5-dimethoxybenzonitrile

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:

2007-11-27 to 2008-04-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 306 (Biodegradability in Seawater)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Test substance:
Mediator SNP (4-hydroxy-3,5-dimethoxybenzonitrile)
Physical description: tan pow der
Storage: ambient

Oxygen conditions:

aerobic

Inoculum or test system:

other: conditioned natural seawater with incumbent microbial population

Details on inoculum:

Microorganisms present in natural seawater used.
Source: Natural Marine Fisheries Services (Galveston, Texas, USA)
Collection Date: 07 Feb 08
Appearance: Clear
Temperature: 23°C
Salinity: 24 ppt
pH: 7.9
Dissolved Oxygen: 8.1mg/L
Mineral Medium Preparation: Natural Seawater was used.

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

other: Dissolved oxygen

Details on study design:

Blank solution, test substance solution, reference solution, and toxicity reference solution were prepared. Each solution was siphoned from the lowest quarter (not the bottom) of the mixing container and transferred to labeled BOD bottles resulting in 8 bottles for each of the four solutions (up to four sampling periods). Only 6 bottles were used for the toxicity reference solution. Day 0 samples were analyzed for concentration of D.O. The remaining bottles were kept in the dark and incubated at 20 ± 1°C. Replicate bottles for each solution were removed at each sampling interval (0, 5, 15 and 28 Days).

Reference substance:

other: sodium acetate trihyrate

Parameter:

% degradation (O2 consumption)

Value:

2

Sampling time:

28 d

Remarks on result:

other: theoretical oxygen demand calculated

Results with reference substance:

A mean value of 134.3% biodegradation was obtained on Day 5 for sodium acetate trihydrate.

Table 1: Dissolved Oxygen Measurements (in mg/L)
Test group	Day 0	Day 5	Day 15	Day 28
Blank	7.1	6.75	6.45	6.2
	7.1	6.75	6.6	6.3
Sodium acetate trihydrate	7.05	5.45	5.35	4.9
	7.05	5.5	5.35	4.85
Test substance (~2 mg/L)	7.05	6.7	6.45	6.2
	7.1	6.7	6.55	6.1
Toxicity Reference	7.05	5.55	5.4	NA
	7.05	5.6	5.4	NA

 

Table 2: Biodegradation Calculations
	Day 5 Calculations	Day 15 Calculations	Day 28 Calculations
Blank Depletion (mg/L)	0.35	0.575	0.85
Sodium Acetate Trihydrate Net D.O. Depletion (mg /L)	1.225	1.125	1.325
Sodium Acetate Trihydrate ThOD (mg O2/mg)	0.47	0.47	0.47
Sodium Acetate Trihydrate Dose (mg/L)	1.94	1.94	1.94
Sodium Acetate Trihydrate Calculated Degradation	134.30%	123.40%	145.30%
Test Substance Net D.O. Depletion (mg/L)	0.025	0	0.075
Test Substance ThOD NH3 (mg O2/mg)	1.607	1.607	1.607
Test Substance ThOD NO2 (mg O2/mg)	1.875	1.875	1.875
Test Substance ThOD NO3 (mg O2/mg)	1.965	1.965	1.965
Test Substance Dose (mg/L)	2.1	2.1	2.1
Test Substance NH3 Calculated Degradation %	0.7	0	2.2
Test Substance NO2 Calculated Degradation %	0.6	0	1.9
Test Substance NO3 Calculated Degradation %	0.6	0	1.8

Validity criteria fulfilled:

yes

Remarks:

Both reference material and toxicity reference material met the criteria.

Interpretation of results:

other: 2% degradation

Conclusions:

A maximum mean of 2.0% biodegradation was obtained on Day 28 for the test substance. Therefore the test substance is not considered biodegradable.

Executive summary:

This study was designed to assess the biodegradability of the test substance, Mediator SNP (4 -hydroxy-3,5- dimethoxybenzonitrile), utilizing the microorganisms present in natural seawater in accordance with OECD Guideline 306. For a biodegradability test to be considered valid, a biodegradation value of >60% of the theoretical oxygen demand (ThOD) must be obtained for the reference material within 5 days. A mean value of 134.3% biodegradation was obtained on Day 5 for sodium acetate trihydrate. A test substance is considered biodegradable if a biodegradation value of >60% of the ThOD is obtained within 28 days. For test substance, Mediator SNP, a maximum mean of 2.0% biodegradation was obtained on Day 28 of the study. Therefore, the test substance is not considered biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-10-15 to 2008-12-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of activated sludge: aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC)
-The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21°C and used on the day of collection.
Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sew age sludge by suction through pre-weighed GF/A filter paper using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionised reverse osmosis water. The filter paper was then dried in an oven
at approximately 105°C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.4 g/L prior to use.

