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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:

From February the 19th to March the 19th, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 17, 1992

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:

The test solutions used in the test were prepared by mechanical dispersion. The test solutions were freshly prepared at the beginning of the experiment, in the testing laboratory.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of 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 19 February 2014.
- Pretreatment: the coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used 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.
- Cultivation: an aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined. Aliquots of washed sludge suspension, corresponding to 5 g dry material per litre, was mixed with reconstituted water and then aerated until use (200 ml inoculum was prepared). After preparation the sludge was filtered through cotton wool.
- pH adjustment: the pH of the activated sludge inoculum after preparation was 6.81. A pH adjustment before use was not performed.
- Preparation of inoculum for exposure: the microbial inoculum was prepared on the day of the test and was not pre-adapted to the test chemical.
- Aeration: the microbial inoculum was continuously aerated (2 l/minute) at the test temperature of until use.
- Concentration of sludge: in general, microbial inoculum of 2.0 ml per litre was added to each preparation bottle.

Duration of test (contact time):

28 d

Initial conc.:

3 mg/L

Based on:

test mat.

Initial conc.:

6.15 other: mg O2/l

Based on:

COD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

RECONSTITUTED WATER - Stock solution
- Composition: in purified, deionised water analytical grade salts were added (referring to 1000 ml): Solution A) KH2PO 48.50 g, K2HPO 421.75 g, Na2HPO4 x 12 H2O 67.16 g, NH4Cl 0.50 g, water ad 1000 ml; Solution B) CaCl2 x 2 H2O 36.40 g, water ad 1000 ml; Solution C) MgSO4 x 7 H2O 22.50 g, water ad 1000 ml; Solution D*) FeCl3 x 6 H2O 0.25 g, water ad 1000 ml (*the “D” stock solution was prepared on the day of the reconstituted water preparation and was not further stored). 10 ml of the stock solutions A) and 1-1 ml of the stock solutions B - D) were combined and filled to a final volume of 1000 ml with deionised water.
- pH: pH of the reconstituted water was 7.69 (prior study start).
- Aeration of dilution water: 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.

TEST CONDITIONS
- Test temperature: 22 ± 2 °C
- pH adjusted: no.
- Continuous darkness: yes.

TEST SYSTEM
- Test vessels: winkler bottles (300 ml, coded) with special neck and glass stoppers.
- Test group: based on the measured chemical oxygen demand (COD) of the test item (2.05 mg O2/mg test item), at the start of the test a suitable volume (50 ml) of the test item solution was thoroughly mixed into the respective volume (5000 ml) of aqueous test medium.
- Number of test flasks: 10 (+2 reserve) bottles containing the test item and inoculum.
- Number of reference substance flasks: 10 (+2 reserve) bottles containing the reference substance and inoculum.
- Number of blank flasks: 10 (+2 reserve) bottles containing only inoculum.
- Number of toxicity control flasks: 10 (+2 reserve) bottles containing the test item and sodium benzoate.

CONTROL AND BLANK SYSTEM
- Inoculum blank: only filtered inoculum (10 ml) was added to the aqueous test medium (5000 ml). Microbial inoculum (2.0 ml per litre) was added to each preparation bottle.
- Abiotic sterile control: test (50 ml) and reference item (50 ml) stock solutions were mixed into the aqueous test medium (5000 ml) corresponding to the test item concentration of 3 mg/l and to 3.6 mg/l concentration of the reference item.
- Toxicity control: based on the theoretical oxygen demand (ThODNH3) of sodium benzoate (1.67 mg O2 per mg), the stock solution (360 mg/l) of sodium benzoate (50 ml) was thoroughly mixed into the respective volume of aqueous test medium (5000 ml).
- Other:

COD DETERMINATION
In a pre-experiment the COD value was determined in three parallel samples using a COD Cell Test (MERCK) with appropriate controls CombiCheck 50.
For determination of the COD of test item, a test item solution of 10 mg/L was prepared in reconstituted water and measured. The measured values: 21.3 mg O2/l; 20.4 mg O2/l; 19.8 mg O2/l; in average 20.5 mg O2/l that corresponds to 2.05 mg O2/mg test item.

