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Ecotoxicological information

Toxicity to microorganisms

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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:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
Few deviations occurred (i.e. T°C and measurement period differed from the study plan). Both reported to be uncritical, normal respiration activity of the control was observed and correlation of the inhibition values within the test replicates was good.
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Diammonium sodium hexakis(nitrito-N)rhodate
EC Number:
264-713-0
EC Name:
Diammonium sodium hexakis(nitrito-N)rhodate
Cas Number:
64164-17-6
Molecular formula:
H4N.1/2N6O12Rh.1/2Na
IUPAC Name:
Diammonium sodium hexakis(nitro-N)rhodate
Constituent 2
Reference substance name:
Diammonium sodium hexakis (Nitrito-N) rhodate
IUPAC Name:
Diammonium sodium hexakis (Nitrito-N) rhodate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
- Method: In the control vessels, 16 mL nutrient solution was mixed with both 234 and 231 mL dilution water (i.e. tap water), without and with the addition of the nitrification inhibitor N-allylthiourea (ATU), respectively. Both the positive control vessels and the treatments were prepared by putting the appropriate amount of positive control solution, test item nominal concentrations, and ATU into the respective test vessel (concentrations of the test item and positive control were calculated using the concentration of the respective stock solution and the dilution factor). Then, 16 mL nutrient solution and water to 250 mL were added. Lastly, 250 mL inoculum was added in 5 minute intervals and the mixtures were aerated.
After 3 hours, the content of the first vessel was poured in a 250 mL narrow-deck bottle and the respiration rate was determined by measurement of the oxygen concentration over a period of max 5 minutes. The following vessels were measured in five minute intervals.
- Differential loading: Test item nominal concentrations were 1000, 100, 10, 1 mg/L in the first experiment, and 1000 mg/L in the second one.
- Controls: Both blank and positive controls were used. Blank controls contained 16 mL nutrient solution, 250 mL inoculum, and either 234 mL or 231 mL, according to the absence or addition of ATU, respectively. 3,5-Dichlorophenol (CAS 591-35-5) was used as positive control. A stock solution in deionised water containing 500 mg/L (nominal) was freshly prepared for each experiment.
- Nutrient solution: Synthetic sewage that was frozen immediately after preparation.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge from a predominantly domestic sewage treatment plant was collated on the day before treatment. Sludge was filtrated, washed in tap water, resuspended in tap water, aerated until usage in the test, and fed daily with 50 mL synthetic sewage feed/L. The dry matter was determined, and volume was adapted to the desired content of dry matter. The dry matter content of the inoculum was also determined on the day of each experiment, and the following results were obtained, respectively:
Exp. 1) The dry matter was determined as 3.06 g suspended solids/L, giving a concentration of 1.53 g suspended solids/L in the test;
Exp. 2) The dry matter was determined as 2.74 g suspended solids/L, giving a concentration of 1.37 g suspended solids/L in the test.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h

Test conditions

Hardness:
-
Test temperature:
First experiment: range from 20.0 to 22.6 °C
Second experiment: range from 20.7 to 21.8 °C

pH:
First experiment: pH range from 7.7 to 8.0
Second experiment: pH range from 8.1 to 8.3
Dissolved oxygen:
-
Salinity:
-
Nominal and measured concentrations:
Test item nominal concentrations of 1000, 100, 10, 1 mg/L were used in the first experiment, and the sole test item nominal concentration of 1000 mg/L was used in the second experiment as a limit test.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers
- Aeration: Purified air, using Pasteur pipettes
- No. of vessels per concentration (replicates): 1 replicate per treatment (first experiment), and 5 replicates per treatment (second experiment).
- No. of vessels per control (replicates): 2 replicates before and at the end of the exposure period, both for the test item and positive control treatments.
- No. of vessels per positive control (replicates): 1 replicate per treatment.
- Biomass loading rate: 250 mL activated sewage inoculum

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water

TEST CONCENTRATIONS
- Spacing factor for test concentrations: A spacing factor of 10 was used
- Test concentrations: Nominal test concentrations of 1000, 100, 10, 1 mg/L were used for the first experiment. A nominal concentration of 1000 mg/L was used in the second experiment, as a limit test.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol (CAS 591-35-5)

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: without ATU
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
Remarks:
respiration rate
Remarks on result:
other: without ATU
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: with ATU
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
Remarks:
respiration rate
Remarks on result:
other: with ATU
Details on results:
The oxygen consumption rate was calculated from the slope of the linear part of the oxygen consumption curve using linear regression.
Results with reference substance (positive control):
The calculated 3h-EC50 values for the positive control were:
First experiment without ATU - 12 mg/L (95% C.I. 9.2 - 16 mg/L), and with ATU - 12 mg/L (95% C.I. 8.4 - 16 mg/L)
Second experiment without ATU - 12 mg/L (95% C.I. 8.4 - 16 mg/L), and with ATU – 12 mg/L (95% C.I. 7.3 - 19 mg/L)
All values lay within the recommended range of 2 - 25 mg/L (total respiration without ATU), and 5 - 40 mg/L (heterotrophic respiration with ATU).
Reported statistics and error estimates:
A statistical determination of the NOEC was carried out, in order to test whether the differences between the related effect level of 1000 mg/L and the control were significant. For this determination, the values of the oxygen consumption were used. Firstly, it was checked whether equality of variance was given, and afterwards a t-test was applied. No significant differences were found between the treatment of 1000 mg/L and the control.
A linear fit on a probability-logarithmic scale was used for evaluating EC50 values.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The NOEC value for both total respiration and heterotrophic respiration was determined to be ≥ 1000 mg/L, corresponding to >=146 mg Rh/L.
Executive summary:

This study evaluated the effect of Diammonium sodium hexakis (Nitrito-N) rhodate on the respiration of activated sludge (Muckle 2015). The study is reliable without restrictions, being GLP compliant and having followed the standard guidelines OECD 209, and EU-Method C.11. The duration of the test was 3 hours. Activated sludge, which was collected from a predominantly domestic sewage treatment plant, was used as inoculum. The substance 3,5-dichlorophenol was used as a positive control. Two experiments were carried out, a range-finding test followed by a limit test. All validity criteria were met.

 

At the nominal concentration of 1000 mg/L, no significant inhibition was observed in treatments with and without the nitrification inhibitor N-allylthiourea (ATU). The respective NOEC and EC10 values for both total respiration and heterotrophic respiration was determined to be ≥ 1000 mg/L nominal concentration (corresponding to >=235 mg Rh/L), and the EC50 value was >1000 mg/L, corresponding to >=146 mg Rh/L..