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

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

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 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

22 July 2010
Council Regulation (EC) No 440/2008, Annex Part C, C.11: Activated Sludge Respiration Inhibition Test, May 30,
2008 (updated on 1st March 2016) and
OCSPP 850.3300: Modified Activated Sludge, Respiration Inhibition Test, January 2012

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

Analytical monitoring:

no

Details on sampling:

The measurement of the respiration rate in a well-mixed aerated sample of each treatment was performed after exactly 3 hours incubation time.

Vehicle:

not specified

Details on test solutions:

Defined amounts of the test item (1 x 3; 1 x 30 mg and 3 x 300 mg test item that
corresponded to the investigated 10, 100 and 1000 mg/L concentrations) were directly
weighed (administered) into each test flask. The subsequent calculations refer to the initial
weighed nominal concentration.
Concentrations in excess of nominal 1000 mg test item/L were not tested.

Test organisms (species):

activated sludge, domestic

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 01 March 2021 (one day before the test).
Preparation of Activated Sludge Inoculum:
The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids
was decanted. 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 (5.533 g wet weight), dried and the
ratio of wet sludge to dry weight (1.3962 g dry weight) determined. Based on this ratio,
calculated amount of wet sludge (30 g dry weight that was equivalent to 119 g wet sludge)
was suspended in isotonic saline solution (ad. 10 L) to yield a concentration equivalent to
about 3 g/L (on dry weight basis).
(In the test containers (300 mL final volume) the final concentration of suspended solids,
containing 150 mL inoculum was 1.5 g/L on dry weight basis.)
The above concentration calculation accounts for the dilution resulting from feeding with
synthetic sewage. The activated sludge was not used on the day of the collection, but
continuously aerated (2 L/minute) at the test temperature (20 ± 2 °C, actual temperature
range: 20.0-21.0 oC) for about 24 hours (one day) and fed with 50 mL synthetic sewage/L
activated sludge.
The pH of the activated sludge inoculum was checked after preparation (pH: 7.65). pH
adjustment of the inoculum was considered not necessary.
Foaming:
In this test the occurring foaming was not significant, controlling was therefore not
necessary during the incubation.

Test type:

static

Water media type:

not specified

Limit test:

no

Total exposure duration:

3 h

Test temperature:

Environmental Conditions
The test was carried out in a controlled environment room (during the incubation, during the
formulation and oxygen concentration measuring) at a temperature of 20 ± 2 °C according
to the guideline. The recorded temperatures in the environmental room varied between:
20.6 - 21.0 °C.
The test flasks were aerated with compressed air (0.5 L/minute).
The pH and the oxygen concentrations were determined at the start and at the end of the
incubation period in all test concentrations, reference item concentrations and controls. The
temperature was measured in the controlled environment room with a min/max thermometer
during the incubation period. The water temperature was recorded during the oxygen
measurement in all test bottles.
The test conditions were measured with suitable instruments and documented in the raw
data.

pH:

The pH of the activated sludge inoculum was checked after preparation (pH: 7.65). pHadjustment of the inoculum was considered not necessary.

Dissolved oxygen:

The test flasks were aerated with compressed air (0.5 L/minute)

Nominal and measured concentrations:

Defined amounts of the test item (1 x 3; 1 x 30 mg and 3 x 300 mg test item that corresponded to the investigated 10, 100 and 1000 mg/L concentrations) were directly weighed (administered) into each test flask.

Reference substance (positive control):

yes

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

Toxicity of 3,5-Dichlorophenol
The following concentrations of the positive reference control 3,5-Dichlorophenol were
tested on the same activated sludge and under identical conditions as the test item: 2, 7 and
24.5 mg/L. In comparison to the blank controls the oxygen consumption rate of the activated
sludge was inhibited by 25.23 % at the lowest concentration of 2 mg/L and at the nominal
concentrations of 7 and 24.5 mg/L, the oxygen consumption rate was inhibited by 38.74 %
and 89.37 %, respectively. The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 6.53
mg/L.