ACCEPTANCE CRITERIA
- Oxygen depletion in the inoculum control does not exceed the 1.5 mg O2/l after 28 days.
- The residual oxygen concentration in the test flasks does not drop below 0.5 mg O2/l at any time.
- 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 is not greater than 20 %.
- The percentage degradation of the reference item reaches the level for ready biodegradability (> 60 %) by exposure day 14.

Reference substance:

benzoic acid, sodium salt

Remarks:

3.6 mg/l (i.e. ThODNH3: 6.012 mg O2/l)

Preliminary study:

In the preliminary experiment, the test item biodegradability, toxicity was investigated for 14 days at the test item concentration of 3 mg/l. No toxic effect of the test item was found at the investigated concentration: in the toxicity control containing both, the test item and the reference item, a mean of 36.8 % biodegradation was noted within 14 days, thus, the test item was assumed not to inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).

Parameter:

% degradation (O2 consumption)

Value:

14.8

Sampling time:

28 d

Details on results:

Under the test conditions, the percentage biodegradation of test item reached a mean of 14.8 % after 28 days based on its COD (the highest value, 14.9 % was calculated on the 21st day of the test). The test item biodegradation reached its plateau already on the 7th day and from this the variations, slight changes were considered as being within the biological variability range of the applied test system. Based on the obtained values the test item can be considered to be not ready biodegradable.

Because of the N-containing test item, the oxidised nitrogen (nitrate and nitrite) concentrations were determined after 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 were below the LOQ in the measured samples throughout the study.
The measured quantity of nitrite was in the measurable range in both parallel vessels of the test item and toxicity control groups on the 0, 7th, 14th, 21st and 28th days; furthermore in the inoculum control group on the 21st and 28th days. The measurable nitrite quantities were nearly the same throughout the test. Any correction of the BOD values with the observed nitrite values was considered as not necessary, because a correction with these low values would not have any influence on the results and conclusion of the study. The corrected BOD values would remain in the biological variability range of the applied test system.

TOXICITY CONTROL
In the toxicity control containing both, the test item and the reference item, a mean of 29.3 % biodegradation was noted within 14 days and 33.3 % 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).

Results with reference substance:

The reference item Sodium benzoate was sufficiently degraded to a mean of 64.5 % after 14 days, and to a mean of 73.0 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.

ACCEPTANCE OF THE STUDY

The study was considered as valid because of the oxygen depletion in the inoculum control was 1.46 in average, not exceeded 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 2.93, it was measured on the 28th day in the procedure control group.

The difference of duplicate values for the degradation at any time during the test was not greater than 20 %. The highest difference between the duplicate values was observed on the 14th day in the procedure control group, it was ~17 %.

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 64.5 % on the 14th day.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Not ready biodegradable.

Executive summary:

The purpose of the study was to determine the ready biodegradability of the test item, according to the Closed Bottle Test method. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

Under the test conditions the percentage biodegradation of test item reached a mean of 14.8 % after 28 days based on its COD (the highest value, 14.9 % was calculated on the 21^stday of the test). The test item biodegradation reached its plateau already on the 7^th day and from this the variations, slight changes were considered as being within the biological variability range of the applied test system. Based on the obtained values the test item can be considered to be not ready biodegradable.

The reference item Sodium benzoate was sufficiently degraded to a mean of 64.5 % after 14 days, and to a mean of 73.0 % after 28 days of incubation, based on ThOD_NH3, thus confirming the suitability of the used activated sludge inoculum.

In the toxicity control containing both, the test item and the reference item, a mean of 29.3 % biodegradation was noted within 14 days and 33.3 % biodegradation after 28 days of incubation. Thus, the test item can be assumed not to inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).

Conclusion

Not ready biodegradable

Description of key information

Not ready biodegradable

Additional information

The ready biodegradability potential of the test item has been investigated according to the Closed Bottle Test method. Under the test conditions the percentage biodegradation of test item reached a mean of 14.8 % after 28 days based on its COD (the highest value, 14.9 % was calculated on the 21^stday of the test). The test item biodegradation reached its plateau already on the 7^thday and from this the variations, slight changes were considered as being within the biological variability range of the applied test system. Based on the obtained values the test item can be considered to be not ready biodegradable.

In the toxicity control containing both, the test item and the reference item, a mean of 29.3 % biodegradation was noted within 14 days and 33.3 % biodegradation after 28 days of incubation. Thus, the test item can be assumed not to inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).