(6R-trans)-7-amino-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl)thio]methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic 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:

2016

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)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

anaerobic

Inoculum or test system:

activated sludge, domestic, 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, seven days before the main test. The prepared activated sludge was continuously aerated (2 L/minute) at the test temperature of 22 ± 2 oC, for about 7 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 with shaking and again centrifuged. This procedure was repeated twice.
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 reconstituted water and then aerated under test conditions (for 7 days) until use. The pH of the activated sludge inoculum after preparation was 7.81, just before use the pH was: 7.26. A pH adjustment of activated sludge inoculum was not performed.

Pre-conditioning of Activated Sludge Inoculum:
Pre-conditioning consisted of aerating (2 L/minute) activated sludge (in mineral medium, reconstituted water for 7 days at the test temperature (the actual temperature: 20.4 – 22.0 °C). 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 10E-2, 10E-3 and 10E-4 dilutions of cultures on nutrient agar plates.
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 10E8-10E9 /L; therefore on the day of the test this inoculum was diluted 1000 x with reconstituted water to reach the necessary 10E5-10E6 cells/L cell concentration. After preparation the sludge was filtered through cotton wool. Pre-conditioning improves the precision of the method. ) for 7 days at the test temperature (the actual temperature: 20.4 – 22.0 °C). 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 10E-2, 10E-3 and 10E-4 dilutions of cultures on nutrient agar plates.
The inoculum was not pre-adapted to the test chemical.

Duration of test (contact time):

28 d

Initial conc.:

5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Formulation:
The test item solubility was determined in the preliminary test. The test item solution (suspension) with a concentration of 400 mg/L was prepared by mechanical dispersion, and about 5 minutes ultrasonic treatment. At this concentration level homogeneous, opalescent suspension was obtained. In the main experiment the test solutions was freshly prepared at the beginning of the experiment, in the testing laboratory as follows: at first the suitable amount (100 mg) of 7-ACT was dissolved in the respective volume (2000 mL) of aqueous test medium and a stock solution with a concentration of 50 mg/L was prepared. The test item stock solution was prepared with short (<10 min.) ultrasonic treatment and adequate stirring to ensure a good dispersion (extra care was taken for avoiding of air bubbles in the stirred solution). During the performance of the test the test solutions were mixed by mechanical stirring to ensure a good dispersion (however during the incubation period the test solutions were not stirred). The stock solution was adequately diluted in the test item containing test groups.

Environmental Conditions:
The test was carried out in a controlled environment room (during the preparation, aeration and incubation of the reconstituted water, preparation of test bottles (units), during the formulation, oxygen and pH measuring) at a temperature of 22 ± 2 °C according to the guideline. The actual temperature range was 20.6 - 21.9 °C.
The test flasks were incubated in incubator at 22 ± 2 °C, in the dark. During the incubation (28 days) of the test units the temperature range was the following: 20.0-20.2 °C.
During the pre-conditioning of activated sludge inoculum the temperature was 20.4-22.0 °C.
Temperature was measured continuously using min/max thermometer (in controlled environment room) or built-in thermometer (in incubator) and noticed at least once a day.
The oxygen concentration of test water (reconstituted water) was in the range of 8-9 mg/L. It was measured at the start of the test and found to be 8.27 mg/L.
The pH was checked prior study start and found to be 7.44. A pH adjustment was considered as not necessary.
Test Units: Type and Size: Winkler bottles (300 mL, coded) with special neck and glass stoppers.

Preliminary Toxicity Test
The test item solubility, behavior, and toxicity were tested in a 14-day preliminary toxicity experiment. The test design was the same as described at the main experiment.
In the preliminary toxicity test the test item was investigated at 4 mg/L concentration and any toxic effect of the test item was not noticed 42.8 % biodegradability occurred within 14 days in the toxicity control group.

The Test Groups
In general: microbial inoculum (2.0 mL per litre) was added to each preparation bottle.
1.) Test Item (flasks 1a and 1b)
2.) Procedure Control, Sodium benzoate (flasks 2a and 2b)
3.) Inoculum Control (flasks 3a and 3b)
4.) Toxicity Control (flasks 4a and 4b): Test (500 mL) and reference item (50 mL) stock solutions were mixed into the aqueous test medium (ad. 5000 mL) corresponding to the 5.0 mg/L test item concentration [chosen based on the calculated ThODNH3 of the test item and the preliminary experiment] and to 3.0 mg/L concentration of the reference item.

