[3R-(3α,3aβ,6α,7β,8aα)]-octahydro-6-methoxy-3,6,8,8-tetramethyl-1H-3a,7-methanoazulene

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

Study period:

10 - 11 September 2015

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

Version / remarks:

(2010)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

(2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Version / remarks:

(2007)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
According to information obtained by the sponsor Cedramber was expected to be volatile based on predicted values of EpiSuite. The vapour pressure was circa 2.5 Pa but the water solubility was predicted to be low 3.1 mg/L and therefore the Henry coefficient was quite high. Therefore, 1-Litre test bottles were filled with 200 mL of test item mixtures in Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. The test item was pipetted below the surface of the Milli-RO water and stirred in closed dark brown bottles briefly to mix (5 minutes). Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in loading rates of 10, 100 and 1000 mg/L. Optimal contact between the test item and test organisms was ensured applying continuous aeration and stirring.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Inoculum: A sample of activated sludge was obtained the day before the start of the tests from the municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was 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 ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The pH was 7.4 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

The temperature continuously measured in the temperature control vessels ranged between 18.0 and 21.3°C during the test.

pH:

Initial (before addition of the sludge): 7.7 - 7.8.
Final (after the 3h exposure period): 7.2 - 8.2

Nominal and measured concentrations:

- Nominal concentrations (0 (control), 10, 100 and 1000 mg/L (loading rates).

Details on test conditions:

TEST SYSTEM:
- Test vessel: All glass open dark-brown bottles
- Fill volume: 500 mL of test solution
- Aeration: Clean, oil-free air, 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.
- No. of vessels per concentration (replicates): Loading rate 10 and 100 mg/L: 1, Loading rate 1000 mg/L: 3
- No. of vessels per control (replicates): 6
- No. of vessels of the nitrification control: 2
- No. of vessels of the highest loading rate with a nitrification inhibitor: 3
- No. of vessels of the abiotic control: 1
- Suspended solids concentration: 1.5 g/L (determined by reviewer: the suspended solids concentration of the sludge was 3.0 g/L. The sludge was diluted twice in the test vessels)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
- Culture medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) 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

- Synthetic medium (sewage feed): In accordance with OECD 209.

OTHER TEST CONDITIONS
- Adjustment of pH: No

EFFECT PARAMETERS MEASURED: rate of oxygen consumption at the end of the period

Reference substance (positive control):

yes

Remarks:

3.5-dichlorophenol (3,5-DCP)

Duration:

3 h

Dose descriptor:

other: EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: An EL50 that is higher than the water solubility can be justified because undissolved test substance is found to be less confounding in microbial tests than in tests with higher organisms (see IR/CSA guidance R7b version 2.0, section R.7.8.17.3)

Key result

Duration:

3 h

Dose descriptor:

other: NOEL (No Observed Loading rate)

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: A NOEL that is higher than the water solubility can be justified because undissolved test substance is found to be less confounding in microbial tests than in tests with higher organisms (see IR/CSA guidance R7b version 2.0, section R.7.8.17.3)

Details on results:

- The inhibition of the total respiration rate was -8%, 13% and 15% (average) at 10, 100 and 1000 mg/L, respectively. No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg/L.
- There was no oxygen uptake from abiotic processes and the result at 1000 mg/L with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition. Based on the results of the total and heterotrophic inhibition, nitrification was calculated to be 19% inhibited.

Results with reference substance (positive control):

- The EC50 for 3,5-DCP was 4.8 mg/L

Reported statistics and error estimates:

An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the blank control revealed significant inhibition of the respiration rate (Two-sample t-test Procedure, α=0.05, one-sided smaller).

The calculations were performed with ToxRat Professional v.3.0.0 (ToxRat Solutions® GmbH, Germany).

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on materials and methods incl. tables'.

Conclusions:

In microorganisms in activated sludge the EL50 and NOEL were >1000 mg/L and 1000 mg/L, respectively.

Executive summary:

In a 3 h activated sludge respiration inhibition study conducted according to OECD guideline 209, microorganisms in activated sludge were exposed to Cedramber (mono-constituent) at loading rates of 10, 100 and 1000 mg/L.

The inhibition of the total respiration rate was -8%, 13% and 15% (average) at 10, 100 and 1000 mg/L, respectively. No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg/L.
There was no oxygen uptake from abiotic processes and the result at 1000 mg/L with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition. Based on the results of the total and heterotrophic inhibition, nitrification was calculated to be 19% inhibited. 

Description of key information

In a 3 h activated sludge respiration inhibition study conducted according to OECD guideline 209, microorganisms in activated sludge were exposed to Cedramber (mono-constituent) at loading rates of 10, 100 and 1000 mg/L. The inhibition of the total respiration rate was -8%, 13% and 15% (average) at 10, 100 and 1000 mg/L, respectively. No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg/L. There was no oxygen uptake from abiotic processes and the result at 1000 mg/L with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition. Based on the results of the total and heterotrophic inhibition, nitrification was calculated to be 19% inhibited.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L