5-potassium hydrogen L-glutamate

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:

2021

Report date:

2021

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

1

Reference substance name:

5-potassium hydrogen L-glutamate

EC Number:

243-094-0

EC Name:

5-potassium hydrogen L-glutamate

Cas Number:

19473-49-5

Molecular formula:

C5H9NO4.K

IUPAC Name:

potassium 5-oxido-5-oxo-L-norvaline

Test material form:

solid

Specific details on test material used for the study:

5.1.1 Name and Data of Test Item
Name: L-Glutamic acid potassium salt monohydrate[1]
Batch number: VG29748063 / Vendor Lot: SLCJ1327
CAS number: 6382-01-0
Description: White powder
Expiry date: 01 December 2022
Purity: 100 %
Storage conditions: Room temperature (15-25 ºC)
[1] Anhydrous substance name: 5-potassium hydrogen L-glutamate, CAS: 19473-49-5
Remark: The theoretical oxygen demand (ThODNH3) value of the test item was calculated based on the
provided information about the test item CAS number.
For calculations the following molecular formula and molecular weight was applied:
Molecular formula: C5H8KNO4 x H2O; molecular weight: 203.23 g/mol (the anhydrous form: 185.22
g/mol); ThODNH3: 0.78 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:

Species:
Activated sludge, microorganisms from a domestic waste water treatment plant.
Origin:
The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in
Balatonfüred, Hungary, on June 25, 2021 (six days before the test). The prepared activated
sludge was continuously aerated (2 L/minute) at the test temperature of
20.0 – 20.8 °C, for about 6 days.
Preparation of Activated Sludge Inoculum:
The activated sludge used for this study 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 (5.688 g wet weight), dried and the
ratio of wet sludge to dry weight (0.5394 g dry weight) was determined. Based on this ratio,
calculated amount of wet sludge (5 g dry weight that was equivalent to 52.725 g wet sludge)
was suspended in mineral medium (Section 5.4; ad. 1000 mL) to yield a concentration
equivalent to about 5 g per litre (on dry weight basis). The prepared activated sludge
inoculum was aerated under test conditions (for 6 days) until use.
The pH of the activated sludge inoculum after preparation was 7.49, just before use the pH
was: 7.28. A pH adjustment of activated sludge inoculum was not performed.
NaCl (for isotonic saline solution): Manufacturer: lach:ner;
Batch Number: PP/2020/00452,
Retest date: 28 August 2022
Pre-conditioning of Activated Sludge Inoculum:
Pre-conditioning consisted of aerating (2 L/minute) activated sludge (in mineral medium1)
for 6 days (from June 25 to July 01, 2021) at test temperature (the actual temperature: 20.0 –
20.8 °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 and 10-4 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.
The approximately cell count of aerated inoculum was ~ 108 - 109/L; therefore, before the
test the inoculum was further diluted 100 000x 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: VM883450, Expiry date: 23 April 2024

Duration of test (contact time):

ca. 28 d

Initial test substance concentration

Initial conc.:

ca. 6.5 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-Glutamic acid potassium 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 3.0 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 6.5 mg/L. The chosen test item
concentration was based on the solubility properties of the test item (investigated in a non-GLP
solubility trial); furthermore, on the calculated ThODNH3 value of the test item, based on the
provided molecular structure (ThODNH3: 0.78 mg O2/mg).
The pre-experiment on solubility was not performed in compliance with the GLPRegulations and are excluded from the Statement of Compliance in the final report, but the
raw data of this test will be archived under the study code of the present study.

