Ethanol, 1-[2-butyne-1,4-diylbis(oxy)][2,2,2-trichloro-

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

The study will be conducted in accordance with the following guidelines:
• OECD Guidelines for the Testing of Chemicals No. 209 adopted 22. Jul. 2010
”Activated Sludge, Respiration Inhibition Test“
• Commission Regulation (EU) No. 2016/266 amending Regulation (EC) No. 440/2008, EU-Method C.11. “Activated Sludge, Respiration Inhibition Test“, adopted 07. Dec. 2015
In case that differing demands are made in these two guidelines, the study is performed adhering to the OECD guideline following the recommendations of the regulatory authority.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP-Conformity All procedures will be according to the principles of GLP (Chemikaliengesetz §19a and §19b and annexes 1 and 2 from 28. Aug. 2013, published in Federal Law Gazette, Germany (BGBl) No. 55/2013 as of 06. Sep. 2013, and further revisions).

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The test material is representative of the registered substance, and no significant differences in the purity profile exist
- Expiration date of the lot/batch: not relevant (stable)
- Purity test date: not relevant, the test material is representative of the registered substance

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble
- Reactivity of the test substance with the solvent: none (contains water)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The test material is representative of the registered substance
- Expiration date of the lot/batch: not relevant
- Purity test date: not relevant

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

4.5.2 Oxygen Measurement
After 3 hours, narrow neck bottles are filled to the rim with an aliquot of the content of each vessel, a magnetic stirrer is added, and the oxygen electrode is tightly fitted into the vessel. Care will be taken that no air can reach the contents of the vessel. The oxygen concentration of the vessel is measured every 5–10 seconds over a period of 3-5 minutes. The interface of the oxygen meter is connected to a personal computer, feeding the meas-ured values with their corresponding times in a table.
Usually, measurement of total oxygen uptake is sufficient according to OECD guideline 209. If inconsistencies in the dose-response curve occur, a second experiment is per-formed in order to discern between inhibition of nitrificators and inhibition of total popula-tion of heterotrophic bacteria. This will, if necessary, be described in an amendment to this study plan.

Test solutions

Vehicle:

no

Details on test solutions:

A range-finding test with the following treatments will be performed:
1000 / 100 / 10 / 1 mg/L. nominal concentration
The real concentration will be stated in the final report.
For the treatment 1000 mg/L, 5 replicates are used; for the other treatments, 1 replicate each. If no inhibition > 20 % is observed at 1000 mg/L a NOEC will be determined by us-ing a one-tailed significance test. If no statistically significant inhibition is present and the NOEC is stated as ≥ 1000 mg/L, the EC50 is stated as > 1000 mg/L and no further testing will be done.
If significant inhibition is observed at 1000 mg/L, a main test will be conducted.

Nutrient Solution see inoculum

The substances are dissolved in deionised water. pH is adjusted to 7.5 ± 0.5, if necessary.
The nutrient solution may be frozen directly after preparation and stored at < -15 °C until use in the test.

The following instruments and devices were used
Scales
Adjustable pipettes with one-way tips
Volume measurement materials
pH-meter
Oxygen meterHeating chamber
PC with interface for the collection of oxygen values
Standard laboratory material will also be used in the test.
A stock solution of the test item containing 10 g/L (± 10 %) was prepared.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Specification and Origin
The microbial inoculum to be used in this study consists of activated sludge from a sewage treatment plant treating predominantly domestic sewage.
Origin: sewage treatment plant Neustadt/W (if unavailable: Edenkoben. or Haßloch)
Date of collection 1 or 2 days before the start of the test
The sludge is filtrated or centrifuged and re-suspended in tap water. The activated sludge is aerated until usage in the test and fed daily with 50 mL synthetic sewage feed/L. Suspended solids 1.0 – 1.6 g/L
On the day of the start of the test the dry matter is determined by drying a volume of 5 mL at 105 ± 1 °C and weighing the remnants. The sludge then is adjusted to a suspended solids level of 2.0 – 3.2 g/L with tap water and aerated until usage in the test.
Before the test, pH will be checked and adjusted to 6.0 – 8.0, if necessary.

All chemicals to be used in the test are suited for microbial purposes or analytical grade or otherwise proved suitable.
Table 3.4 a Composition of Nutrient Solution
Parameter Concentration in g/L
Peptone 16.0
Meat Extract 11.0
Urea 3.0
NaCl 0.7
CaCl2*2H2O 0.4
MgSO4*7H2O 0.2
K2HPO4 2.8

The substances are dissolved in deionised water. pH is adjusted to 7.5 ± 0.5, if necessary.
The nutrient solution may be frozen directly after preparation and stored at < -15 °C until use in the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

The respiration rates of samples of activated sludge fed with synthetic sewage are measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours.

Post exposure observation period:

After 3 hours, narrow neck bottles are filled to the rim with an aliquot of the content of each vessel, a magnetic stirrer is added, and the oxygen electrode is tightly fitted into the vessel. Care will be taken that no air can reach the contents of the vessel. The oxygen concentration of the vessel is measured every 5–10 seconds over a period of 3-5 minutes. The interface of the oxygen meter is connected to a personal computer, feeding the meas-ured values with their corresponding times in a table.
Usually, measurement of total oxygen uptake is sufficient according to OECD guideline 209. If inconsistencies in the dose-response curve occur, a second experiment is per-formed in order to discern between inhibition of nitrificators and inhibition of total popula-tion of heterotrophic bacteria.

Test conditions

Hardness:

Not specified but the following concentrations reported:
CaCl2*2H2O 0.4
MgSO4*7H2O 0.2

Test temperature:

20.0 +/- 2.0 °C

pH:

The substances are dissolved in deionised water. pH is adjusted to 7.5 ± 0.5

Dissolved oxygen:

Aeration by purified air, using Pasteur pipettes (no data measured), usually between 7.0 and 2.0 mg O2/L

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

nominal

Details on test conditions:

Suspended solids 1.0 – 1.6 g/L

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (1,3-Dichloro-5-hydroxybenzene, C6H4Cl2O, CAS-No. 591-35-5)

Results and discussion

Effect concentrations

Key result

Duration:

3 h

Dose descriptor:

IC50

Effect conc.:

1 g/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

inhibition of heterotrophic respiration

Details on results:

VALIDITY Criteria
• The oxygen uptake rate in the controls should not be less than 20 mg oxygen per gram of activated sludge (dry weight of suspended solids) in 1 hour.
• The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of the test.
• EC50 of the positive control should lie between 2 and 25 mg/L (total respiration)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

IC50 = 1 g/L

Executive summary:

Testing according to OECD 209 indicates an IC50 = 1 g/L for the Test item.