5,7-Di-t-butyl-3-[3,5-dimethyl-4-[(1,3,7,9-tetra-t-butyl-5-methyl-5H-benzo[d][1,3,2]benzodioxaphosphocin-11-yl)oxy]phenyl]-3H-benzofuran-2-one

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

Study period:

2016

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Application of the test substance: Direct addition by initial weight into the test flasks. The test substance was weighed in the required amounts for the test concentrations directly to the test vessels.

The test substance was added in the required amounts according to the test concentrations directly to the test vessels with 234 mL deionized water. Aliquots of the reference substance stock solution were dosed to the test vessels and made up with deionized water to a volume of 234 mL. 16 mL synthetic medium were dosed to each test vessel with test substance and reference substance afterwards. To prepare the blank control assays 234 mL of deionized water and 16 mL synthetic medium were mixed. The pH-values in all test vessels were checked. An adjustment was not necessary. After addition of 250 mL of inoculum suspension (3 g/L DW) the incubation was started by aeration of the test vessels with pressured air. The vessels for the blank control assays were prepared to the same procedure without addition of test- or reference substance. After 3 hours incubation at 20 ± 2°C the mixtures in the test vessels were placed subsequently for oxygen measuring. The temperature was measured for seven times in a separate vessel filled with aerated deionized water during incubation phase. The content of oxygen at the start of the measurements was > 7 mg/L with the exception described in remark. The oxygen uptake was measured for a period of about 8 to 10 with the exception described in remark. The total oxygen consumption was measured in the sequence BC1-3 and RS1, RS2-5, RS6 and TS1-3, TS4-7, TS8-11 and TS12-15. The oxygen consumption of the blank control BC4-6 were measured at last. No abiotic control was tested. The value for oxygen consumption of test assays TS3 was set as an outlier by expert judgment and was not used for the evaluation of the test results.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Origin: aeration tank of the wastewater treatment plant of Mannheim, Germany
Collection of the test system: 03 Aug 2016
Arrival in the test facility: 03 Aug 2016
After arrival of the activated sludge suspension in the test facility the suspension was sieved with a fine woven mesh (mesh size about 1 mm). This suspension was pre-aerated over night at room temperature. At the next day the sludge suspension was washed once with drinking water and the suspension was adjusted to 3 g/L Dw.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

19.7 – 20.6 °C

Dissolved oxygen:

Oxygen concentration during aeration: > 2 mg/L
Oxygen concentration immediately before measurement: > 7 mg/L

Nominal and measured concentrations:

1000, 500, 250, 125, 62.5 mg/L as nominal concentration based on test substance without correction of purity and blank controls.

Details on test conditions:

Test concentrations:
1000, 500, 250, 125, 62.5 mg/L as nominal concentration based on test substance without correction of purity and blank controls.
100, 10, 1 mg/L as nominal concentration based on reference substance.

Test replicates:
6 replicates for the control,
3 replicates for each test substance concentration,
2 replicates for each reference substance concentration.

Reason for selection of test concentrations:
According to the test guidelines, at least 5 concentrations in a geometric series with a separation factor of ≤ 3.2 should be used, preferably encompassing the range from 10 – 80% inhibition .

Incubation time: 3 hours
Test temperature: 19.7 – 20.6 °C
Test vessels: Glas-beakers (nominal volume 1L)
Test volume: 500 mL
Synthetic medium: 16 mL/test vessel of 100-fold concentrated OECD medium
Oxygen concentration during aeration: > 2 mg/L
Oxygen concentration immediately before measurement: > 7 mg/L
Duration of the measurement of oxygen consumption: About 8 - 10 min
Sludge concentration in the test vessel: 1.5 g/L Dw

Reference substance (positive control):

yes

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

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

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

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

No monotone concentration response exists because no inhibition was determined. Therefore the ECx calculation is not meaningful and the EC10 value is assumed to be greater than 1000 mg/L.

Results with reference substance (positive control):

In order to verify that the microorganisms are responding normally to toxic stress, tests with a reference substance are conducted according to OECD 209 guideline. The results from the reference substance test are compared to EC50 values published in OECD 209 guideline, which represent the typical response range.
According to the test guideline OECD 209 the EC50 values of the reference substance 3,5- dichlorophenol should be in the range of 2-25 mg/L after 3 hours of incubation.
The EC50 of the reference substance 3,5-dichlorophenol was in the range of 2-25 mg/L in 3 hours.
These results indicate that the microorganisms are responding normally to toxicant stress.

Validity criteria fulfilled:

yes

Remarks:

cf. Overall remarks, attachments: Overall remarks

Description of key information

The inhibition of the respiration activity of activated sludge was tested in a study according to OECD TG 209 and under consideration of GLP. The study was conducted as static test with an exposure duration of 3h. Activated sludge from a municipal sewage treatment plant was used which was not adapted. Nominal concentrations of 62.5, 125, 250, 500 and 1000 mg/L test substance were used without correction for purity. After an exposure time of 3h no effects on the respiration activity of activated sludge could be recorded. The EC10, EC20, EC50 and EC80 values were determined to be > 1000 mg/L (nominal).

The results in this study were consistent with the validity criteria with one exception. The mean oxygen uptake in the blank controls was lower than 20 mg/g*h (16 mg/g*h). Because the reference substance shows an EC50in the specified range (usual range of EC50 of the last 20 values (with the values in this study) in the laboratory was 3.4 – 15.5 mg/L) and the measured oxygen uptake from the test substance concentrations showed a good curve progression the study is classified as valid. The used activated sludge which was collected from the same waste water treatment plant showed a maximum oxygen consumption of 36.5 mg/g*h and minimum oxygen consumption of 8.2 mg/g*h of the last 20 measurements (with the values in this study). For this the study is classified as valid.