Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Sampling and analysis

Analytical monitoring:
no

Test solutions

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 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.

Test organisms

Test organisms (species):
activated sludge, domestic
Details on inoculum:
Age: 1 day
Origin: aeration tank of the wastewater treatment plant of Mannheim, Germany
Collection of the test system: 19 Feb 2018
Arrival in the test facility: 19 Feb 2018
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.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h

Test conditions

Test temperature:
19.4 – 20.4°C
pH:
7.4-7.5
Dissolved oxygen:
Oxygen concentration during aeration: > 2 mg/L
Oxygen concentration immediately before measurement: > 7 mg/L (start oxygen concentration for TS15 @1000mg/L was approx. 6.8 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.
100, 10, 1 mg/L as nominal concentration based on reference substance.
Details on test conditions:
Test vessels: Glas-beakers (nominal volume 1L)
Test volume: 500 mL
Sludge concentration in the test vessel: 1.5 g/L Dw
Synthetic medium: 16 mL/test vessel of 100-fold concentrated OECD medium

Preparation of the test:
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. The total oxygen consumption of 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 oxygen uptake was measured for a period of about 8 to 10 minutes.
Reference substance (positive control):
yes
Remarks:
3,5 dichlorophenol

Results and discussion

Effect concentrations
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
Details on results:
EC10 >1000 mg/L, EC20 >1000 mg/L, EC50 >1000 mg/L, EC80 >1000 mg/L were found in the activated sludge respiration inhibition test with 3 hour residence time for the test substance Neopentylglycol diacetate. The test substance showed a increasing stimulation of the oxygen consumption of the activated sludge of a about 50% at the highest test concentration with 1000 mg/L.
Results with reference substance (positive control):
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.
Reported statistics and error estimates:
The consumption rates were used for the determination of the ECx for the reference substance by the probit method based on Finney (1971) with the software TOXRAT Professional 2.10. The effect concentrations were given with an accuracy of 2 significant digits.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In conclusion the 3 hours effect concentrations for Neopentylglycol diacetate in the Activated Sludge Respiration Inhibition Test are:
The following effect concentrations (mg/L) were obtained:
EC10 > 1000 mg/L
EC50 > 1000 mg/L
No monotone concentration response exists. Therefore the ECx calculation is not meaningful and the EC10 value is assumed to be greater than 1000 mg/L. The results in this study are consistent with all validity criteria and the test is valid according to
the guidelines used for this study. The test substance showed a increasing stimulation of the oxygen consumption of the activated sludge of a about 50% at the highest test concentration with 1000 mg/L.