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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:
from 26 jul 2006 to 29 nov 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Toxicity data obtained from OECD 301F ready biodegradation test.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD 301F
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of inoculum/activated sludge: from wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland).
- Preparation of inoculum for exposure: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was
decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g
(±10%) dry material per liter. During holding, the sludge was aerated at room temperature until use. Prior to use, the sludge was first thoroughly
mixed and then diluted with test water to a concentration of 1 g per liter (dry weight basis). Based on the determined dry weight of this diluted
activated sludge, defined amounts were added to test water to obtain a final concentration of 30 mg dry material per liter.
- Concentration of sludge: 30 mg/L
- Water filtered: no
Test type:
static
Water media type:
freshwater
Total exposure duration:
28 d
Test temperature:
22°C
pH:
measured in all flasks at the start: in the rabge 7.2 to 7.3
At the end of exposure: 7.3-7.7 were measured
Nominal and measured concentrations:
nominal: 100 mg/l
Details on test conditions:
- Composition of medium: The test water was prepared according to the testing guidelines. Analytical grade salts were dissolved in purified water to obtain the following stock solutions:

a) KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4 x 2H2O 33.40 g/L
NH4Cl 0.50 g/L
The pH of this solution was 7.4.

b) MgSO4 x 7H2O 22.50 g/L

c) CaCl2 x 2H2O 36.40 g/L

d) FeCl3 x 6H2O 0.25 g/L, stabilized with one drop of concentrated HCl per liter

To obtain the final test water, 10 mL of stock solution a) and 1 mL each of stock solutions b and d were combined and made up to 1000 mL with
purified water. The pH was adjusted from 8.0 to 7.4 with a diluted hydrochloric acid solution.
- Test temperature: 22°C, maintained with a built-in thermostat and checked once per week
- pH: 7.1 to 7.4
- pH adjusted: yes
- Aeration of dilution water: no
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: not relevant
- Number of culture flasks/concentration: 8: 2 for test substance (100 mg/L), 2 for inoculum control (30 mg/L), 2 for reference (100 mg/L), 1
abiotic control (100 mg/L) and 1 toxicity control (100 mg/L)
- Method used to create aerobic conditions: during holding, the sludge was aerated at room temperature until use.
- Measuring equipment:
Apparatus: The test flasks (500-mL Erlenmeyer flasks, labeled with the necessary information to ensure unmistakable identification) were incubated under continuous stirring in a SAPROMAT D12 (Voith GmbH, Heidenheim, Germany). Oxygen consumption was recorded manually by taking a daily
reading at least on each working day.

Principle: Electro-chemical analysis process: the biodegradation process consumes the dissolved oxygen in the liquid and generates CO2. The CO2
is adsorbed by soda lime and the total pressure decreases in the airtight test flasks. The pressure drop is detected and converted into an electrical
signal by means of an electrode type manometer. The consumed oxygen is replaced by electrolytically generated oxygen from a copper sulfate
solution.

SAMPLING: not relevant

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Abiotic sterile control: 1
- Adsorption control: 0
- Toxicity control: 1
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
In the toxicity controls, the biochemical oxygen demand was similar to the two procedure controls, containing only the reference item, until about exposure day 8. Thereafter, the BOD was significantly higher than in the two procedure controls until the end of the 28-day exposure period. Within 14 days of exposure, biodegradation amounted to 74%.
Thus according to the test guidelines, paradimethoxybenzene had no inhibitory effect on activated sludge micro-organisms at the tested concentration of 100 mg/l because biodegradation in the toxicity control was >25% within 14 days.
Validity criteria fulfilled:
yes
Conclusions:
Thus according to the test guidelines, paradimethoxybenzene had no inhibitory effect on activated sludge micro-organisms at the tested concentration of 100 mg/l because biodegradation in the toxicity control was >25% within 14 days.
Executive summary:

The test item Paradimethoxybenzene was investigated for its ready biodegradability in a manometric respirometry test over 28 days according to EU Commission Directive 92/69 EEC, C.4-D (1992) and OECD Guideline for Testing of Chemicals No. 301 F (1992).

The percent biodegradation of the test item was calculated based on the theoretical oxygen demand (ThOD) of 2.20 mg O2/mg test item.

Paradimethoxybenzene was found to be biodegradable under the test conditions within 28 days. Moreover, the pass level for ready biodegradability, i.e. biodegradation of at least 60% of the ThOD in a 10-day window within the 28-day period of the test, was reached.

Consequently, Paradimethoxybenzene was found to be readily biodegradable under the test conditions within28 days andhad no inhibitory effect on the activity of activated sludge microorganisms at the testedconcentrationof 100 mg/L.

Description of key information

The test item Paradimethoxybenzene was investigated for its ready biodegradability in a manometric respirometry test over 28 days according to EU Commission Directive 92/69 EEC, C.4-D (1992) and OECD Guideline for Testing of Chemicals No. 301 F (1992).
The percent biodegradation of the test item was calculated based on the theoretical oxygen demand (ThOD) of 2.20 mg O2/mg test item.
Paradimethoxybenzene was found to be biodegradable under the test conditions within 28 days. Moreover, the pass level for ready biodegradability, i.e. biodegradation of at least 60% of the ThOD in a 10-day window within the 28-day period of the test, was reached.
Consequently, Paradimethoxybenzene was found to be readily biodegradable under the test conditions within28 days and had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information