Measurement of Total Oxidized N (Nitrite and Nitrate):
Because of the nitrogen content of the test item, samples for nitrate and nitrite analysis were taken from all vessels (of test item, inoculum control and toxicity control group) and the oxidized nitrogen (nitrate and nitrite) concentrations were measured. For technical reason the nitrate and nitrite analysis of the start (day 0) and 7-day samples was performed together on the 7th day of the test, the analysis of 14-day samples was performed one week later, together with the 21-day samples, on the 21st day of the test. The start (day 0) and 14-day samples were adequately stored in freezer until determination of nitrate and nitrite. The 7-, 21- and 28-day samples were analysed directly after oxygen measurements.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

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 3 mg/L. No toxic effect of the test item was found at this investigated concentration.

Test performance:

Correction for Oxygen Uptake for Interference with Nitrification
Errors due to not considering nitrification in the assessment by oxygen uptake of the biodegradability of test substances not containing N are marginal (not greater than 5%), even if oxidation of the ammonium N in the medium occurs erratically as between test and blank vessels. However, for test substances containing N, serious errors can arise if the observed oxygen uptake is not corrected for the amount of oxygen used in oxidising ammonium to nitrite and nitrate. For that reason at this N-containing test item, the oxidised 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 measured quantities of nitrate in the inoculum control, test item and toxicity control samples were below the LOQ throughout the study and any correction of the oxygen consumption values was considered as not necessary.
The nitrite concentration of the test item and toxicity control samples was below the LOQ throughout the study and in the inoculum control samples on day 0, on 7th, 14th and on 21st day of the study; however in the inoculum control samples in average 0.09 mg/L nitrite was detected in the 28-day samples.
At the biological results, at dissolved oxygen concentration measurements the oxygen uptake resulting from ammonium oxidation did not appear, therefore any correction was considered as not necessary; the measured relatively higher nitrite concentration values were caused likely by a technical effect.

Key result

Parameter:

% degradation (O2 consumption)

Value:

12.4

Sampling time:

28 d

Details on results:

Biodegradation of the Test Item:
Under the test conditions the percentage biodegradation of 7-ACT reached a mean of 12.4% after 28 days based on its ThODNH3. Therefore the test item can be considered to be not ready biodegradable.

Results with reference substance:

Biodegradation of the Reference Item
The reference item Sodium benzoate was sufficiently degraded to a mean of 67.6 % after 14 days, and to a mean of 78.0 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.

Biodegradation of the Toxicity Control
In the toxicity control containing both, the test item and the reference item, a mean of 33.4 % biodegradation was noted within 14 days and 34.7 % 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).

Validity of the Study:

The study was considered as valid because of the oxygen depletion in the inoculum control was 1.24 in average, did not exceed the 1.5 mg O2/L after 28 days. The residual oxygen concentration in the test flasks did not drop below 0.5 mg O2/L at any time. The lowest value was 3.07 mg O2/L, it was measured on the 28th day in the procedure control.

The difference of duplicate values for the degradation at the plateau or at the end of the test was not greater than 20 %.

At the biodegradation plateaus (test item, procedure control, and toxicity control groups) or at the end of the test the highest difference (~16 %) between duplicate values for degradation was calculated in the toxicity control group, it was observed on the 21st day.

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 67.6 % on the 14th day.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The biodegradation of the test item reached 12.4 % after 28 days.

Executive summary:

A Closed Bottle Test was performed.

The biodegradation of the test item reached 12.4 % after 28 days.

The measured quantities of nitrate in the inoculum control, test item and toxicity control samples were below the LOQ throughout the study. The measured quantities of nitrite in the inoculum control on the 28th day samples did not account for any correction of the oxygen consumption values.

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 34.7 %, i.e.,higher than 25 %, within 28 days.

The percentage biodegradation of the reference item, 78.0 % in 28 days, confirms the suitability of the used activated sludge inoculum.

Description of key information

The biodegradation of the test item reached 12.4 % after 28 days.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable