1-Propanaminium, 3-amino-N-ethyl-N,N-dimethyl-, N-rape-oil acyl derivs., Et sulfates

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:

From June 29, 2017 to September 04, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Activated sludge was obtained from Totnes Sewage Treatment Works, Totnes, Devon, UK. This works treats sewage of predominantly domestic origin. At the laboratory, the activated sludge was kept aerated at room temperature and the pH maintained at 7.0 ± 1.0.
Seven days prior to the exposure start the activated sludge was centrifuged, washed and re-suspended in the mineral medium and the solids concentration determined. This sludge was then diluted in medium, added to test bottles and stirred until required for use. The seeded mineral medium was pre-conditioned for seven days to reduce the blank oxygen uptake readings in the test.
The mineral medium was made up according to the OECD and EC guidelines and contained with nutrients in reverse osmosis (RO) water.

Duration of test (contact time):

60 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test apparatus
The measurement of oxygen uptake was conducted in the Oxitop respirometers (Wissenschaftlich-Technische Werkstätten, GmbH, Germany). Each individual unit consisted of a dark glass 500 mL bottle with an Oxitop bottle top containing a piezoresistive electronic pressure sensor. Bottles were situated on a magnetic stirrer in a constant temperature incubator. Carbon dioxide produced by microbial respiration was absorbed by potassium hydroxide solution (KOH) placed in a seal cup in the neck of each bottle, and the oxygen taken up was measured as a decrease in pressure. The Oxitop controller collected the pressure values from the measuring tops and calculated the BOD (as mg/L).

Experimental design
All test bottles contained the prepared activated sludge inoculum in mineral medium. Following the pre-conditioning the test bottles were set up for the test
. Inoculum blanks contained no test or reference substance, in order to demonstrate there was no other carbon source in the medium. Positive controls contained the reference substance, sodium benzoate at 100 mg/L, to demonstrate the viability of the inoculum. The test substance bottles contained the test substance at 100 mg/L, to determine its biodegradation. Toxicity controls contained the test and reference substances, both at 100 mg/L, and were used to show if there had been any inhibition of the inoculum by the test substance. Each set of bottles was prepared in triplicate. Additional single replicate of the test substance and toxicity control bottles were prepared for determination of pH at day 0, so undissolved test substance was not removed by the pH probe.
Due to its low aqueous solubility the bottles containing test substance were prepared by directly weighing the required quantity to the relevant test bottles. Sodium benzoate was dosed as a 1000 mg/L stock solution, prepared by dissolving sodium benzoate in RO water to give a clear and colourless solution, this was prepared on the day of exposure start.
Oxygen uptake was recorded automatically every 240 minutes during the 60-day experimental period. Oxygen uptake values were corrected for the inoculum blank and the biodegradation was calculated as a percentage of the chemical oxygen demand for the substance under test and as a percentage of the theoretical oxygen demand for the reference substance.

Reference substance:

benzoic acid, sodium salt

Test performance:

At the end of the 60-day test period, the pH values ranged from 7.1 to 7.2 in the inoculum blank bottles, from 6.8 to 6.9 in the sodium benzoate bottles and from 7.5 to 7.7 in the test substance bottles and from 6.8 to 6.9 in the toxicity control bottles.
Temperature measurements recorded in several of the test bottles on days 0 and 60 indicated the temperature was within the range 22 ± 1°C. Continuous monitoring of the incubator temperature showed it to have remained within the range 22 ± 1°C throughout the study.

Key result

Parameter:

% degradation (O2 consumption)

Value:

3

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

4

Sampling time:

60 d

Details on results:

Validity Criteria
The validity requirements of the OECD guideline state:
• the difference between extremes of replicate biodegradation values should be less than 20% at the end of 10-day window, at the plateau or at the end of the test;
• the positive control should achieve > 60% biodegradation by Day 14.
• the oxygen consumption of the inoculum blank should not exceed 60 mg/L in 28 days.
Mean oxygen uptake of the inoculum blank was below 60 mg/L, as required in the OECD guideline.

The difference between replicate of the test substance extremes was < 20% after the 10-day window, at plateau, and on day 60. Sodium benzoate reached a mean 68% biodegradation by Day 14. The average of the mean oxygen consumed in the inoculum blanks was 11.3 mg/L after 28 days and 18.9 mg/L after 60 days. Therefore, this test has satisfied all the validity criteria.

Key result

Parameter:

COD

Value:

2.52 g O2/g test mat.

Key result

Parameter:

BOD5

Value:

ca. 0.1 g O2/g test mat.

Results with reference substance:

The ThOD of sodium benzoate was calculated as 1.67 g O2/g of substance. Biodegradation of sodium benzoate has been calculated on the basis of calculated ThOD. Sodium benzoate attained a maximum mean level of biodegradation (based on the BOD:ThOD ratio) of 71%, and the results showed good replication.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the study conditions test substance is considered as not readily biodegradable.

Executive summary:

A study was conducted to determine the ready biodegradability of the test substance, ‘C18-unsatd. and C22-unsatd. AAP EDM-ES’ (active: 101.3%), using Manometric respirometry method, according to OECD Guideline 301F, in compliance with GLP. Following the pre-conditioning, the test bottles containing prepared activated sludge inoculum in mineral medium were set up for the test according to the experimental design. In the study, Inoculum blanks, positive control (sodium benzoate at 100 mg/L), test substance (100 mg/L) as well as toxicity controls (test and reference substances, both at 100 mg/L), each were prepared in triplicate, except inoculum blanks which were prepared in duplicate. Additional single replicate test substance and toxicity control bottles were prepared for determination of pH at Day 0, so undissolved test substance was not removed by the pH probe. Oxygen uptake was recorded automatically every 240 minutes during the 60 d experimental period. Oxygen uptake values were corrected for the inoculum blank and the biodegradation was calculated as a percentage of the chemical oxygen demand (COD) for the substance under test and as a percentage of the theoretical oxygen demand (ThOD) for the reference substance. The chemical oxygen demand (COD) of test substance was determined using spectrophotometer for evaluation. The test substance attained a mean level of biodegradation (based on the BOD:COD ratio) of 3% after 28 d and 4% after 60 d. The test substance results showed good replication. Less than 60% degradation was achieved within 28 d, so test substance was classified as inherently biodegradable. Sodium benzoate attained a mean level of biodegradation (based on the BOD:ThOD ratio) of 71%, and the results showed good replication. More than 60% degradation was achieved within the 10-d window as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms. The mean toxicity control degradation achieved on Day 14 was 22% (based on combined ThOD/COD), as this was < 25% the test substance was assumed to be inhibitory at this concentration. The test met all the all validity criteria. Under the study conditions, the test substance was considered to be not readily biodegradable (Scymaris, 2017).

Description of key information

Based on the study results, test substance was considered to be not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

Study 1: A study was conducted to determine the ready biodegradability of the test substance, ‘C18-unsatd. and C22-unsatd. AAP EDM-ES’ (active: 101.3%), using Manometric respirometry method, according to OECD Guideline 301F, in compliance with GLP. Following the pre-conditioning, the test bottles containing prepared activated sludge inoculum in mineral medium were set up for the test according to the experimental design. In the study, Inoculum blanks, positive control (sodium benzoate at 100 mg/L), test substance (100 mg/L) as well as toxicity controls (test and reference substances, both at 100 mg/L), each were prepared in triplicate, except inoculum blanks which were prepared in duplicate. Additional single replicate test substance and toxicity control bottles were prepared for determination of pH at Day 0, so undissolved test substance was not removed by the pH probe. Oxygen uptake was recorded automatically every 240 minutes during the 60 d experimental period. Oxygen uptake values were corrected for the inoculum blank and the biodegradation was calculated as a percentage of the chemical oxygen demand (COD) for the substance under test and as a percentage of the theoretical oxygen demand (ThOD) for the reference substance. The chemical oxygen demand (COD) of test substance was determined using spectrophotometer for evaluation. The test substance attained a mean level of biodegradation (based on the BOD:COD ratio) of 3% after 28 d and 4% after 60 d. The test substance results showed good replication. Less than 60% degradation was achieved within 28 d, so test substance was classified as inherently biodegradable. Sodium benzoate attained a mean level of biodegradation (based on the BOD:ThOD ratio) of 71%, and the results showed good replication. More than 60% degradation was achieved within the 10-d window as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms. The mean toxicity control degradation achieved on Day 14 was 22% (based on combined ThOD/COD), as this was < 25% thetest substance was assumed to be inhibitory at this concentration. The test met all the all validity criteria. Under the study conditions, the test substance was considered to be not readily biodegradable (Scymaris, 2017).

Study 2: A study was conducted to determine the ready biodegradability of the test substance, 'C18-unsatd and C22-unsatd. AAP EDM-ES' (active: 104%), according to the OECD Guideline 310, Ready Biodegradability – CO2 in sealed vessels (Headspace Test) method, in compliance with GLP. Aerobic sludge inoculum was prepared with mixed treatment plant of urban (66%) and industrial (34%) liquid sewage and in order to have a concentration of suspended solid of about 4 mg/L. In the study, for Inoculum blanks, positive control (reference substance: sodium benzoate at 22.72 mg/L) and test substance (27.02 mg/L) groups, 29 inoculum bottles replica each, whereas for toxicity control (test and reference substances) group, 11 inoculum bottles replica were used. All treated samples were kept at the temperature of 20°C for 56 d. The calculation of biodegradation as total inorganic carbon (TIC) were determined at each sampling time (0, 1, 7, 14, 21, 28, 35, 42, 49 and 56 d) for the reference substance, test sample and blank. For toxicity control, calculation of biodegradability as total inorganic carbon (TIC) was determined on Day 1, 28 and 56. The trend of the inorganic carbon and related biodegradation percentages in the bottle contained both the test sample and the reference substance, which confirmed the absence of inhibitory effect of the test sample on inoculum. At 28 d, the biodegradability of the test substance was determined to be 1%. At 14 d biodegradability for reference substance was determined to be 85%. The mean amount of TIC present in the blank controls at the end of the test was < 3 mg C/L. Based on the study results, the test had satisfied all the validity criteria. Under the study conditions, the test substance was determined to be not readily biodegradable (Eurofins, 2018).

[Type of water: freshwater]