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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 June 2016 - 04 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: ISO International Standard 9439 “Water Quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - carbon dioxide evolution test.
Version / remarks:
1999
Deviations:
no
Qualifier:
according to
Guideline:
other: ISO International Standard 10634 "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium".
Version / remarks:
1995
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Test material form:
solid
Details on test material:
Identification: Nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide
Appearance: Light yellow lumps
Test item storage: At room temperature; Store in closed container
Specific details on test material used for the study:
Solubility in water: 5-10%
Stability in water: stable

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: 'Waterschap Aa en Maas', Heeswijk-Dinther, The Netherlands, receiving predominantly domestic sewage.
- Storage conditions: The freshly obtained sludge was preconditioned to experimental conditions by continuous aeration until further treatment.
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle (44 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.
- Concentration of sludge: 3.4 g suspended solids/L in concentrated sludge
- Water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
19 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral medium according to OECD 301B
- Test temperature: 21.4 - 22.9 °C
- pH: start: 7.5-7.6, end: 7.2-7.8
- pH adjusted: yes, the pH of test solutions was adjusted to 7.6 at the start of the test, the pH of the toxicity control was adjusted to 7.5 at the start of the test. (For both adjustments 1 M HCl was used.
- Aeration of dilution water: yes, continuously
- Suspended solids concentration: Final solutions contained 10 mL of supernatant liquid, which resulted after a settling period of freshly obtained sludge, per 2 L of solution. Suspended solids concentration in the supernatant liquid was not reported.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2 litre brown coloured glass bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: aeration with synthetic air (CO2 <1 ppm) at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Measuring equipment: Three CO2 absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle. The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dulution from 1 M HCl). Phenolphthalein (1% solution in ethanol) was used as pH indicator.

SAMPLING
- Sampling frequency: titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made over a period of at least 14 days.
- Sampling method: at each sampling point, the CO2 absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series.
- Sample storage before analysis: not applicable

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 2 replicates
- Abiotic sterile control: no
- Toxicity control: yes, 1 replicate
- Other: 1 positive control

STATISTICAL METHODS: none
Reference substance
Reference substance:
acetic acid, sodium salt
Remarks:
12 mg TOC/L; Purity: 99.1%

Results and discussion

% Degradationopen allclose all
Key result
Parameter:
% degradation (CO2 evolution)
Value:
9
Sampling time:
28 d
Remarks on result:
other: Bottle 1
Key result
Parameter:
% degradation (CO2 evolution)
Value:
13
Sampling time:
28 d
Remarks on result:
other: Bottle 2
Details on results:
- The criterion for ready biodegradability (> 60%) was not met, therefore the test item was determined to be not readily biodegradable.
- The ThCO2 of the test item was calculated to be 2.29 mg CO2/mg, the ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.
- In the toxicity control, less than 25% biodegradation occurred within 14 days (3%, based on ThCO2). Therefore, the test item was assumed to possibly inhibit microbial activity. OECD 301B states that in such case the test should be repeated with a lower concentration of test substance and/or a higher concentration of inoculum. According to OECD 301B, test item concentrations should be between 10-20 mg TOC/L and concentration of inoculum max 30 mg solids/L. The test item concentration (12 mg TOC/L) in the performed test was already close to the lower limit, and the used concentration of inoculum was also close to the maximum allowed concentration. Altogether, the chosen experimental conditions were close to the prescribed OECD 301B boundary settings, thus repetition of the test is not considered to provide significantly different results.
- Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve

BOD5 / COD results

Results with reference substance:
61% after 14 days

Any other information on results incl. tables

The following acceptability criteria were met, therefore the study was considered to be valid:

1. The positive control item was biodegraded by at least 60% (i.e. 61%) within 14 days.

2. The difference of duplicate values for %-degradation of the test item was always less than 20% (i.e. ≤ 4%).

3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (i.e. 39.3 mg CO2 per 2 litres of medium, corresponding to 19.7 mg CO2/L).

4. The Inorganic Carbon content (IC) of the test item (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis, IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See 'any other information on results' for details.
Interpretation of results:
not readily biodegradable
Conclusions:
Under the conditions of the modified Sturm test, according to OECD 301B, Nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide was not readily biodegradable. In addition, the test item was concluded to possibly have an inhibiting effect on microbial activity.
Executive summary:

In a test performed according to OECD 301B (modified Sturm test) and under GLP conditions, Nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide was tested in duplicate for readily biodegradability. Results showed degradation levels of 9% and 13% after 28 days. Therefore, the test item is designated as not readily biodegradable under the experimental conditions of the test. In addition, the toxicity control degraded for 3% and therefore the test item was concluded to possibly have an inhibiting effect on microbial activity. All criteria for acceptability of the test were met and the study was considered to be valid.