Registration Dossier

Diss Factsheets

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:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
yes
Remarks:
see "Principles of method if other than guideline"
Principles of method if other than guideline:
Two deviations from the study plan and the test guidelines was realized.
1. The start value of oxygen content for measurement of oxygen consumption was lower than 7 mg/L.
2. The measuring time for oxygen consumption higher than 10 minutes.
An influence to the results of this study can be excluded.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Amines, C10-14-branched and linear alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)
EC Number:
285-083-3
EC Name:
Amines, C10-14-branched and linear alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)
Cas Number:
85029-58-9
Molecular formula:
C34H24CrN8O6.C10-14H21-29NH2
IUPAC Name:
Amines, C10-14-branched and linear alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)
Test material form:
solid
Details on test material:
Appearance
- physical state: solid
- color: brown
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient at room temperature

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
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.

Test organisms

Test organisms (species):
activated sludge, domestic
Details on inoculum:
Reason for the selection of the test system: required by the test guidelines
Age: 1 day
Origin: aeration tank of the wastewater treatment plant of Mannheim, Germany
Collection of the test system: 24 May 2016
Arrival in the test facility: 24 May 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.

Study design

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

Test conditions

Test temperature:
18.8 – 19.6 °C
pH:
7.4 - 7.5
Dissolved oxygen:
Oxygen concentration during aeration: > 2 mg/L
Oxygen concentration immediately before measurement: > 7 mg/L with the exceptions (see below)
Nominal and measured concentrations:
1000, 500, 250, 125, 62.5 mg/L as nominal concentration without correction of purity and blank controls.
100, 10, 1 mg/L as nominal concentration based on reference substance.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glas-beakers (nominal volume 1L) with 500 mL test volume
- Material, size, headspace, fill volume: 500 mL test volume
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per reference substance (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Synthetic medium: 16 mL/test vessel of 100-fold concentrated OECD medium

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod:
- Light intensity:
- Details on termination of incubation:

TEST CONCENTRATIONS
- Spacing factor for 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

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 with the exception described in table 1 (see below).
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 ( BC = blank control; RS = reference substance; TS = test substance). 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 concentrationsopen allclose all
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:
Validity criteria:
The coefficient of variation of the six replicates of blank control was 10.7 % O2 consumption.
The EC50 of 3,5-dichlorophenol was 4.2 mg/L
The mean oxygen uptake of the blank controls was 20 mg/g*h
In conclusion, the test conformed to all validity criteria and is valid.
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 7.4 mg/L and, tehrefore, in the range of 2-25 mg/L in 3 hours.
These results indicate that the microorganisms are responding normally to toxicant stress.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The inhibition of the degradation activity of activated sludge is not anticipated when introduced to biological treatment plants in appropriate low concentrations.

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