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Environmental fate & pathways

Biodegradation in water: screening tests

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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:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
January 1998
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
Part VI: “Closed Bottle Test (Method C.4-E)”. Dated May 30, 2008
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
“Ready Biodegradability: Closed Bottle Test”, adopted July 17, 1992
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Name:LEVOTHYROXINE ACID Batch Number:B486066 Active component: 100 % Appearance:White, slightly creme, powder Expiry date: 27 August 2020 Storage: At room temperature, protected from light Remark: Based on the provided CAS Number (51-48-9 ) the chemical formula of the test item is C15H11I4NO4
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Activated sludge, microorganisms from a domestic waste water treatment plant: the (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 17 January 2019 (six days before the main test). The prepared activated sludge was continuously aerated (2L/minute) at the test temperature of 22±2°C, for about 6 days until use.

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 (see Section 5.4) and then aerated under test conditions until use. The pH of the activated sludge inoculumafter 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
Duration of test (contact time):
ca. 28 d
Initial conc.:
ca. 9 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test Item Concentration
The chosen test item concentration of 9.0 mg/L to be investigated in the main test was based on the calculated theoretical oxygen demand (ThODNH3) and available information about the toxicity of the test item (based on preliminary toxicity test results).
For the preparation of test item test solutions, at first the suitable amount (180 mg) of LEVOTHYROXINE ACID was suspended (using ultrasonic bath for about 2 min.) in the respective volume (1000 mL) of aqueous test medium (mineral medium) to prepare a 180 mg/L stock solution (homogeneous opalescent suspension). The test item stock solution (homogeneous opalescent suspension) was continuously stirred until use to ensure a good dispersion and homogeneity (extra care was taken to avoid air bubbles in the stirred solution). During the incubation period the test solutions were not stirred. The stock solution was adequately diluted in the test item containing test groups. The test solutions were freshly prepared in the testing laboratory at the beginning of the experiment.


Stock Solutions
In water (purified, deionized) analytical grade salts were added to prepare the following stock solutions:
A) Solution:
KH2PO48.50 g
K2HPO421.75 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

(The “D” stock solution was prepared on the day of the mineral medium preparation and was not further stored).

Ratio of Ingredients
1 mL of each of the stock solutions A - D) were combined and filled to a final volume of 1000 mL with water (purified deionised). The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature. The dissolved oxygen concentration was checked and found 8.92 mg/L. The pH of the mineral medium was 7.38.

Environmental Conditions
The test was carried out in a controlled environment room (during the formulation and oxygen measuring) at a temperature of 22 ± 2°C according to the guideline. The test bottles were incubated in a controlled environment room at 22 ± 2°C, in the dark. Temperature was measured continuously using min/max thermometer and recorded every day. During pre-conditioning of activated sludge inoculum the temperature was 20.0-20.9 °C; during the preparation, aeration and incubation of the mineral medium, preparation of test bottles (units), the temperature was 20.0-21.1°C. During the incubation (28 days) of the test units the temperature range was the following: 19.9-20.1°C. The oxygen concentration of test water (mineral medium) was in the range of 8-9 mg/L. It was measured at the start of the test and found to be 8.92 mg/L.The pH was checked prior study start and found to be 7.38.

Test Procedure

The Test Groups
1.) Test item bottels (1a and 1b)
250 mL test item stock solution (180 mg/L):[theoretical oxygen demand (ThODNH3 assuming that no nitrification occurs) of 0.58 mg O2/mg test item]
10 mL activated sludge inoculum ad. 5000 mL mineral medium
In 9.0 mg/L test item concentration, respectively the ThODNH3 corresponds to about 9.0 x 0.58 = 5.22 mg O2/L.

2.) Procedure Control, Sodium benzoate (bottles 2a and 2b):
50 mL reference item stock solution (300 mg/L):(ThODNH3 of reference item: 1.67 mg O2 per mg),
10 mL activated sludge inoculum ( final concentration: 2 mL/L),
ad. 5000 mL mineral medium

3.) Inoculum Control (bottles 3a and 3b):
10 mL activated sludge inoculum ( final concentration: 2 mL/L),
ad. 5000 mL mineral medium;

4.)Toxicity Control (bottles 4a and 4b)
250 mL test item stock solution (180 mg/L),
50 mL reference item stock solution (300 mg/L),
10 mLactivated sludge inoculum (final concentration: 2 mL/L),
ad. 5000 mL mineral medium;

Preliminary study:
Preliminary Experiment
The test item solubility, behavior, and toxicity were tested in a 9-day preliminary experiment. In the preliminary experiment the test item was investigated at the concentration of 10.0 mg/L. Unequivocal toxic effect of the test item was not noticed at this investigated concentration level; but at slightly lowered concentration level (9 mg/L) no toxicity can be guaranteed.
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.
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).

Oxygen Measurements
The incubation period of the closed bottle test was 28 days. The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method]. Oxygen concentration measurements were performed in all duplicate bottles in the test item, procedure control, inoculum control and toxicity control groups on days 0, 7, 14, 21 and 28.
Key result
Parameter:
% degradation (O2 consumption)
Value:
> 16.6 - < 17.3
Sampling time:
28 d
Key result
Parameter:
BOD5
Value:
ca. 10 mg O2/g test mat.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the test conditions the percentage biodegradation of LEVOTHYROXINE ACID reached a mean of 17.0 % after 28 days based on its ThODNH3. Therefore, the test item can be considered to be not ready biodegradable.
Executive summary:

The purpose of this study was to determine the ready biodegradability of the test itemLEVOTHYROXINE ACID. 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 9.0 mg/L investigated in the main test was based on the calculated theoretical oxygen demand (ThODNH3 assuming that no nitrification occurs) of 0.58 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 LEVOTHYROXINE ACID reached a mean of 17.0 % after 28 days based on its ThODNH3. 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 26.4 % biodegradation was noted within 14 days and 29.7 % 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.

Description of key information

According to the test READY BIODEGRADABILITY IN A CLOSED BOTTLE TEST the percentage biodegradation of LEVOTHYROXINE ACID reached a mean of 17.0 % after 28 days based on its ThODNH3. Therefore, the test item can be considered to be not ready biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable
Type of water:
freshwater

Additional information

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