Tris(2-hydroxyethyl)ammonium salts of tall-oil fatty acids

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

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)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Totnes Sewage Treatment Works, Totnes, Devon, UK
- Method of cultivation / Preparation of inoculum for exposure: 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 resuspended 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.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Initial conc.:

259 mg/L

Based on:

other: ThOD(NO3)/L

Initial conc.:

244 mg/L

Based on:

other: ThOD(NH3)/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: mineral salts medium according to guidelines, prepared using reverse osmosis (RO) water.
- Test temperature: 22 ± 1°C. The temperature of the solutions in a selection of test bottles was measured on days 0 and 28. The temperature of the incubator was continuously monitored throughout the study.
- pH: 7.4 at the start of the test (measured in an extra bottle) and 6.6-6.7 at the end (measured in the test substance bottles)
- pH adjusted: the pH of the seeded mineral medium was adjusted as necessary to 7.4 ± 0.2 prior to use.
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing 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).
- Number of culture flasks/concentration: 3

SAMPLING
- Sampling frequency: oxygen uptake was recorded automatically every 112 minutes during the 28 day experimental period.

CONTROL AND BLANK SYSTEM
- Inoculum blank (3 flasks): inoculated mineral medium
- Reference (3 flasks): inoculated mineral medium + sodium benzoate (100 mg/L)
- Toxicity control: not performed

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

calculation based on ThOD(NO3)

Value:

65

St. dev.:

1.7

Sampling time:

28 d

Details on results:

The test material attained a maximum mean level of biodegradation (based on the BOD:ThOD(NO3) ratio) of 65% (see Table 1).
Greater than 60% degradation was achieved within the 10-day window, so the test material can be classified as readily biodegradable.

Nitrate and nitrite analysis:
At the end of the 28 day test period, no nitrite was measurable in any of the test or control bottles. Approximately 16 mg/L nitrate was measured in the inoculum blanks, and 22 mg/L in the test material bottles. These results indicate that nitrification occurred in the test bottles, as nitrate was produced from the test material.
There was an increased concentration of nitrate of 6.0 mg/L in the test material bottles over the blanks, equivalent to 1.35 mg nitrate-N/L. According to the OECD test guideline the oxygen consumed in nitrifying this amount of nitrogen (and the associated ammoniacal hydrogen) would be 4.57 times the increase in nitrate-N, or 6.2 mg O2/L. If this oxygen uptake is subtracted from the Day 28 BOD result the mean oxygen uptake, due to carbon oxidation, becomes 162 mg O2/L. When the biodegradation is calculated using the ThOD(NH3) (without nitrification), the percentage degradation of carbon is 66%, compared to 65% measured using the ThOD(NO3).

Results with reference substance:

Sodium benzoate attained a maximum mean level of biodegradation (based on the BOD:ThOD ratio) of 67% (see Table 2).
More than 60% degradation was achieved within the 10 day window as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms.

Table 1: Biodegradation of the test material

Day	% Biodegradation based on ThOD(NO3)*
	Flask 1	Flask 2	Flask 3	Mean
5	44	42	42	43
10	60	59	58	59
15	64	63	61	63
20	64	64	63	64
28	66	66	63	65

*: the ThOD(NO3) of the test material was calculated as 2.59 g O₂/g

 

Table 2: Biodegradation of the reference substance

Day	% Biodegradation based on ThOD*
	Flask 1	Flask 2	Flask 3	Mean
5	60	58	60	59
10	68	63	66	66
15	69	65	66	67
20	70	65	65	67
28	68	65	65	66

*: the ThOD of sodium benzoate was calculated as 1.67 g O₂/g

Validity criteria fulfilled:

yes

Remarks:

rate of degradation of sodium benzoate was 60% of its ThOD within 10 days; difference between replicates was <20%

Interpretation of results:

readily biodegradable

Description of key information

Biodegradation in water: screening tests: aerobic biodegradation 65% (ThOD(NO3)) in 28 days (OECD 301F, EU C.4-D)

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

Nitrate was produced from the test material. When the biodegradation is calculated using the ThOD(NH3) (without nitrification), the percentage degradation of carbon is 66%.