L-aspartic acid, sodium salt

Biodegradation in water: screening tests

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

17th July 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

sodium 2-amino-3-carboxypropanoate

Cas Number:

323194-76-9

Molecular formula:

C4H7NO4.xNa

IUPAC Name:

sodium 2-amino-3-carboxypropanoate

Test material form:

solid: particulate/powder

Details on test material:

Name: L-aspartic acid, sodium salt monohydrate 1
Batch/Lot number: Z201125/ VG29848563
CAS number: 323194-76-9
Anhydrous substance name: L-aspartic acid, sodium salt, CAS: 17090-93-6
Appearance: Solid, white, crystalline powder
Purity: 98%
Expiry date: 24 November 2022
Storage conditions: Room temperature (15-25 ºC), protected from humidity (tight closed
container)

Specific details on test material used for the study:

Test item: L-aspartic acid, sodium salt monohydrate
Lot No.: Z201125 / VG29848563
CAS No.: 323194-76-9 *
Purity: 99.9 %
Molecular formula: C4H6NO4Na • H2O
Molecular weight: 173.1 g/mol
Appearance: Solid, white crystalline powder
Expiry date: 24 November 2022
Storage conditions: At room temperature, protected from humidity (tightly
closed container)
Safety precautions: According to the SDS
* The L-aspartic acid sodium salt (anhydrous) CAS Number is: 17090-93-6.
Test item information is based on written information provided by the Sponsor.
Remark: The theoretical oxygen demand (ThODNH3) value of the test item was calculated based on the
provided molecular formula: C4H6NO4Na.
For calculations this molecular formula and the following molecular weight was applied:
molecular weight: 155 g/mol; ThODNH3: 0.62 mg O2/mg test item.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Remarks:

The inoculum was not pre-adapted to the test chemical

Details on inoculum:

Test item: L-aspartic acid, sodium salt monohydrate
Lot No.: Z201125 / VG29848563
CAS No.: 323194-76-9 *
Purity: 99.9 %
Molecular formula: C4H6NO4Na • H2O
Molecular weight: 173.1 g/mol
Appearance: Solid, white crystalline powder
Expiry date: 24 November 2022
Storage conditions: At room temperature, protected from humidity (tightly
closed container)
Safety precautions: According to the SDS
* The L-aspartic acid sodium salt (anhydrous) CAS Number is: 17090-93-6.
Test item information is based on written information provided by the Sponsor.
Remark: The theoretical oxygen demand (ThODNH3) value of the test item was calculated based on the
provided molecular formula: C4H6NO4Na.
For calculations this molecular formula and the following molecular weight was applied:
molecular weight: 155 g/mol; ThODNH3: 0.62 mg O2/mg test item.
Pre-conditioning of Activated Sludge Inoculum:
Pre-conditioning consisted of aerating (2 L/minute) activated sludge (in mineral medium1)
for 7 days (from August 04−11, 2022) at test temperature (the actual temperature: 20.0 –
21.5 °C). Before use the cell count of inoculum was checked as follows: the viability of the
cultured sludge was determined by plating of 0.1 mL undiluted sample and different, 10-1, 10-2,
10-3, 10-4 and 10-5 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.
The approximately cell count of aerated inoculum was ~ 108 /L; therefore, before the test the
inoculum was further diluted adequately with mineral medium to reach the necessary ~104 –
106 cells/L cell concentration. After preparation the sludge was filtered through cotton wool.
Pre-conditioning (see above) improves the precision of the method.
The inoculum was not pre-adapted to the test chemical.
Nutrient agar: Supplier: MERCK; Lot Number: VM923850, Expiry date: 01 April 2025

Duration of test (contact time):

ca. 28 d

Initial test substance concentration

Initial conc.:

ca. 10 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The purpose of this study was to determine the ready biodegradability of the test item
L-aspartic acid, sodium salt monohydrate.
The test item was exposed to activated sludge from the aeration tank of a domestic waste
water treatment plant in completely full and closed bottles in the dark at controlled
temperature (22 ± 2 oC) for 28 days. The biodegradation was followed by the oxygen uptake
of the microorganisms during exposure. As a reference item Sodium benzoate (at a
concentration of 3 mg/L) was tested simultaneously under the same conditions as the test
item, and functioned as a procedure control (reference control). Additionally, inoculum
(containing the filtered inoculum, only) and toxicity (containing both the test item and
reference item) controls were examined.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

Test Item Concentration
The test item was investigated at the concentration of 10 mg/L. The chosen test item
concentration was based on the calculated ThODNH3 value of the test item, based on the
provided molecular structure (ThODNH3: 0.62 mg O2/mg).

Test performance:

The study was considered as valid since the oxygen depletion in the inoculum control was
1.13 mg O2/L on average, and did not exceed the validity criteria of 1.5 mg O2/L after
28 days.
The residual oxygen concentration in the test bottles did not drop below 0.5 mg O2/L at any
time. The lowest value was 0.50 mg O2/L, which was measured on the 28th day in the
toxicity control.
The difference of duplicate values for the degradation at the plateau or at the end of the test
was not greater than 20 %.
The differences between the duplicate biodegradability values in the test item, procedure
and toxicity control groups varied between 0.3-15.1 % throughout the experiment.
The percentage degradation of the reference item reached the level for ready
biodegradability (> 60 %) by exposure day 14. The percentage degradation of the reference
item was 79.4 % on the 14th day.
All validity criteria were met as required by the test guideline OECD 301.

