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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:

November 17 2020 to December 15 2020

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Not specified

Oxygen conditions:

anaerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Test System
Species:
Activated sludge, microorganisms from a domestic waste water treatment plant.
Origin:
The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in
Balatonfüred, Hungary, on 13 November 2020 (four days before the test). The prepared
activated sludge was continuously aerated (2 L/minute) at the test temperature of
20.0 – 20.9 °C, for about 4 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 by shaking
and was again centrifuged. This procedure was repeated twice.
An aliquot of the final sludge suspension was weighed (5.144 g wet weight), dried and the
ratio of wet sludge to dry weight (0.3835 g dry weight) was determined. Based on this ratio,
calculated amount of wet sludge (5 g dry weight that was equivalent to 67.07 g wet sludge)
was suspended in mineral medium (Section 5.4; ad. 1000 mL) to yield a concentration
equivalent to about 5 g per litre (on dry weight basis). The prepared activated sludge
inoculum was aerated under test conditions (for 4 days) until use.
The pH of the activated sludge inoculum after preparation was 7.38, just before use the pH
was: 7.28. A pH adjustment of activated sludge inoculum was not performed.
NaCl (for isotonic saline solution): Manufacturer: lach:ner; Batch Number: PP/2018/05962,
Retest date: 31 January 2021

Pre-conditioning of Activated Sludge Inoculum:
Pre-conditioning consisted of aerating (2 L/minute) activated sludge (in mineral medium1)
for 4 days (from November 13 to 17, 2020) at test temperature (the actual temperature: 20.0
– 20.9 °C). Before use 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, 10-2, 10-3, 10-4 and 10-5
dilutions of cultures on nutrient agar plates.
Plates were incubated at 37 °C for 24 hours. The viable cell number of the cultures was
determined by these plating experiments by manual colony counting.
The approximately cell count of aerated inoculum was ~108 - 109/L; therefore, before the
test the inoculum was further diluted 40 000x with mineral medium to reach the necessary
~104 – 106 cells/L cell concentration. After preparation the sludge was filtered through
cotton wool. Pre-conditioning (see above) improves the precision of the method.
The inoculum was not pre-adapted to the test chemical.

Duration of test (contact time):

28 d

Initial conc.:

4 mg/L

Based on:

other: ThODNH3

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The test item was investigated at the concentration of 4 mg/L. The chosen test item
concentration was based on the solubility properties of the test item (investigated in a non-GLP
solubility trial); furthermore, on the ThODNH3 (informal) values of the test item, calculated on
the basis of the provided molecular structure (ThODNH3: 1.2 - 1.4 mg O2/mg). At the start of
the test the chemical oxygen demand (COD) of the test item was determined (1.39 mg O2/mg).

sufficient number of Winkler bottles 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. (The volume of a completely filled
bottle is approximately 300 mL.) Then they were tightly closed with glass stoppers.

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 measurements were performed in all
duplicate bottles in all groups on days 0, 2, 5, 7, 12, 14, 21 and 28.

Temperature Measurements
Temperature was measured continuously (with a built-in thermometer) in the incubator
(during the 28-day incubation period) and recorded twice a day.

