Reaction mass of 1-(3,3-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one and 1-(5,5-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one

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:

27 August - 25 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:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The batch of GALBASCONE tested was a clear colorless liquid. No correction was made for the purity/composition of the test item.

- 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 (tap-water purified by reverse osmosis; Millipore Corp., USA) with initial loading rates of 2.5 times the final loading rate. The test substance was added by pipetting the required amount just below the surface of the test medium. These mixtures were briefly stirred (ca. 5 minutes) in closed dark brown bottles. 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 applying continuous aeration and stirring.
- Controls: Blank control = test medium without test item

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 Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: On the day of collection, the sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and adjusted 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). Synthetic sewage (50 mL/L) was added to the stock of activated sludge and this was aerated at test temperature overnight and until use. The pH was 7.6 on the day of testing.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

Combined limit/range-finding test: 19 - 22 °C
Final test: 18- 21 °C

pH:

Combined limit/range-finding test: 7.5-7.6 (start), 7.4-8.3 (end)
Final test: 7.8-7.9 (start), 7.5- 8.3 (end)

Nominal and measured concentrations:

- Nominal concentrations (loading rates):
* combined limit/range-finding test: blank control (0), 10, 100 and 1000 mg/L
* final test: blank control (0), 46, 100, 220, 460 and 1000 mg/L

Details on test conditions:

TEST SYSTEM (Definitive test):
- Test vessel: 1 L bottle
- Type: open
- Headspace, fill volume: 500 mL of test solution (including 250 mL activated sludge)
- Aeration: yes; clean, oil-free compressed air - 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): 5
- No. of vessels per control (replicates): 6
- Suspended solids concentration in final test solution: 1.5 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water purified by reverse osmosis (Millipore Corp., USA)
- Test medium: adjusted ISO medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature - continuously in a control vessel, pH - at the start and the end of the test period

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): rate of oxygen consumption at the end of the period

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 2.2
* Range finding study
- Test concentrations (loading rates): Blank control, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

3.5-dichlorophenol (3,5-DCP)

Key result

Duration:

3 h

Dose descriptor:

other: EL50

Effect conc.:

ca. 960 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Remarks:

(loading rate)

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

other: NOEL

Effect conc.:

46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(loading rate)

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

other: EL10

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(loading rate)

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL: 0.31 - 75 mg/L (loading rate)

Details on results:

Evaluation was based on the inhibition of the total respiration.

- Combined limit/range-finding test:
The test showed no inhibition, 9 and an average of 37% inhibition of the respiration rate at a loading rate of 10, 100 and 1000 mg/L, respectively.

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 46%. Based on the results of the total and heterotrophic respiration, nitrification was calculated not to be inhibited. Therefore, N-allylthiourea was not used in the final test.

- Final test:
The effects observed were in agreement with what was expected based on the results of the combined limit/range-finding test.
No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 46 mg test substance per litre. At higher loading rates the inhibitory effect of the test substance on aerobic waste water (activated sludge) bacteria increased with increased loading rate, ranging from 22% inhibition at 100 mg/L to 47% at 1000 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- The EC50 for 3,5-DCP was 5.5 mg/L (definitive test)

Reported statistics and error estimates:

- ECx and ELx

For the reference item, calculation of EC50 values was based on probit analysis using linear maximum likelihood regression with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item.

For the test substance, calculation of ELx values was based on probit analysis using linear maximum likelihood regression with the percentages of respiration inhibition versus the logarithms of the corresponding loading rates of the test item.

- NOEL determination

An effect was considered to be significant if statistical analysis of the data obtained for the test loading rates compared with those obtained in the blank control revealed significant inhibition of the respiration rate (Welch-t test for Inhomogeneous Variances with Bonferroni-Holm Adjustment, α=0.05, one-sided, smaller).

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

Final test – Overview of the results

Treatment	Loading rate (mg/L)	pH		Mean respiration rate		% Inhibition of the respiration rate (mean value)
		Start	End	(mg O2/L h)	(mg O2/g h)¹
Blank control		7.8	7.5 – 8.0	44	30
T1	46	7.8	7.7 – 7.9	41	27	7
T2	100	7.8	7.7 – 7.8	35	23	22*
T3	220	7.8	7.5 – 7.9	27	18	39*
T4	460	7.8	7.5 – 7.9	27	18	40*
T5	1000	7.8	7.5 – 7.7	24	16	47*

¹ The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control

Validity criteria fulfilled:

yes

Remarks:

mean blank control O2 uptake rate >20 mg O2/g act. sludge/h, CV O2 uptake in blank control < 30%; EC50 reference item within acceptable range

Conclusions:

Under the conditions of the test the substance was not toxic to waste water bacteria (activated sludge) at a loading rate of 46 mg/L (NOEL). In the final test, the highest tested concentration (loading rate of 1000 mg/L) caused 47% inhibition of the total respiration rate. The EL50 of the test substance was estimated to be 960 mg/L.

Executive summary:

In a 3 h activated sludge respiration inhibition study conducted according to OECD guideline 209 and GLP, microorganisms in activated sludge were exposed to a blank control and 5 concentrations of the test substance at loading rates of 46, 100, 220, 460 and 1000 mg test substance/L. The EL50 of the test substance was estimated to be 960 mg test substance/L (based on loading rate) and the NOEL was 46 mg test substance/L.

Description of key information

In a 3 h activated sludge respiration inhibition study conducted according to OECD guideline 209 and GLP, microorganisms in activated sludge were exposed to a blank control and 5 concentrations of the test substance at loading rates of 46, 100, 220, 460 and 1000 mg test substance/L. The EL50 of the test substance was estimated to be 960 mg test substance/L (based on loading rate) and the NOEL was 46 mg test substance/L.

Key value for chemical safety assessment

EC50 for microorganisms:

960 mg/L

EC10 or NOEC for microorganisms:

46 mg/L