2,4,8,10-tetraoxa-3λ6,9λ6-dithiaspiro[5.5]undecane 3,3,9,9-tetraoxide

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

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:

17th July 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item: 2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]undecane,
3,3,9,9-tetraoxide
CAS No.: 201419-80-9
Lot number: AZ08AVL1
Assay (by GC): 99.9 %
Appearance: crystalline solid, white
Expiration date: 21 September 2021
Storage conditions: room temperature, protected from humidity, well-closed
container
Safety precautions: According to the MSDS

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Remarks:

The inoculum was not pre-adapted to the test chemical

Details on inoculum:

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 February 26, 2021 (six days before the test). The prepared
activated sludge was continuously aerated (2 L/minute) at the test temperature of
20.0 – 21.9 °C, for about 6 days

Duration of test (contact time):

ca. 28 d

Initial conc.:

ca. 6 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

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 February 26, 2021 (six days before the test). The prepared
activated sludge was continuously aerated (2 L/minute) at the test temperature of
20.0 – 21.9 °C, for about 6 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 (5.252 g wet weight), dried and the
ratio of wet sludge to dry weight (0.4804 g dry weight) was determined. Based on this ratio,
calculated amount of wet sludge (5 g dry weight that was equivalent to 54.66 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 6 days) until use.
The pH of the activated sludge inoculum after preparation was 7.63, just before use the pH
was: 7.56. A pH adjustment of activated sludge inoculum was not performed.
NaCl (for isotonic saline solution): Manufacturer: lach:ner;
Batch Number: PP/2020/00452,
Retest date: 28 August 2022
Pre-conditioning of Activated Sludge Inoculum:
Pre-conditioning consisted of aerating (2 L/minute) activated sludge (in mineral medium1)
for 6 days (from February 26 to March 04, 2021) at test temperature (the actual temperature:
20.0 – 21.9 °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 ~107 – 108/L; therefore, before the
test the inoculum was further diluted 60 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.
Nutrient agar: Supplier: MERCK; Lot Number: VM883450, Expiry date: 23 April 2024

Reference substance:

benzoic acid, sodium salt

Remarks:

Sodium benzoate (at a concentration of 3.0 mg/L)

Preliminary study:

The test item was investigated at the concentration of 6 mg/L. The chosen test item
concentration was based on the performed preliminary experiments: a 7-day toxicity test and a
preliminary experiment on solubility properties of the test item; furthermore, on the calculated
ThODNH3 value of the test item, based on the provided CAS number (ThODNH3: 0.74 mg
O2/mg).

Test performance:

The study was considered as valid since the oxygen depletion in the inoculum control was
1.37 mg O2/L on average, and did not exceed the validity criteria of 1.5 mg O2/L after
28 days.
The residual oxygen concentration in the test bottles did not drop below 0.5 mg O2/L at any
time. The lowest value was 1.70 mg O2/L, which was measured on the 28th day in the
toxicity control.
The difference of duplicate values for the degradation at the plateau or at the end of the test
was not greater than 20 %.
The differences between the duplicate biodegradability values in the procedure control and
toxicity control groups were 0-13 % throughout the experiment. In the test item group
higher values were obtained: 48 % on the 7th day, 16 % on the 14th day, 18 % on the 21st day
and 19 % on the 28th day, at the end of the test.
The high, 48 % difference was considered not contradictive with the validity of the study since the test item
biodegradability was minimal, a biodegradation curve cannot be established. The difference between the
corresponding measured dissolved oxygen concentration values (on the 7th day: 6.73 and 6.67 mg/L, see Table
2 of Appendix 1) was minimal, about 1 %; therefore, this result does not affect the validity of the study.
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 70.1 % on the 14th day.
All validity criteria were met as required by the test guideline OECD 301.

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 2.4

Sampling time:

28 d

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item, 2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]undecane, 3,3,9,9-tetraoxide, was
considered to be not readily biodegradable (2.4 % biodegradation on day 28).
According to the test guidelines the pass level for ready biodegradability is 60 % of
ThODNH3.
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 37.0 % within 14 days, and therefore higher than 25 %.
The percentage biodegradation of the reference item (70.1 % 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 item2,4,8,10-
Tetraoxa-3,9-dithiaspiro[5.5]undecane, 3,3,9,9-tetraoxide.
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 ± 2oC) 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 6 mg/L. The chosen test item
concentration was based on the performed preliminary experiments: a 7-day toxicity test and a
preliminary experiment on solubility properties of the test item; furthermore, on the calculated
ThODNH3value of the test item, based on the provided CAS number (ThODNH3: 0.74 mg
O2/mg).

Under the applied test conditions, no ready biodegradation of this test item was noticed.
The percentage biodegradation of 2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]undecane, 3,3,9,9-
tetraoxide reached a mean of 2.4 % on the 28thday of the test based on its ThODNH3. The
highest biodegradability value of 4.2% was noticed on the 14thday of the test.
The test item is considered to be not readily biodegradable, since the pass level for ready
biodegradability is the removal of 60 % theoretical oxygen demand (ThODNH3) in a 10-day
window.
The reference item sodium benzoate was sufficiently degraded to a mean of 70.1 % after
14 days, and to a mean of 83.2 % 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
37.0 % biodegradation was noted within 14 days and the calculated biodegradation was
42.7 % after 28 days of incubation (the biodegradation values reached a plateau on about the
7thday 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.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

other: activated sludge