2,4-Hexadienoic acid, 3-(trimethoxysilyl)propyl ester, (2E,4E)-

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:

2012-01-19 to 2012-02-16

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:

The activated sludge was obtained from the Wareham Wastewater Treatment Plant, Wareham, Massachussets, which receives primarily domestic waste. Approximately 8 L of activated sludge was collected on 18 January 2012, and transported to the laboratory. Upon arrival at the laboratory, the sludge was passed through a 2 mm sieve, recombined and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, the sludge was washed with mineral media (1000 mL) and the contents were centrifuged at least once again and the supernatant discarded. The moisture content of the activated sludge was determined, using an automated moisture analyser (Sartorius MA-150) to be 94.90% and the percent solid determined to be 5.1%. A 15 mg solids/mL inoculum solution was prepared (1.5 g dry weight sludge brought to 100 mL with mineral media) and aerated until used. The test substance flasks, the blank flasks, the procedural control flask and the toxicity control flask all received 6 mL of the inoculum to produce an activated sludge concentration of 30 mg/L.

In addition, a 50 g aliquot of fresh soil, a 25 g aliquot of Weweantic River sediment and a 25 g aliquot of Taunton River sediment were collected near the laboratory. The soil and sediment were suspended in 1.0 L of Weweantic River water. The suspension was filtered through glass wool prior to use. A 3.0 mL aliquot of the soil/sediment filtrate was added to each test vessel containing 2991 mL of mineral medium and 6.0 mL of activated sludge as shown in Table 2.

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test design:
Each test unit consisted of a 4 L glass bottle with a rubber stopper into which one stainless steel needle with a Luer-Lok connection and two pieces of glass tubing were inserted. Prior to test initiation, the test vessels were acid washed and rinsed repeatedly with reagent grade water. The stainless steel needle was extended through the stopper into the test solution serving as a sampling port for solution samples. A rubber cap was used to cover the top of the sample port. The glass tubing provided the inlet and outlet ports for air exchange. CO2-free air was pumped under positive pressure through a hydration flask before entering the test system. The outlet port of each system was connected to two CO2 effluent gas traps, the first consisting of 200 mL of 0.2 N potassium hydroxide (KOH) and the second trap containing 100 mL of 0.2 N KOH. The test vessels were identified with project number, replicate number and treatment type. Each test vessel was placed on a magnetic stir plate located in a dark environmental chamber set to maintain a temperature of 22±2°C.

Test initiation:
Six test vessels were established: two for the test substance, two inoculum blanks, one sodium benzoate procedural control and one toxicity control (see Table 2).

On day 1, each 4 L vessel was established by adding 2991 mL of mineral medium (see Table 1). A 6 mL aliquot of the activated sludge inoculum and 3 mL of soil inoculum were added to each vessel for a total volume of 3 L per vessel. All test vessels were attached to a CO2-free compressed air tank and aerated under positive pressure. The vessels were mixed and purged with CO2-free air until day 0 to reduce carbon in the test system from the inoculum prior to test initiation.

On day 0, the test substance replicate vessels (A and B) were dosed with 0.0552 g aliquots of the test substance, which were first each added to 100 mg portions of silica gel, then transferred to 4 L flasks. The total nominal fortification was 10 mg C/L in the test substance vessels. The inoculum blank control vessels only received 100 mg portions of untreated silica gel.

The toxicity control vessel, which was fortified with 0.0552 g of the test substance, also received 3.0 mL of the 10.0 mg C/mL sodium benzoate stock solution. The total fortification was 20 mg C/L (test and reference substance) in the toxicity control vessel.

The sodium benzoate procedural control was fortified with 3.0 mL of the sodium benzoate stock solution for a final concentration of 10 mg C/L.

A summary of the dosing procedure is given in Table 2. DOC samples were not taken at test initiation because of the insoluble nature of the test substance.

Test maintenance and sampling:
All test systems were aerated continuously for 28 days under positive pressure using CO2-free air in order to provide oxygen for the microbes and to capture evolved carbon dioxide. The temperature of the environmental chamber was recorded daily throughout the exposure period using a digital minimum-maximum thermometer (VWR).