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.
The nitrite and nitrate concentrations were determined using photometric method with
Nitrite and Nitrate Cell Tests (MERCK®).
For technical reason the nitrate and nitrite analysis of the 7-day samples was performed
together with the 14-day samples on the 14th day of the test, the analysis of 21-day samples
was performed one week later, together with the 28-day samples, on the 28th day of the test.
The 7-day and 21-day samples were adequately stored in freezer (at -30 ± 5 °C) until
determination of nitrate and nitrite. The 0-, 14- and 28-day samples were analyzed directly
after oxygen measurements.
Measurement of Chemical Oxygen Demand (COD)
The given molecular structure allowed a preliminary ThODNH3 estimation; however, for
correct calculation of biodegradability values, the COD value of the test item was
determined using a COD Cell Test (MERCK®) and CombiCheck 50.
The COD expresses the amount of oxygen originating from potassium dichromate that
reacts with the oxidizable substances contained in 1 L of water under working conditions of
the specified procedure. 1 mol K2Cr2O7 is equivalent to 1.5 mol O2. The results are
expressed as mg/L COD (mg/L O2). The mg O2/mg test item unit is calculated from the
known test item solution concentration.
For determination of the COD of the test item a solution with a concentration of 10 mg/L
was prepared (dilution of stock solution, Section 5.1.3) and measured. The COD of the test
item solution was determined by parallel measurements with appropriate controls
(CombiCheck 50).
The measured values for a 10 mg/L solution were 13.9 mg O2/L; 14.0 mg O2/L and 13.7 mg
O2/L, in average 13.9 mg O2/L. For the mg O2/mg test item unit the above value was divided
with the known test item solution concentration (10 mg/L). In summary, the COD of the test
item was measured and calculated as 1.39 mg O2/mg test item.

Reference substance:

benzoic acid, sodium salt

Test performance:

Under the test conditions the percentage biodegradation of Dye-2019 reached a mean of
12.8 % after 28 days based on its COD. The highest biodegradability value of 14.7 % was
noticed on the 21st day of the test. Based on the dissolved oxygen depletion, the calculated
biodegradation values reached a plateau on about the 7th day of the experiment. From this
day on, the slight subsequent changes were considered as being within the biological
variability range of the applied test system.

Key result

Parameter:

other: % biodegradation based on dissolved oxygen depletion

Value:

14.7

Sampling time:

21 d

Details on results:

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 oxidizing
ammonium to nitrite and nitrate. For that reason, at this N-containing test item, the oxidized
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 nitrite concentration was below the LOQ in the start, 7-, and 14-day samples (test item,
inoculum control and toxicity control). Furthermore, the nitrite concentrations remained
below the LOQ in the 21-day and 28-day toxicity control samples. In the 21-day inoculum
control samples in average 0.41 mg/L nitrite concentrations, in the 21-day test item samples
in average 0.11 mg/L nitrite concentrations were measured.
In the 28-day inoculum control samples in average 0.69 mg/L in the test item samples in
average 0.83 mg/L nitrite concentrations were measured.
The nitrate concentration was less than 0.4 mg/L in all measured start, 7-day, 14-day and 21-
day samples; furthermore, in the 28-day toxicity control samples, but the nitrate
concentration was in average 0.8 mg/L in the 28-day inoculum control and test item samples.
According to the referred OECD 301 guideline (Annex V) the oxygen consumed in nitrite
formation approximates 3.43 x increase of nitrite-N concentration, and the oxygen
consumed in nitrate formation is 4.57 x increase of nitrate-N concentration.
Detailed calculations (see below) were conducted with 21-, and 28-day test item samples.
Accordingly, in the test item containers the calculated oxygen consumed by ammonium
oxidation processes on the 21st day was 0.11 x 3.43 = 0.38 mg/L (nitrite). In contrast, the
measured average oxygen depletion (not corrected with inoculum control) from 14th day to
21st day of the test was in average 0.25 mg/L.
On the 28th day of the test, in the test item containers, the oxygen consumed by ammonium
oxidation processes was 0.83 x 3.43 = 2.85 mg/L (nitrite) and 0.8 x 4.57 = 3.7 mg/L (nitrate).
But, the corresponding measured average dissolved oxygen concentrations (not corrected
with inoculum control) from 21st day to 28th of the test showed slight increase, 0.06 mg/L
(within the biological variability range of the applied test system).
In this study the change of the measured dissolved oxygen concentrations in the test item
bottles did not correspond to the consumed oxygen of ammonium oxidation processes.
Because of the relationship between oxygen uptake resulting from a possible ammonium
oxidation and oxygen uptake of applied microbial population was equivocal, any correction
of the measured dissolved oxygen concentrations was considered as not possible. The
calculated oxygen consumed by ammonium oxidation processes was significantly higher,
than the measured oxygen depletions. The measured higher nitrite and nitrate concentration
values were caused likely by a technical effect (possible discoloration of the solutions and/or
turbidity).

