Phosphoric acid, 2-ethylhexyl ester, sodium salt

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:

weight of evidence

Study period:

from 2019-05-09 to 2019-07-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: sewage plant for domestic sewage in Balatonfüred, Hungary
- Storage conditions: continuously aerated (2 L/minute) at the test temperature of 20.0 – 20.7 °C
- Storage length: 7 days
- Preparation of inoculum for exposure: The activated sludge was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution by shaking and was again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed (5.603 g wet weight), dried and the ratio of wet sludge to dry weight (0.4727 g dry weight) was determined. Based on this ratio, calculated amount of wet sludge (5 g dry weight that was equivalent to 59.27 g wet sludge) was suspended in mineral medium (ad. 1000 mL) to yield a concentration equivalent to about 5 g per litre (on dry weight basis). The pH of the activated sludge inoculum after preparation was 7.44, just before use the pH was: 7.31. A pH adjustment of activated sludge inoculum was not performed.
- Pretreatment: Before use the cell count of inoculum was checked as follows: the viability of the cultured sludge was determined by plating 0.1 mL of the different, 1E-1, 1E-2, 1E-3 and 1E-4 dilutions of cultures on nutrient agar plates. Plates were incubated at 37 °C for 24 hours. The viable cell number of the cultures was determined by these plating experiments by manual colony counting.
- Concentration of sludge: 5 g per litre (on dry weight basis)
- Initial cell/biomass concentration: ~1E8 - 1E9/L, before the test the inoculum was further diluted 10 000x with mineral medium to reach the necessary ~1E4 – 1E6 cells/L cell concentration
- Water filtered: no
- Type and size of filter used: cotton wool

Duration of test (contact time):

28 d

Initial conc.:

3 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

other: Chemical Oxygen Demand (COD)

Details on study design:

TEST CONDITIONS
- Composition of medium:
A) Solution: KH2PO4 8.50 g + K2HPO4 21.75 g + Na2HPO4 x 12H2O 67.16 g + NH4Cl 0.50 g + Water ad 1000 mL
B) Solution: CaCl2 x 2 H2O 36.40 g + Water ad 1000 mL
C) Solution: MgSO4 x 7 H2O 22.50 g + Water ad 1000 mL
D) Solution: FeCl3 x 6 H2O 0.125 g + Water ad 500 mL
- Test temperature: 20.0 - 20.6 °C
- pH: 7.42
- pH adjusted: no
- Aeration of dilution water: for 20 minutes
- Initial oxygen concentration: 8.81 mg/L

TEST SYSTEM
- Culturing apparatus: Narrow necked, Winkler bottles with glass stoppers
- Number of culture flasks/concentration: 16
- Measuring equipment: O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method]

SAMPLING
- Sampling frequency: in all duplicate bottles in all groups on days 0, 2, 5, 7, 12, 14, 21 and 28

CONTROL AND BLANK SYSTEM
- Inoculum blank: only inoculum
- Toxicity control: the test item, sodium benzoate and inoculum

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

26

Sampling time:

28 d

Details on results:

Under the test conditions the percentage biodegradation of the test item reached a mean of 26.0 % after 28 days based on its COD. Therefore, the test item can be considered to be not readily biodegradable but primarily biodegradable.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to a mean of 69.3 % after 14 days, and to a mean of 72.9 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.

Correction for Oxygen Uptake for Interference with Nitrification

For that reason at this N-containing test item, the oxidized nitrogen (nitrate and nitrite) concentrations were determined following each oxygen measurement with photometric method using nitrite and nitrate cell tests. The LOQ (Limit Of Quantification) of the measurements was 0.03 mg NO2/L and 0.4 mg NO3/L, respectively. The nitrite concentration in the start, 7-, 14-, and 21-day samples (test item, inoculum control and toxicity control) was below the LOQ. Furthermore, the nitrite concentrations remained below the LOQ in the 28-day inoculum control and toxicity control samples. In the 28-day test item samples 0.13 mg/L nitrite concentrations were measuered in both parallels. The nitrate concentration of the samples was less than 0.4 mg/L in all measurement occasions, throughout the study.

In the test item containers the oxygen consumed by ammonium oxidation processes was 0.13 x 3.43 = 0.45 mg/L. The measured average oxygen depletion (not corrected with inoculum control) from 21st day to 28th day of the test was in average 0.32 mg/L. The change of the measured dissolved oxygen concentrations in the test item bottles did not correspond to the consumed oxygen of ammonium oxidation processes. Because of the relationship between oxygen uptake resulting from a possible ammonium oxidation and oxygen uptake of applied microbial population was equivocal, any correction of the measured dissolved oxygen concentrations was considered as not possible. The calculated oxygen consumed by ammonium oxidation processes was significantly higher, than the measured oxygen depletions. The measured higher nitrite concentration values were caused likely by a technical effect (possible discoloration of the solutions and/or turbidity).

Biodegradation of the Toxicity Control

In the toxicity control containing both, the test item and the reference item, a mean of 31.2 % biodegradation was noted within 14 days and 40.9 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

The test item was considered to be not readily biodegradable (26 % biodegradation on day 28). As the pass level of 20 % was achieved, the test item is considered to be primarily inherently biodegradable.