On test days 1, 4, 7,10, 13, 18, 22 and 25, a 7 mL sample was removed from the first KOH CO2 trap on each test system and analysed for CO2 evolution. on Day 28, 1 mL of concentrated HCl was added to each test vessel following pH measurements to terminate biological activity. Aeration was continued overnight to drive any residual inorganic carbon from the test vessels. After overnight aeration, 7 mL samples for analysis of CO2 evolution were removed from the first and second traps. The amount of evolved CO2 in each trap was determined using a Shimadzu TOC V-CPH Carbon Analyser.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

42.6

St. dev.:

0.83

Sampling time:

28 d

Details on results:

The mean cumulative net percent CO2 evolved (percent biodegradation) from the aqueous test medium fortified with the test substance at 10 mg C/L was 42.63% on day 28.

The toxicity control on day 13 was 51.60%, which indicates that the test substance was not toxic to the inoculum in the test medium.

Results with reference substance:

The cumulative net percent C02 evolved from the sodium benzoate procedural control was 73.14% of theoretical by day 10, thus exceeding the "pass" criteria of the test (reaching 60% or greater CO2 evolution within a 10-day window of reaching 10% biodegradation). This rapid biodegradation of sodium benzoate confirmed the presence of an active microbial population and system integrity.

Test conditions:

During the 24 hour incubation period designed to purge CO₂ from the test vessels, the temperature ranged from 21.3 -21.8°C. The temperature in the environmental chamber was recorded daily during the study and ranged from 20.7 -22.5°C. The pH measurements are presented in Table 3. The pH of the test medium was measured to be 7.42 on day 1 and ranged from 7.19 to 7.32 on day 28.

Table 3: pH measurements taken at termination (day 28) of the 28-day biodegradation study

Test vessel	Replicate	pH* on day 28
Test substance	A B	7.25 7.25
Inoculum	A B	7.19 7.23
Procedural control	A	7.32
Toxicity control	A	7.30

*The pH was measured using a Yellow Springs Instrument (YSI) pH 100 meter pH meter. The pH of the medium on day 1 was 7.42. The pH measurements on day 28 were taken prior to acidification.

CO₂ evolution:

The total inorganic carbon measured in the KOH traps (Table 4) was used to calculate the cumulative CO₂ evolved from the test vessels (Table 5). The mean cumulative CO₂ from the test substance, procedural control, toxicity control and blank control test vessels was 41.71, 51.44, 69.19 and 26.08 mg/L respectively at day 28. The cumulative net percent CO₂ production (blank control values substracted), or percent ultimate biodegradation for the test substance, procedural control, and toxicity control was calculated to be 42.63, 69.16 and 58.78% respectively. The data is summarised in Table 6. The test cumulative net CO₂ evolved from the toxicity control was 51.60% on day 13 and therefore the test substance was not considered toxic to the inoculum based on the OECD 301B Guideline (i.e.>25% CO₂ evolution by day 14 is not considered toxic). The cumulative net CO₂ evolved from the sodium benzoate procedural control was 73.14% theoretical by day 10, thus exceeding the 'pass' criteria of the test (reaching 60% or greater CO₂ evolution within a 10 -day window of reaching 10% biodegradation). This rapid biodegradation of of sodium benzoate confirmed the presence of an active microbial population and system integrity.

Table 4: Total inorganic carbon (mg C/L) measured in KOH traps during the 28 day biodegradation study

Vessel No	Test vessel	Day 1	Day 4	Day 7	Day 10	Day 13	Day 18	Day 22	Day 25	Day 28	Day 28
										Trap 1	Trap 2
1	Test substance	19.78	68.14	98.85	125.00	155.50	176.50	190.00	203.30	219.00	24.12
2	Test substance	19.58	65.36	92.73	119.60	147.30	168.70	181.60	196.10	213.60	35.43
3	Blank	14.28	37.39	58.60	73.02	85.49	102.40	109.40	117.50	124.60	26.52
4	Blank	15.91	42.40	62.41	78.43	89.51	109.80	119.70	130.80	138.50	21.35
5	Procedural control	23.50	129.90	176.70	198.30	223.50	245.20	257.60	269.10	278.30	20.10
6	Toxicity control	55.47	165.90	211.80	248.20	267.50	303.10	323.50	341.10	358.70	49.52

 

Table 5: Cumulative CO₂(mg/L) evolved from the test substance test vessels during the 28 day biodegradation study*