Test performance:

The study was considered as valid since the oxygen depletion in the inoculum control was
1.48 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 0.76 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, at the end of the test or
at the end of the 10-d window was not greater than 20 %.
The highest difference between the duplicate biodegradability values was 17 % (noticed in
the test item group, on the 28th day of the test), was lower than 20 %.
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 75.0 % 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. 61.7

Sampling time:

7 d

% Degradation 2

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 75.5

Sampling time:

28 d

Details on results:

Under the test conditions the percentage biodegradation of L-Glutamic acid potassium salt
monohydrate reached a mean of 75.5 % after 28 days based on its calculated ThODNH3.
In this test the test item biodegradation curve reached its plateau about on the 14th day, from
that day slight increases in biodegradability values (the slight changes were considered as
being within the biological variability range of the applied test system) occurred. For
informative reason the test was not stopped before the 28th day.
The percentage biodegradation of L-Glutamic acid potassium salt monohydrate reached a
mean of 61.7 % (based on its ThODNH3) already on the 7th day of the test. The 10-day
window criterion for ready biodegradation was unequivocally fulfilled, 10 % biodegradation of
the test item (derived from the biodegradation curve) was reached on about the 1st day of the test
and the biodegradation reached the 60 % level between the 6th and 7th days of the test.
The pass level for biodegradability, 60 % removal of ThODNH3, was reached in about 6 days
from the attainment of 10 % biodegradation.
The 10-day window calculation is based on the biological observations and the values were
derived from the biodegradation curve. Based on the obtained values the test item is
considered to be readily biodegradable.

BOD5 / COD results

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to a mean of 75.0 % after
14 days, and to a mean of 78.3 % after 28 days of incubation, based on ThODNH3, thus
confirming the suitability of the used activated sludge inoculum.

Any other information on results incl. tables

In the toxicity control containing both, the test item and the reference item, a mean of
48.3 % biodegradation was noted within 14 days and the calculated biodegradation was
48.8 % after 28 days of incubation (the biodegradation values reached a plateau on about the
7thday of the experiment and from this day on, the slight subsequent changes were
considered as being within the biological variability range of the applied test system).
Thus, the test item can be assumed to not inhibit the activated sludge microorganisms
(higher than 25 % degradation occurred within 14 days).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item, L-Glutamic acid potassium salt monohydrate is considered to be ready
biodegradable since fulfilled the pass level for ready biodegradability that is the
removal of 60 % of ThODNH3.
The relationship between oxygen uptake resulting from a possible ammonium oxidation
(measured exclusively in the start test item containing samples) and oxygen uptake of
applied microbial population was equivocal, any correction of the measured dissolved
oxygen concentrations was not possible.
Likely technical effects (possible discoloration of the solutions and/or turbidity) influenced
the nitrite concentration determinations.
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 48.3 % within 14 days, and therefore higher than 25 %.
The percentage biodegradation of the reference item (75.0 % 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-Glutamic acid potassium 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 ± 2oC) 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 3.0 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 6.5 mg/L. The chosen test item
concentration was based on the performed preliminary experiment: on the solubility properties
of the test item; furthermore, on the calculated ThODNH3value of the test item, based on the
provided molecular structure (ThODNH3: 0.78 mg O2/mg).
Under the applied test conditions, ready biodegradability of L-Glutamic acid potassium salt
monohydrate was noticed. The percentage biodegradation ofL-Glutamic acid potassium
salt monohydratereached a mean of 61.7 % (based on its ThODNH3) already on the 7thday
of the test. The 10-day window criterion for ready biodegradation was unequivocally fulfilled,
10 % biodegradation of the test item (derived from the biodegradation curve) was reached on
about the 1stday of the test and the biodegradation reached the 60 % level between the 6thand
7thdays of the test. The pass level for biodegradability, 60 % removal of ThODNH3, was
reached in about 6 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.
The concurrently conducted analytical determination of possible nitrite and nitrate development
showed slight changes in nitrite concentrations in start (0-day) test item and toxicity control
samples, while the nitrite and nitrate concentrations remained below the quantification limits
(LOQ) in all other samples taken and measured throughout the test. Likely technical effects
(turbidity and/or discoloration) influenced the nitrite concentration determination at the start
samples. At the biodegradability calculations any correction, based on the measured nitrite
and/or nitrate content was not necessary.
The reference item sodium benzoate was sufficiently degraded to a mean of 75.0 % after
14 days, and to a mean of 78.3 % 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
48.3 % biodegradation was noted within 14 days and the calculated biodegradation was
48.8 % 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.