% Degradation

% Degradation 1

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 67.1

Sampling time:

12 d

% Degradation 2

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 75.8

Sampling time:

28 d

Details on results:

Under the applied test conditions, ready biodegradability of L-aspartic acid, sodium salt
monohydrate was noticed. The percentage biodegradation of L-aspartic acid, sodium salt
monohydrate reached a mean of 67.1 % (based on its ThODNH3) on the 12th day of the test.
The 10 % biodegradation of the test item was reached after the 2nd day of the test and the
biodegradation reached the 60 % level before the 12th day of the test, on about the 10th day
(derived from the biodegradation curve), hence the 10-day window criterion for ready
biodegradation was unequivocally fulfilled, The pass level for biodegradability, 60 % removal
of ThODNH3, was reached in about 8 days from the attainment of 10 % biodegradation.
The test item is considered to be ready biodegradable, since fulfilled the pass level for
ready biodegradability that is the removal of 60 % theoretical oxygen demand
(ThODNH3) in a 10-day window.

BOD5 / COD results

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to a mean of 79.4 % after
14 days, and to a mean of 82.4 % after 28 days of incubation, based on ThODNH3.
The percentage biodegradation of the reference item confirms the suitability of the
used activated sludge inoculum.

Any other information on results incl. tables

Under the applied test conditions, ready biodegradability of L-aspartic acid, sodium salt
monohydrate was noticed. The percentage biodegradation of L-aspartic acid, sodium salt
monohydrate reached a mean of 67.1 % (based on its ThODNH3) on the 12th day of the test.
The 10 % biodegradation of the test item was reached after the 2nd day of the test and the
biodegradation reached the 60 % level before the 12th day of the test, on about the 10th day
(derived from the biodegradation curve), hence the 10-day window criterion for ready
biodegradation was unequivocally fulfilled, The pass level for biodegradability, 60 % removal
of ThODNH3, was reached in about 8 days from the attainment of 10 % biodegradation.
The test item is considered to be ready biodegradable, since fulfilled the pass level for
ready biodegradability that is the removal of 60 % theoretical oxygen demand
(ThODNH3) in a 10-day window.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item, L-aspartic acid, sodium salt monohydrate is considered to be ready
biodegradable since fulfilled the pass level for ready biodegradability that is the
removal of 60 % of ThODNH3.
Based on the results of the analytical determination of possible nitrite and nitrate
development, oxygen uptake resulting from a possible ammonium oxidation did not
influence the amount of oxygen taken up by microbial population. Consequently, the
biodegradability value of the test item was calculated based on its calculated ThODNH3 any
correction was not performed.
According to the test guidelines the test item can be assumed as not inhibitory on the
activated sludge microorganisms because the degradation in the toxicity control group
was 43.3 % within 14 days, and therefore higher than 25 %.
The percentage biodegradation of the reference item (79.4 % after 14 days) confirms
the suitability of the used activated sludge inoculum.

Executive summary:

The purpose of this study was to determine the ready biodegradability of the test item
L-aspartic acid, sodium salt monohydrate.
The test item was exposed to activated sludge from the aeration tank of a domestic waste
water treatment plant in completely full and closed bottles in the dark at controlled
temperature (22 ± 2 oC) for 28 days. The biodegradation was followed by the oxygen uptake
of the microorganisms during exposure. As a reference item Sodium benzoate (at a
concentration of 3 mg/L) was tested simultaneously under the same conditions as the test
item, and functioned as a procedure control (reference control). Additionally, inoculum
(containing the filtered inoculum, only) and toxicity (containing both the test item and
reference item) controls were examined.
The test item was investigated at the concentration of 10 mg/L. The chosen test item
concentration was based on the calculated ThODNH3 value of the test item, based on the
provided molecular structure (ThODNH3: 0.62 mg O2/mg).
Under the applied test conditions, ready biodegradability of L-aspartic acid, sodium salt
monohydrate was noticed. The percentage biodegradation of L-aspartic acid, sodium salt
monohydrate reached a mean of 67.1 % (based on its ThODNH3) on the 12th day of the test.
The 10 % biodegradation of the test item was reached after the 2nd day of the test and the
biodegradation reached the 60 % level before the 12th day of the test, on about the 10th day
(derived from the biodegradation curve), hence the 10-day window criterion for ready
biodegradation was unequivocally fulfilled, The pass level for biodegradability, 60 % removal
of ThODNH3, was reached in about 8 days from the attainment of 10 % biodegradation.
The test item is considered to be ready biodegradable, since fulfilled the pass level for
ready biodegradability that is the removal of 60 % theoretical oxygen demand
(ThODNH3) in a 10-day window.
Based on the results of the analytical determination of possible nitrite and nitrate
development, oxygen uptake resulting from a possible ammonium oxidation did not
influence the amount of oxygen taken up by microbial population. Consequently, the
biodegradability value of the test item was calculated based on its calculated ThODNH3; any
correction, based on the measured nitrite and nitrate content was not performed.
The reference item sodium benzoate was sufficiently degraded to a mean of 79.4 % after
14 days, and to a mean of 82.4 % after 28 days of incubation, based on ThODNH3.
The percentage biodegradation of the reference item confirms the suitability of the
used activated sludge inoculum.
In the toxicity control containing both, the test item and the reference item, a mean of
43.3 % biodegradation was noted within 14 days and the calculated biodegradation was
42.8 % after 28 days of incubation.
According to the test guidelines the test item can be assumed as not inhibitory at the
applied concentration level on the activated sludge microorganisms because the
degradation in the toxicity control group was higher than 25 % within 14 days.