Duration of test (contact time):

28 d

Initial conc.:

10 other: mg Carbon/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The culture medium used in this study was that recommended in the OECD Guidelines.
- Test temperature: ~21°C
- Storage: in the dark

TEST SYSTEM
- Culturing apparatus: 5 litre glass culture vessels each containing 3 litres of solution
- Number of culture flasks/concentration: 2

Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 mL of culture medium and 26.5 mL of inoculum and aerated overnight. On Day 0 the test and standard materials were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium. The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 mL/minute and stirred continuously by magnetic stirrer. The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb~ granules).
The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.

SAMPLING for CO2 analysis
- Sampling frequency: Day 0, 1, 2, 3, 5, 7, 9, 11, 14, 16, 18, 20, 23, 27, 28 and 29 (for the first absorber vessel). Day 0 and 29 for the second absorber vessel. The samples taken on Days 11, 18 and 27 were stored at approximately -20°C. The rest were assayed immediately for CO2.
Analysed by: Tekmar-Dohrmann Apollo 9000 TOC analyser and Shimadzu TOC-VcsH TOC analyser.

Sampling for DOC
Samples (20 mL) were removed from the test material and toxicity control vessels on Day 0 prior to the addition of the test material in order to calculate the Inorganic Carbon content in the test media. The samples were filtered through Gelman 0.45 µm Acrocap filters (approximately 5 mL discarded) prior to DOC analysis. DOC analysis of the test material dispersions after dosing was not possible due to the limited solubility of the test material in culture medium. On Days 0 and 28 samples (20 mL) were removed from the control and standard material vessels and filtered through Gelman 0.45 µm Acrocap filters (approximately 5 mL discarded) prior to DOC analysis. The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser.

CONTROL AND BLANK SYSTEM
- Inoculum blank: consisted of inoculated culture medium.
- Toxicity control: containing the test material and sodium benzoate, was prepared in order to assess any toxic effect of the test material on the
sewage sludge micro-organisms used in the test.

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

90

Sampling time:

28 d

Details on results:

The test material attained 91% degradation after 28 days and satisfied the 10-day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%.

Results with reference substance:

Sodium benzoate attained 109% degradation after 14 days and 101% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test material attained 91% degradation after 28 days and satisfied the 10-day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 B.

Executive summary:

The form in the supply chain for 4-hydroxy-3,5-dimethoxybenzonitrile is in conjunction with laccase enzyme, adipic acid, and monosodium phosphate, a ready-to-use product in the textile industry for denim bleaching applications (Tradename: PrimaGreen EcoFade LT100). This

composition is referred to here as RTU blend. In order to examine the biodegradability of the product in an aerobic aqueous medium, this study was conducted in accordance with OECD 301B guideline.

The test material, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days. The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

The test material attained 91% degradation after 28 days and satisfied the 10-day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. The test material can therefore be considered to be readily

biodegradable under the strict terms and conditions of OECD Guideline No 301 B.

Description of key information

Biodegradability of 4-hydroxy-3,5-dimethoxybenzonitrile by itself and as a ready to use product (RTU) produced by Genencor International is described below.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

The form in the supply chain for 4-hydroxy-3,5-dimethoxybenzonitrile is in conjunction with laccase enzyme, adipic acid, and monosodium phosphate, a ready-to-use product in the textile industry for denim bleaching applications (Tradename: PrimaGreen EcoFade LT100). This

composition is referred to here as RTU blend.

In order to examine the biodegradability of the 4 -hydroxy-3,5 -dimethoxybenzonitrile (subject of this registration) as well as the ready-to-use commercial product, two separate studies were conducted in accordance with OECD 306 and 301B respectively.

28-day closed bottle biodegradation test (OECD 306)

4-hydroxy-3,5-dimethoxybenzonitrile was tested for biodegradability in seawater (closed bottle test) and was found to achieve 2% biodegradation on day 28. This classifies the chemical as “not readily biodegradable.”

Biodegradation test (CO2 evolution/modified sturm test)(OECD 301B)

The RTU blend biodegradability was determined by calculating the amount of CO2 released by inoculated medium. Under the conditions of this assay, the RTU blend attained 8% degradation after 1 day and 71% degradation after 9 days thereby satisfying the 10-day window validation criterion. After 28 days the RTU blend achieved 91% degradation, classifying the blend as “readily biodegradable.”

The RTU blend is the only form of 4 -hydroxy-3,5 -dimethoxybenzonitrile that will be commercialized by Genencor International for use in textiles bleaching. Exposure of the 4 -hydroxy-3,5 -dimethoxybenzonitrile by itself to the aquatic environment is practically non-existent. After use, the mixture (from the RTU blend) is released into the environment through the sewage system and any impact to the environment would result from exposure to this discharged mixture and not to 4-hydroxy-3,5 –dimethoxybenzonitrile by itself. Using a weight-of-evidence approach, it can be concluded that any reaction products from 4-hydroxy-3,5-dimethoxybenzonitrile in the discharge water after textiles application is readily biodegradable.