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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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Appearance: white yellowish weakly odorous powder
Active component: ca. 98.0 %
Storage conditions: In refrigerator at 5±3 °C, protected from light
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, on the day of test. The prepared activated sludge was continuously aerated (2 L/minute) at the test temperature of 22 ± 2 °C, until use.

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 mineral medium and then aerated under test conditions until use. The pH of the activated sludge inoculum after preparation was 7.36. A pH adjustment of activated sludge inoculum was not performed.
The microbial inoculum was prepared on the day of the test and was not pre-conditioned and pre-adapted to the test chemical.
The microbial inoculum was continuously aerated (2 L/minute) at the test temperature of until use.
Based on the laboratory’s experience the prepared microbial inoculum was diluted with mineral medium 10,000 x just before use to reach the necessary 10E4-10E6 cells/L cell concentration. After preparation the sludge was filtered through cotton wool.
The viable cell number of the applied inoculum was determined by plating 0.1 mL of the microbial inoculum itself, and by plating its 10E-1, 10E-2 and 10E-3 dilutions on nutrient agar plates. The viable cell counts were determined by manual colony counting.
Duration of test (contact time):
28 d
Initial conc.:
4.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
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.

Environmental Conditions
The test was carried out in a controlled environment room (during the preparation, aeration and incubation of the mineral medium, 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 20.4 – 20.9 oC.
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: 20.0-20.1 oC.
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 (mineral medium) was in the range of 8-9 mg/L. It was measured at the start of the test and found to be 8.60 mg/L.
The pH was checked prior study start and found to be 7.38. A pH adjustment was considered as not necessary.

Preparation of the Test Solutions
The test substance stock solution with a concentration of 450 mg/L was prepared just before the test. The stock solution was adequately stirred to ensure a good dispersion and homogeneity. The stock solution was adequately diluted in the test item containing test groups. During the incubation period the test solutions were not stirred.

The Test Groups
1.) Test Item (flasks 1a and 1b):
Test Item (bottles 1a and 1b):
60 mL test item stock solution (450 mg/L, according to the calculated theoretical oxygen demand ThODNH3 assuming that no nitrification occurs of 1.24 mg O2/mg test item), 12 mL activated sludge inoculum, ad. 6000 mL mineral medium. 4.5 mg/L test item concentration corresponded to about 4.5 x 1.24 = 5.58 mg O2/L.
2.) Procedure Control, Sodium benzoate (flasks 2a and 2b):
3.0 mg/L reference item concentration corresponded to about 3.0 x 1.67 = 5.01 mg O2/L.
3.) Inoculum Control (flasks 3a and 3b):
Only inoculum was added to the aqueous test medium.
4.) Toxicity Control (flasks 4a and 4b):
Test and reference item stock solutions were mixed into the aqueous test medium corresponding to the 4.5 mg/L test item concentration and to 3.0 mg/L concentration of the reference item.
In general: microbial inoculum (2.0 mL per litre) was added to each preparation bottle.
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
32.1
Sampling time:
28 d
Details on results:
Biodegradation of the Test Item:
Under the test conditions the percentage biodegradation of the test item reached a mean of 32.1 % after 28 days based on its ThODNH3. Therefore the test item can be considered to be not ready biodegradable.
See also the attachment.
Results with reference substance:
The reference item Sodium benzoate was sufficiently degraded to a mean of 85.1 % after 14 days, and to a mean of 85.5 % 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 47.4 % biodegradation was noted within 14 days and 47.3 % 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).

Correction for Oxygen Uptake for Interference with Nitrification:

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 nitrate concentration of all samples was less than 0.4 mg/L on day 0, 14 and 21. In the 28-day test item and toxicity control samples the nitrate concentration was less than 0.4 mg/; however in the 28-day inoculum samples in average 1.0 mg/L nitrate was detected.

The nitrite concentration in the toxicity control samples was below the LOQ throughout the study; furthermore in the inoculum control and test item samples on day 0, on 7th and 14th days of the study. However the nitrite concentration of the inoculum control was in average 0.08 mg/L in the 21-day samples and 0.92 mg/L in the 28-day samples and the nitrite concentration of the test item samples was in average 0.10 mg/L in the 21-day samples and 0.15 mg/L in the 28-day samples.

See the attached results.

According to the referred OECD 301 guideline the oxygen consumed in nitrate formation approximates 4.57 x increase of nitrate-N concentration, and the oxygen consumed in nitrite formation is 3.43 x increase of nitrite-N concentration. In this study the change of 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 or not possible. The measured relatively higher nitrite-nitrate concentration values were caused likely by a technical effect (possible discoloration of the solutions and/or turbidity).

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The biodegradation of the test item reached 32.1 % after 28 days.
Executive summary:

The biodegradability of the test item was investigated according to the OECD 301 guideline with the closed bottle test, measuring the O2-consumption.

The biodegradation of the test item reached 32.1 % after 28 days.

The concurrently conducted analytical determination of possible nitrite and nitrate development demonstrated that no nitrification occurred. 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 85.1 % after 14 days, and to a mean of 85.5 % after 28 days of incubation, based on ThODNH3.

In the toxicity control containing both, the test item and the reference item, a mean of 47.4 % biodegradation was noted within 14 days and 47.3 % biodegradation after 28 days of incubation. 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.

Description of key information

The biodegradation of the test item reached 32.1 % after 28 days.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

[Type of water: freshwater]