An additional nitrification control was examined in the test with three parallels to check the
possible nitrification potential of the applied activated sludge batch. With the applying of the
nitrification control the differentiation between the total, heterotrophic and nitrification
respiration was possible. The total respiration (RT) was 45.74 mg/Lh, the heterotrophic
respiration (RH) was 45.09 mg/Lh, the nitrification respiration (RN): 0.65 mg/Lh was
calculated according to the following equation: RN = RT - RH.
The obtained 0.65 mg/Lh was considered as not significant difference within a biological
variability range of the applied test system, and lower than the 5 % of RT (2.29 mg/Lh) in
blank controls.
The above calculation confirmed the assumption, that the heterotrophic oxygen uptake
equals the total uptake.

Reported statistics and error estimates:

The EC50 value of the reference item was calculated using Probit analysis.
The analysis was done using the IBM® SPSS® Statistics, Version 25 (2017) statistical
software program.
The specific respiration rates of the highest test item concentration level were compared to
the blank control values by 2-Sample t-Test (2-sided, α=0.05) by IBM® SPSS® Statistics,
Version 25 (2017) statistical software program.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the performed Activated Sludge Respiration Inhibition Test,
the EC10 and EC50 values of the test item were determined as greater than 1000 mg/L.
Based on the statistical evaluation in this test the NOEC was 1000 mg/L.
In conclusion, this preliminary test demonstrated the absence of inhibition of oxygen
consumption by the test substance up to and including the limit concentration of
1000 mg/L. Therefore, in line with OECD 209 guideline, a definite test is not required.

Executive summary:

The purpose of the test was to evaluate the influence of the2,4,8,10-Tetraoxa-3,9-
dithiaspiro[5.5]undecane, 3,3,9,9-tetraoxideon the activity of the activated sludge by
measuring the respiration rate under defined conditions.
The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples
of activated sludge fed with synthetic sewage were measured in an enclosed cell containing
an oxygen electrode after a contact time of 3 hours.
This pre-test is used to estimate the range of concentrations of the test item needed in a possible
definite test for determining the inhibition of oxygen consumption or to serve as stand-alone
(limit test) according to OECD 209.
2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]undecane, 3,3,9,9-tetraoxidewas investigated in this
study at the nominal concentrations of 10, 100 and 1000 mg/L. Defined amounts of the test item
were added directly into the test vessels.
Triplicate vessels were prepared and investigated at the highest examined test item
concentration.
In parallel with the test item treatments 3,5-Dichlorophenol as positive reference control at
concentrations of 2, 7 and 24.5 mg/L; furthermore, blank (inoculum) control, nitrification
controls and abiotic controls were investigated.
Abiotic controls (investigated in three parallels) were prepared containing the test item at
1000 mg/L, synthetics sewage feed, but without inoculum. In this test no abiotic oxygen
consumption was noticed.
The experiment was performed without pH adjustment. The test item did not have any
influencing effect on the pH within the test system and additional neutralization step of test
item containing mixtures before inoculum addition was therefore not necessary.
All validity criteria of the study were met. The average specific respiration rate of the blank
was 30.49 mg O2/ g activated sludge (based on dry weight) in an hour (unequivocally
higher than 20 mg/gh) with a coefficient of variation of 6.66 % (clearly below the 30 % as
required in the validity criteria). The 3-hour EC50of the reference item 3,5-Dichlorophenol
was 6.53 mg/L within the range of 2 mg/L to 25 mg/L, that was required for total respiration
(in this study the differentiation between heterotrophic respiration and nitrification was
considered as not necessary as the parallel investigated nitrification control, containing Nallylthiourea confirmed the equality of heterotrophic and total oxygen uptake).
The observed oxygen consumption rates and consequently the specific respiration rates in all
examined test item concentrations remained in the range of the blank controls (the average
specific respiration rate at 1000 mg/L: 2.17 mg O2/gh). No inhibitory effect of the test item
was observed.
Under the conditions of the performed Activated Sludge Respiration Inhibition Test,
the EC10and EC50values of the test item were determined as greater than 1000 mg/L.
Based on the statistical evaluation in this test the NOEC was 1000 mg/L.
In conclusion, this preliminary test demonstrated the absence of inhibition of oxygen
consumption by the test substance up to and including the limit concentration of
1000 mg/L. Therefore, in line with OECD 209 guideline, a definite test is not required.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L