(2S)-2-amino-3-[3,5-diiodo-4-(4-methoxyphenoxy)phenyl]propanoic acid

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

Study period:

30 April 2019

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: O-METHYL-L-DIIODOTHYRONIN
Batch Number: B488840
Appearance: White powder
Expiry date: 24 September 2020
Storage: At room temperature, protected from light

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Species:
The (controlled) activated sludge was supplied by the sewage plant for domestic sewage: the prepared activated sludge was continuously aerated (2L/minute) at the test temperature of 22±2oC, for about 6 days until use.
Origin:
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 with shaking and again centrifuged. This procedure was repeated twice.
Preparation of Activated Sludge Inoculum:
An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 5 g dry material per litre was mixed with mineral medium and then aerated under test conditions until use. The pH of the activated sludge inoculum after preparation was: 7.29, just before use: 7.38. A pH adjustment of activated sludge inoculum was not performed.
NaCl (for isotonic saline solution):
Manufacturer: lach:ner; Batch Number: PP/2017/00996, Retest date: 24 November 2019
Pre-conditioning consisted of aerating (2L/minute) activated sludge (in mineral medium
Pre-conditioning of Activated Sludge Inoculum:
The viable cell number of the cultures was determined by these plating experiments by manual colony counting. The approximately cell count of aerated inoculum fell in the range of ~108 - 109 cells/L; therefore, on the day of the test this inoculum was diluted 100000 x with mineral medium to reach the necessary ~104-105cells/L cell concentration. After preparation the sludge was filtered through cotton wool. Pre-conditioning improves the precision of the method. The inoculum was not pre-adapted to the test chemical. ) for 6 days at the test temperature. During the aeration 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, usually 10-1, 10-2, 10-3 and 10-4 dilutions of cultures on nutrient agar plates.
Nutrient agar: Supplier: MERCK; Lot Number: VM817950, Expiry date: 12 February 2023
The approximately cell count of applied, aerated inoculum was 1.75 x 106/L, fell in the range of 104-106/L.

Duration of test (contact time):

ca. 28 d

Initial conc.:

ca. 5 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation:

other: nitrite and nitrate concentrations

Details on study design:

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.
The chosen test item concentration of 5.0 mg/L investigated in the main test was based on the calculated theoretical oxygen demand (ThODNH3 assuming that no nitrification occurs) of 0.98 mg O2/mg test item and based on the information about the toxicity of the test item (based on preliminary toxicity test results).
Under the test conditions ready biodegradation of this test item was not noticed, the percentage biodegradation of O-METHYL-L-DIIODOTHYRONIN reached a mean of 13.8 % after 28 days based on its ThODNH3. The highest biodegradability value of 14.7 % was noticed on the 21st day of the test.
The concurrently conducted analytical determination of possible nitrite and nitrate development demonstrated that no nitrification occurred (the slight changes in nitrite and nitrate concentrations in the 28-day samples were caused likely by a technical effect: turbidity and/or discoloration). Therefore, the biodegradability value of the test item was calculated based on its ThODNH3; any correction, based on the measured nitrite and/or nitrate content was not performed.
The reference item Sodium benzoate was sufficiently degraded to a mean of 73.6 % after 14 days, and to a mean of 80.9 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum. The biodegradability reached its plateau on about the 7th day and from this day the slight changes were considered as being within the biological variability range of the applied test system.
In the toxicity control containing both, the test item and the reference item, a mean of 35.7 % biodegradation was noted within 14 days and 36.2 % 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). The biodegradability in the toxicity control reached its plateau on about the 7th day and from this day the slight changes were considered as being within the biological variability range of the applied test system.

Preliminary study:

In the preliminary toxicity test the test item was investigated at 5.0 mg/L concentration for 9 days and no toxic effect of the test item was noticed. The test item solubility, behavior, and toxicity were tested in a 9-day preliminary experiment. The test design was the same as described at the main experiment. In the preliminary experiment the test item was investigated at the concentration of 5.0 mg/L. No toxic effect of the test item was noticed at this investigated concentration level.

Test performance:

A sufficient number of Winkler flasks were cleaned with 5 – 10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing standing for about 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the previously described test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly closed with glass stopper.
Sulphuric acid: Supplier: AMRESCO, LLC; Batch Number: 1257C318, Expiry date: 30 June 2020;
Potassium iodide: Supplier: REANAL (lach:ner); Batch Number: PP/2017/06465, Retest date: 12 January 2019;
Iodine: Supplier: Alfa Aesar; Batch Number: 10191462; Retest date: 18 June 2025.
The number of test bottles was the following:
 10 (+2 reserve) bottles containing the test item and inoculum (source: bottle 1a and 1b);
 10 (+2 reserve) bottles containing the sodium benzoate and inoculum (procedure control) (source: bottle 2a and 2b);
 10 (+2 reserve) bottles containing only inoculum (inoculum control) (source: bottle 3a and 3b);
 10 (+2 reserve) bottles containing the test item, sodium benzoate and inoculum (toxicity control) (source: bottle 4a and 4b).

Key result

Parameter:

% degradation (O2 consumption)

Value:

>= 13.1 - <= 14.7

Sampling time:

28 d

Key result

Parameter:

BOD5

Value:

> 0.13 - < 0.14 mg O2/g test mat.

Results with reference substance:

The reference item Sodium benzoate was sufficiently degraded to a mean of 73.6 % after 14 days, and to a mean of 80.9 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item was considered to be not ready biodegradable. According to the test guidelines the pass level for ready biodegradability is 60 % of ThODNH3.

Executive summary:

The measured dissolved oxygen concentrations in the inoculum control, test item and procedure control bottles did not correspond to the consumed oxygen of ammonium oxidation processes. The oxygen uptake resulting from a possible ammonium oxidation did not influence the amount of oxygen taken up by microbial population. Therefore, any correction of the measured dissolved oxygen concentrations was considered as not necessary. The measured nitrite-nitrate concentration values were caused likely by a technical effect (possible discoloration of the solutions and/or turbidity).

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 higher than 25 % within 14 days.

The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.

Description of key information

The test item was considered to be not ready biodegradable. According to the test guidelines the pass level for ready biodegradability is 60 % of ThODNH3.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater