[No public or meaningful name is available]

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:

other: GLP and Guideline compliant study with good documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

Due to the limited amount of tet item, some concentrations were set up with a reduced number of replicates. Based on the clear dose response relationship incl. low variation, this is considered to have no effect on the validity of the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test item concentrations diluted out of the stock solution with 16 g/L.
- Controls:Test medium without test or reference item

Test organisms (species):

activated sludge of a predominantly domestic sewage

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.4

pH:

The pH value of the synthetic waste water was determined prior to use. Adjustment to 7.5 ± 0.5 was not necessary.

Nominal and measured concentrations:

Control. 62.5 - 125 - 250 - 500 - 1000 mg/L.(nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flasks, DIN 12380
- Material, size, headspace, fill volume: material: glass; fill voulume: 300 mL
- Aeration: Shaking of flasks at 125 rpm to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.
- No. of organisms per vessel: 1.51 g/L dry sludge in test solution
- No. of vessels per concentration (replicates): 1000 mg/L: 1 replicate; 500 mg/L: 4 replicates; 250 – 125 – 62.5 mg/L: 5 replicates
- No. of vessels per control (replicates): 6
- Biomass loading rate: 1.51 g/L dry sludge in test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic waste water according to OECD-Guideline 209

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: not provided
- Light intensity: not provided

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : respiration

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: none
- Range finding study
- Test concentrations: 1000 mg/L
- Results used to determine the conditions for the definitive study: Inhibition of 36% at 1000 mg/L.

Reference substance (positive control):

yes

Remarks:

copper (II) sulphate pentahydrate

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

1 204 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50 95.9 mg/L, historical data 53-155 mg/L
- Other: none

Reported statistics and error estimates:

The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) and Dunnett’s test was used for NOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha=0.05.

The EC-values of the test item were calculated by sigmoidal dose-response regression and the EC50-value of the reference item by linear regression using software GraphPadPrism. Calculations of the confidence intervals for the EC-values were carried out using standard procedures.

Validity criteria fulfilled:

yes

Remarks:

for discussion of lower number of replicates in the two highest concentrations see text

Executive summary:

In the Klimisch 1 GLP study from Fiebig (2013) the respiration inhibition of Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18 unsaturated alkyl) tetrasodium salts on activated municipal sludge was determined according to OECD 209. Based on the outcome of a pre-test, the test was performed at following concentrations Control, 62.5 - 125 - 250 - 500 - 1000 mg/L. The test groups were set up in with following number of replicates: 6 (Control), 5 (62.5, 125, 250 mg/L), 4 (500 mg/L) and 1 (1000 mg/L). Except the lower number of replicates in the two higher test concentrations, all validity parameters as outlined in the OECD 209 were fulfilled. The lower number of replicates in the two higher test concentrations which was chosen due to the limited amount of test item, is considered to have no impact on the outcome of the study, especially since the outcome is furthermore supported by the range-finding test. Statistical evaluation showed that the test item has an EC50 of > 1000 mg/L , extrapolated to 1204 mg/L.

The results of this study are considered relevant and reliable for the risk assessment.

Description of key information

3 h EC50 (OECD 209): 1204 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:

1 204 mg/L

Additional information

In the Klimisch 1 GLP study from Fiebig (2013) the respiration inhibition of Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18 unsaturated alkyl) tetrasodium salts on activated municipal sludge was determined according to OECD 209. Based on the outcome of a pre-test, the test was performed at following concentrations Control, 62.5 - 125 - 250 - 500 - 1000 mg/L. The test groups were set up in with following number of replicates: 6 (Control), 5 (62.5, 125, 250 mg/L), 4 (500 mg/L) and 1 (1000 mg/L). Except the lower number of replicates in the two higher test concentrations, all validity parameters as outlined in the OECD 209 were fulfilled. The lower number of replicates in the two higher test concentrations which was chosen due to the limited amount of test item, is considered to have no impact on the outcome of the study, especially since the outcome is furthermore supported by the range-finding test. Statistical evaluation showed that the test item has an EC50 of > 1000 mg/L, extrapolated to 1204 mg/L.

The results of this study are considered relevant and reliable for the risk assessment.