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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:
2016-07-19 to 2016-09-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Analytical purity: 97.5% (based on chromatographic purity GC)
- Source and lot/batch No.of test material: A15JB2997
- Expiration date of the lot/batch: 2016-10-21 (expiry date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: solubility in water: 0.2 g/L
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, 1-Litre test bottles were filled with 200 mL of test item mixtures in Milli-RO water with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for approximately 24 hours. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
- Controls: test medium without test item and treated in the same way as the test item solutions.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: predominantly domestic sewage treatment plant, "Waterschap Aa en Maas", 's-Hertogenbosch, The Netherlands.
- Preparation of inoculum for exposure: coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ~105°C to determine the amount of suspended solids
- Pretreatment: The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium was added per L of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Initial biomass concentration: 3.0 g/L of sludge
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
After 3 h of exposure, oxygen consumption was recorded for approximately 10 minutes.
Test temperature:
The temperature continuously measured in the temperature control vessels ranged between 18 and 23°C during the test, and was slightly outside the range prescribed by the study plan (20 ± 2°C). At the start of the exposure phase of the final test the temperature was slightly too high for a maximum of 15 minutes. This only affected the first two blank control vessels and the reference substance vessels.
Evaluation: The validity criteria for the blank controls and reference item were met and the temperature spike lasted for 15 minutes or less. Therefore, this deviation is considered not to have influenced the sludge.
pH:
combined limit/range-finding test:
* pH before addition of sludge: 7.6-7.7
* pH after 3-h exposure period: 7.1-7.7

final test:
* pH before addition of sludge in control: 7.6
* pH before addition of sludge in the test item replicates: 2.2-7.5
* pH after 3-h exposure period in control: 8.0-8.2.
* pH after 3-h exposure period in the test item replicates: 4.2-8.3.

Significant pH decrease was observed with increasing concentration.
Dissolved oxygen:
The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
Salinity:
Not applicable
Nominal and measured concentrations:
Loading rate for the test substance:
* Combined limit/range-finding test: 10, 100 and 1000 mg/L
* Final test: 10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass open bottles/vessels
- Aeration: The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
- Number of replicates: For final test 5 replicates per test group
- Number of control replicates: 6
- Biomass loading rate: initial loading ca. 3.0 g dw/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: adjusted ISO medium, formulated using RO-water (tap water purified by reverse osmosis) with the following composition:
CaCl2.2H2O: 211.5 mg/L
MgSO4.7H2O: 88.8 mg/L
NaHCO3: 46.7 mg/L
KCl: 4.2 mg/L

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Determination of oxygen was performed with multiple oxygen probes connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system. After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes. Respiration rate was calculated from the measurements.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 10,100, 1000 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
other: NOELR
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
other: ELR50
Effect conc.:
272 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% confidence intervals: 197-378 mg/L
Details on results:
- Blank controls oxygen uptake rate:24.88 mg O2/L.h
- Coefficient of variation of oxygen uptake rate in control replicates: 6.33
Results with reference substance (positive control):
3-h EC50 = 6.3 mg/L for limit test (this was in the accepted range of 2 to 25 mg/L for total respiration)
Reported statistics and error estimates:
The 3-h EC50 values were based on Probit Analysis.
The NOEC determination was based on the Welch-t test for inhomogeneous variances with Bonferroni-Holm Adjustment, alpha=0.05, one sided, smaller.
Validity criteria fulfilled:
yes
Conclusions:
An activated sludge respiration inhibition test (according OECD guideline 209) with activated sludge from a predominantly domestic sewage treatment plant resulted in a 3-h ELR50 = 272 mg T000749 /L (95% confidence interval: 195.8 – 377.0 mg/L). Under the conditions of this present test, the test item was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 32 mg/L (NOELR). The results of the study can be considered reliable without restriction.

Description of key information

Based on the OECD 209 guideline study (Desmares-Koopmans, 2017), to investigate the toxicity of the test substance to microoganisms, it was concluded that the test item was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 32 mg/L (NOELR).

Under the conditions of this present test, the ELR50 was estimated to be at a loading rate of 272 mg/L.

The results of the study can be considered reliable without restriction.

Key value for chemical safety assessment

EC50 for microorganisms:
272 mg/L
EC10 or NOEC for microorganisms:
32 mg/L

Additional information