Vessel No	Test vessel	Day 1	Day 4	Day 7	Day 10	Day 13	Day 18	Day 22	Day 25	Day 28
1	Test substance	4.84	16.07	22.47	27.35	32.69	35.60	36.69	37.52	41.50
2	Test substance	4.79	15.42	21.08	26.17	30.97	34.02	35.07	36.19	41.93
	Mean Std dev	4.81 0.03	15.75 0.46	21.78 0.98	26.76 0.84	31.83 1.22	34.81 1.11	35.88 1.15	36.86 0.94	41.71 0.31
5	Procedural control	5.74	30.64	40.17	43.39	46.99	49.45	49.75	49.67	51.44
3	Blank	3.49	8.82	13.32	15.98	17.97	20.65	21.13	21.69	25.17
4	Blank	3.89	10.00	14.19	17.16	18.82	22.15	23.12	24.14	26.99
	Mean Std dev	3.69 0.28	9.41 0.84	13.76 0.61	16.57 0.84	18.40 0.60	21.40 1.06	22.12 1.41	22.91 1.74	26.08 1.28
6	Toxicity control	13.56	39.14	48.15	54.31	56.24	61.13	62.48	62.96	69.19

*Corrected for removal of 7 mL KOH sample from each trap at each sampling interval

The rounded values presented in the table were calculated based on unrounded experimental results

Table 6: Cumulative net percent CO2 evolved (ultimate biodegradation) during the 28 day biodegradation study

Vessel No	Test vessel	Day 0	Day 1	Day 4	Day 7	Day 10	Day 13	Day 18	Day 22	Day 25	Day 28
1	Test substance	0	3.12	18.17	23.77	29.40	38.99	38.72	39.74	39.84	42.04
2	Test substance	0	2.99	16.38	19.98	26.18	34.29	34.43	35.31	36.21	43.22
	Mean Std dev	0 -	3.06 0.09	17.28 1.26	21.88 2.68	27.79 2.28	36.64 3.32	36.58 3.03	37.53 3.13	38.03 2.56	42.63 0.83
5	Procedural control	0	5.60	57.90	72.04	73.14	77.97	76.51	75.34	72.96	69.16
6	Toxicity control	0	13.46	40.53	46.90	51.46	51.60	54.18	55.02	54.60	58.78

The rounded values presented in the table were calculated based on unrounded experimental values. All values were corrected for mean blank CO₂production.

Validity criteria fulfilled:

yes

Interpretation of results:

other: not readily biodegradable

Conclusions:

A ready biodegradation value of 42.6% in 28 days was obtained for the substance in accordance with a relevant test method and in compliance with GLP. The result is considered to be reliable.

Executive summary:

This study was performed to determine the potential for ultimate biodegradation of the test substance in water by the carbon dioxide evolution method following OECD Test Guideline 301B. The amount of carbon dioxide (CO₂) released upon biodegradation of the test substance and a reference substance, sodium benzoate, was measured to assess the potential for ultimate biodegradation. Two blank controls (containing inoculum), a procedural control (containing inoculum and reference substance) and a toxicity control (containing inoculum, reference and test substances) were established to account for background CO2 production, inoculum viability and toxicity ofthe test substance, respectively. Test flasks were incubated aerobically in the dark for a period of 28 days.

The mean cumulative net percent CO₂ evolved (percent biodegradation) from the aqueous test medium fortified with the test substance at 10 mg C/L was 42.63% on day 28. The toxicity control on day 13 was 51.60%, which indicates that the test substance was not toxic to the inoculum in the test medium. The cumulative net percent CO₂ evolved from the sodium benzoate procedural control was 73.14% of theoretical by day 10, thus exceeding the "pass" criteria of the test (reaching 60% or greater CO₂ evolution within a 10-day window of reaching 10% biodegradation). This rapid biodegradation of sodium benzoate confirmed the presence of an active microbial population and system integrity.

Description of key information

Biodegradation in water: screening tests: 42.6% biodegradation in 28 days.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The key study is the only biodegradation study available for the substance. In a test conducted in accordance with OECD 301B (CO₂ Evolution Method) and in compliance with GLP, the test substance attained 42.6% degradation in 28 days.

3-(Trimethoxysilyl)propyl-(2E,4E)-hexa-2,4-dienoate hydrolyses within the timescale of the ready biodegradation study (4.7 hours at 25°C) to 3-(trihydroxysilyl)propyl-(2E,4E)-2,4-hexadienoate and methanol. The biodegradation observed in the study is mainly attributable to the biodegradation of the methanol hydrolysis product.

Methanol is readily biodegradable (OECD, 2004).

No significant biodegradation is expected for the silanol hydrolysis product.

Reference:

OECD (2004): SIDS Initial Assessment Report for SIAM 19, Berlin, Germany, 18-20 October 2004, Methanol, CAS 67-56-1.