Executive summary:

The ready biodegradability of the test item was determined with a non-adapted domestic activated sludge over a test period of 28 days in the Closed bottle test according to OECD 301D, Regulation (EC) No. 440/2008 Method C.4 -E and EPA OPPTS 835.3110 under GLP. The test item was tested at a concentration of 3 mg/L, corresponding to a chemical oxygen demand (COD) of 1.78 mg O2/L. The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method]. The total oxidised nitrogen (nitrite and nitrate) was measured in all vessels using Nitrite and Nitrate Cell Tests (MERCK®). The change of the measured dissolved oxygen concentrations in the test item bottles did not correspond to the consumed oxygen of ammonium oxidation processes. Because of the relationship between oxygen uptake resulting from a possible ammonium oxidation and oxygen uptake of applied microbial population was equivocal, any correction of the measured dissolved oxygen concentrations was considered as not possible. The biodegradation of the reference item was not inhibited by the test item in the toxicity control. The biodegradation of the test item came to 26.0 % after 28 days. In conclusion, the test item is not readily biodegradable as the pass level of 60 % degradation was not reached in a 10-d-window within the 28 days period of the study but a pass level of 20 % was reached and the test item is considered to be primarily inherently biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Juni - September 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)

Version / remarks:

May 12, 1981

GLP compliance:

no

Oxygen conditions:

not specified

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum: wastewater sewage plant ARA-Birsig, Therwil/Switzerland

Duration of test (contact time):

28 d

Initial conc.:

104 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: The test medium (30 mL per flask) was prepared according to the OECD Guideline 301 E
- Test temperature: 20 - 25 °C
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 50 mL Erlenmeyer flasks
- Number of culture flasks/concentration: 2
- Measuring equipment: DOC-analyses were performed with the various filtrates using a Technicon Carbon Analyser (Autoanalyser Ilj Technicon Industrial Method No. 451 - 76U) equipped with an Automatic Data Processing System (Dr. H.v. Meyenburg AS, CH-8712 Stafa (ZH) Switzerland)
- Test performed in open system: no, loosely covered with an aluminium foil

SAMPLING
- Sampling frequency: day 0, 7, 14 and 28
- Samples were centrifuged or filtered (pore size: 0.2 micrometres) and thereafter directly analysed. If analyses were performed later, 0.05 mL of a 1 % HgC12 solution per 10 mL filtrate were added. Thereafter, the samples were stored for 24 hours at 2 - 4 °C or for a longer period of time at -18 to 20 °C.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Reference substance: aniline (26.3 mg DOC/L)

STATISTICAL METHODS:
Degradation was stated as the percentage DOC-removal within 28 days with respect to the test article (% DOC-removal)

Reference substance:

aniline

Key result

Parameter:

% degradation (DOC removal)

Value:

55.4

Sampling time:

28 d

Details on results:

After 14 days a degradation of 34.4 % was determined, after 28 days a degradation of 55.4 %.

Results with reference substance:

In parallel preparations with the reference substance, aniline showed a degradation of 95 % was achieved within 14 days.

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable, fulfilling specific criteria

Conclusions:

Within the test period of 28 days, a degradation of 55.4 % was determined for the test item.

Executive summary:

The biodegradation of the test item was determined in a test according to OECD 301E. The test substance is suspended in mineral medium, inoculated with a mixed population of aquatic microorganisms and incubated for 28 days in the dark at 20 - 25 °C. During this period, the biodegradation of the test substance is determined on the basis of the reduction of DOC. Within the test period of 28 days, a degradation of 55.4 % was determined. The degradation of the toxicity control was 95 % after 14 days.

Description of key information

The biodegradability potential of the test item was determined in an OECD 301E and OECD 301D studies, where the test item reached 55.4 % and 26 % biodegradation in 28 days, respectively. Thus, the test item is considered to be primarily inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, fulfilling specific criteria

Type of water:

freshwater

Additional information

OECD 301D

The ready biodegradability of the test item was determined with a non-adapted domestic activated sludge over a test period of 28 days in the Closed bottle test according to OECD 301D, Regulation (EC) No. 440/2008 Method C.4 -E and EPA OPPTS 835.3110 under GLP. The test item was tested at a concentration of 3 mg/L, corresponding to a chemical oxygen demand (COD) of 1.78 mg O2/L. The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method]. The total oxidised nitrogen (nitrite and nitrate) was measured in all vessels using Nitrite and Nitrate Cell Tests (MERCK®). The change of the measured dissolved oxygen concentrations in the test item bottles did not correspond to the consumed oxygen of ammonium oxidation processes. Because of the relationship between oxygen uptake resulting from a possible ammonium oxidation and oxygen uptake of applied microbial population was equivocal, any correction of the measured dissolved oxygen concentrations was considered as not possible. The biodegradation of the reference item was not inhibited by the test item in the toxicity control. The biodegradation of the test item came to 26.0 % after 28 days. In conclusion, the test item is not readily biodegradable as the pass level of 60 % degradation was not reached in a 10-d-window within the 28 days period of the study but a pass level of 20 % was reached and the test item is considered to be primarily inherently biodegradable.

OECD 301E

The biodegradation of the test item was determined in a test according to OECD 301E. The test substance is suspended in mineral medium, inoculated with a mixed population of aquatic microorganisms and incubated for 28 days in the dark at 20 - 25 °C. During this period, the biodegradation of the test substance is determined on the basis of the reduction of DOC. Within the test period of 28 days, a degradation of 55.4 % was determined. The degradation of the toxicity control was 95 % after 14 days.

Based on the results from the two studies, it was concluded that the test item is primarily inherently biodegradable in accordance with ECHA guidance R7B (2017).