Key result

Parameter:

COD

Value:

12.8 other: %

Results with reference substance:

Biodegradation of the Reference Item
The reference item sodium benzoate was sufficiently degraded to a mean of 77.4 % after
14 days, and to a mean of 78.7 % 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
39.4 % biodegradation was noted within 14 days and the calculated biodegradation was
40.8 % after 28 days of incubation (the biodegradation values reached a plateau on about the
7th day of the experiment and from this day on, the slight subsequent changes were
considered as being within the biological variability range of the applied test system). Thus,
the test item can be assumed to not inhibit the activated sludge microorganisms (higher than
25 % degradation occurred within 14 days).

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item, Dye-2019, was considered to be not readily biodegradable (12.8 %
biodegradation on day 28). According to the test guidelines the pass level for ready
biodegradability is 60 % of COD.
the test item can be assumed as not inhibitory on the
activated sludge microorganisms because the degradation in the toxicity control group
was 39.4 % within 14 days, and therefore higher than 25 %.
The percentage biodegradation of the reference item (77.4 % after 14 days) confirms
the suitability of the used activated sludge inoculum.

Executive summary:

The purpose of this study was to determine the ready biodegradability of the test item Dye-2019.
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 test item was investigated at the concentration of 4 mg/L. The chosen test item concentration
was based on the solubility properties of the test item (investigated in a non-GLP solubility trial);
furthermore, on the ThODNH3 (informal) values of the test item, calculated on the basis of the
provided molecular structure (ThODNH3: 1.2 - 1.4 mg O2/mg). At the start of the test the following
chemical oxygen demand (COD) of the test item was determined: COD: 1.39 mg O2/mg.
Under the applied test conditions, no ready biodegradation of this test item was noticed.
The percentage biodegradation of Dye-2019 reached a mean of 12.8 % on the 28th day of the test based on its COD (the highest biodegradability value of 14.7 % was noticed on the 21st day of the test). (The calculated biodegradation values reached a plateau on about the 7th day of the experiment. From this day on, the slight subsequent changes were considered as being within the biological variability range of the applied test system.)
The test item is considered to be not readily biodegradable, since the pass level for ready biodegradability is removal of 60 % chemical oxygen demand (COD) in a 10-day window.
The concurrently conducted analytical determination of possible nitrite and nitrate development showed increases in nitrite concentrations in the 21-day and 28-day inoculum control and test item samples, furthermore in nitrate concentrations in the 28-day inoculum control and test item samples; however, the measured dissolved oxygen concentrations did not correspond to the consumed oxygen of ammonium oxidation processes. Likely technical effects (turbidity and/or discoloration) influenced the nitrite and nitrate concentration determinations. Therefore, the biodegradability value of the test item was calculated based on its COD; 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 77.4 % after 14 days, and to a mean of 78.7 % after 28 days of incubation, based on ThODNH3.
The percentage biodegradation of the reference item confirms the suitability of the
used activated sludge inoculum.
In the toxicity control containing both, the test item and the reference item, a mean of 39.4 % biodegradation was noted within 14 days and the calculated biodegradation was 40.8 % after 28 days of incubation (the biodegradation values reached a plateau on about the 7th day of the experiment and from this day on, the slight subsequent changes were considered as being within the biological variability range of the applied test system).
According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level 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 test item, Dye-2019, was considered to be not readily biodegradable (12.8 %
biodegradation on day 28). According to the test guidelines the pass level for ready biodegradability is 60 % of COD.

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 39.4 % within 14 days, and therefore higher than 25 %.
The percentage biodegradation of the reference item (77.4 % after 14 days) confirms the suitability of the used activated sludge inoculum.

 

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

other: Dechlorinated tap water