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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-06-07 to 2018-10-30
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)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ESIM Chemicals, batch LBOP6A1025
- Expiration date of the lot/batch: 08 Feb 2019
- Purity test date: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: cool (1–10 °C), dark, dry
- Stability under test conditions: Stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None
- Preliminary purification step (if any): None
- Final dilution of a dissolved solid, stock liquid or gel: N.a.
- Final preparation of a solid: Direct weighing
Analytical monitoring:
not required
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item solutions were prepared individually by weighing the test item directly for each of the incubation vessels.
- Controls: Blank control, Reference control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Activated sludge from the municipal waste water treatment plant of Pforzheim (Germany), collected from the exit or near the exit of the aeration tank, was used as the microbial inoculum for the test.
This plant is predominantly treating domestic sewage.
- Method of cultivation: Activated sludge is kept under aerobic conditions at room temperature and under laboratory diffused light until test start.
- Preparation of inoculum for exposure: Right before inoculation, the activated sludge is mixed thoroughly to ensure homogeneity.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2 °C
pH:
7.5 ± 0.5
Dissolved oxygen:
The inoculated test mixtures were aerated for three hours. At the end of aeration, dissolved oxygen (DO) was quantified and sludge respiration rates derived from the rate of DO depletion. Dissolved oxygen concentration was always above 60-70 % saturation.
Nominal and measured concentrations:
1 / 3.1 / 9.8 / 31.3 / 100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: Volumetric flasks, filled with 250 mL permanently mixed test media, and respective chemicals. During the test phase, all incubation vessels kept in temperature-controlled laboratory with a constant air supply, ensuring aeration and mixing. At the end of the three-hour incubation period, aliquots of each test media preparation were transferred to the appropriate sample tube. The DO electrodes were sealed in the neck of the tube, ensuring air is completely excluded. The sample tube was stirred at a constant rate during DO measurements. DO was measured over a period of up to 10 minutes. The DO content was measured using electronic oxygen probes.
- Aeration: Yes
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): reference control: 1 ; blank control: 8 replicates
- Sludge concentration (weight of dry solids per volume): 3 g/L activated sludge (suspended solids) (± 0.3 g/L)
- Test medium: The incubation vessels each contained 250 mL of medium. Before use, the suspended solids concentration was determined and adjusted as necessary, to reach a concentration of 1.5 ± 0.3 g/L. Each vessel also contained a synthetic sewage feed, dechlorinated tap water and the test or reference item as appropriate.
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Yes
- Test concentrations: 10, 100 and 1000 mg/L during the range-finder
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
76.6 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.:
33 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of respiration due to nitrification
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
9.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
3.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
31.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of respiration due to nitrification
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No precipitation of the test substance was observable in any of the test vessels. The test substance was optically determined to be homogeneously dispersed in the test vessel.
- Effect concentrations exceeding solubility of substance in test medium: no
- Adsorption (e.g. of test material to the walls of the test container): no
- Blank controls oxygen uptake rate: 43 mg/g/h
- Coefficient of variation of oxygen uptake rate in control replicates: Coefficient of variation of the blank control respiration rate was 10 %
Results with reference substance (positive control):
- Results with reference substance valid? yes
The EC50 values of the reference item 3,5-dichlorophenol were as follows:
• 5.5 mg/L for total respiration (valid if between 2 and 25 mg/L)
• 9.3 mg/L for heterotrophic respiration (valid if between 5 and 40 mg/L)
• 1.8 mg/L for nitrification respiration (valid if between 0.1 and 10 mg/L)
Reported statistics and error estimates:
The EC50 was estimated using Probit analysis following normal distribution.
Validity criteria fulfilled:
yes
Conclusions:
In a 3 h activated sludge respiration inhibition test according to OECD guideline 209 the toxicity of the test item towards microorganisms was tested. All validity criteria were fulfilled during testing. The determined ECx values are:
Total respiration
NOEC = 9.8 mg/L
EC50 = 76.6 mg/L (95 % CI: 48.9–143.2 mg/L)
Heterotrophic respiration
NOEC = 3.1 mg/L
EC50 = 33.0 mg/L (95 % CI: 24.2–48.2 mg/L)
Nitrification
NOEC = 31.3 mg/L
EC50 > 100 mg/L
Executive summary:

A respiration inhibition test with activated sludge according to OECD guideline 209 was carried out with the test item o-phthalaldehyde. The test system was activated sludge collected from a municipal sewage treatment plant, predominantly receiving domestic sewage. The test was carried out under static conditions with the nominal test item concentrations 1 / 3.1 / 9.8 / 31 / 100 mg/L with and without allyl thiourea (ATU). The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The coefficient of variation of the blank control respiration rate (10 %) was lower than 30 %, while the respiration rate (43 mg/g/h) was higher than 20 mg/g/h. The total (5.5 mg/L), heterotrophic (9.3 mg/L) and nitrification (1.8 mg/L) respiration values for the reference item 3,5-dichlorophenol were between their respective ranges. All validity criteria were met and therefore the data are considered valid.

The test substance o-phthalaldehyde had a statistically significant effect on respiration observed during the course of the study when compared to the blank control. ln the range finding test there was significant inhibition at 100 mg/L and 1000 mg/L from 71 to 99 % at all respiration values. From this test the definitive rates were derived. As direct addition was utilised in the test media preparations the total nominal values are used in the evaluation and no further analytical evaluation was carried out. The determined EC50 value of o-phthalaldehyde for total respiration was determined to be 76.6 mg/L (95 % CI = 48.9–143.2 mg/L). For the endpoint organic carbon oxidation (i.e. heterotrophic respiration) the EC50 was determined to be 33.0 mg/L (95 % CI: 24.2–48.2 mg/L) and regarding ammonium oxidation (i.e. nitrification) as 50 % was not reached, the EC50 value was determined to be higher than the highest tested concentration of 100 mg/L, under these test conditions. The respective no-observed effect concentration (NOEC) values were 9.8 mg/L, 3.1 mg/L and 31.3 mg/L for total respiration, heterotrophic respiration and nitrification; respectively.

Results synopsis: 

Test organism: Activated sludge from domestic sewage

Test type: Static

 

Test item concentration in mg/L

ECX after 3 h of exposure

Total respiration

Heterotrophic respiration

Nitrification

NOEC

9.8

3.1

31.3

LOEC

31.3

9.8

100

EC50 (95 % CI)

76.6 (48.9–143.2)

33.0 (24.2–48.2)

> 100*

 

 

 

 

 

Reference item – 3,5-Dichlorophenol concentration (mg/L)

EC50 (95 % CI)

5.5 (3.0–11.2)

9.3 (4.8–22.3)

1.8 (1.1–3.0)

*The value of 50 % of nitrification respiration was not reached; therefore the EC50 is determined to be higher than the highest concentration tested.

 

Endpoint(s) effected: Total respiration, organic carbon oxidation (heterotrophic), ammonium oxidation (nitrification)

Description of key information

In a 3-h activated sludge respiration inhibition test according to OECD guideline 209 the toxicity of the test item towards microorganisms was tested. All validity criteria were fulfilled during testing. The determined ECx values are:

Total respiration

NOEC = 9.8 mg/L

EC50 = 76.6 mg/L (95 % CI: 48.9–143.2 mg/L)

Heterotrophic respiration

NOEC = 3.1 mg/L

EC50 = 33.0 mg/L (95 % CI: 24.2–48.2 mg/L)

Nitrification

NOEC = 31.3 mg/L

EC50 > 100 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:
33 mg/L
EC10 or NOEC for microorganisms:
3.1 mg/L

Additional information