1-(2,4-dimethylcyclohex-3-en-1-yl)propan-1-ol

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:

29 September 2016 to 04 October 2016

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:

(version 2010)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(version 2015)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples for analytical measurement were taken at the beginning after a stirring period of approx. 5 minutes and at the end of the test.

Vehicle:

no

Details on test solutions:

SYNTHETIC NUTRIENT SOLUTION
- Peptone: 16.0 g
- Meat Extract: 11.0 g
- Urea: 3.0 g
- NaCl: 0.7 g
- CaCl2·2H2O: 0.4 g
- MgSO4·7H2O: 0.2 g
- K2HPO4: 2.8 g
- Deionised water: ad 1000 mL
The pH of the solution was 7.0 and therefore within the range of 7.5 ± 0.5. The nutrient solution was frozen immediately after preparation.

- Positive control: A stock solution in deionised water containing 500 mg/L was freshly prepared for the experiment.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source: The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant in D-67435 NW-Lachen-Speyerdorf.
- Pre-treatment: Upon arrival in the test facility, the sludge was filtrated, washed with tap water 3 times and re-suspended in tap water. The activated sludge was aerated until usage in the test and fed daily with 50 mL synthetic sewage feed /L.
- pH: 7.6
- Dry matter of sludge: 3.20 g suspended solids/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

21.5 - 23.2 °C

pH:

7.4 - 7.7

Nominal and measured concentrations:

- Nominal concentrations: 0 (control)*, 3.2, 10, 32, 100 and 320 mg/L
- Measured concentrations (geometric mean): 0, 0.9, 2.3, 14.8, 25.5 and 55.1 mg/L (see 'Any other information on materials and methods incl. tables')

* In the blank solution, a substance was detected at the same retention time as the test item. The concentration was determined at 0.33 mg/L. However, as the shape of the peak in the test solution and the peak in the blank control is completely different, it was concluded that no test item was present in the blank control.

Details on test conditions:

TEST SYSTEM
- Test vessels: 2000 mL SCHOTT-flasks. They were cleaned before use.
- Measuring flasks: Narrow-neck glass bottles with flat bottoms (250 mL).
- Preparation of sludge: On the day before the experiment, the inoculum was taken from its source, washed, aerated and the dry matter was determined. Volume was adapted to the desired content of dry matter. The nutrient solution was thawed and the sludge was fed with 50 mL/L sludge. On the day of the experiment, the dry matter was determined once more and determined at 1.60 g suspended solids/L.
- Replicates: 1 replicate (all concentrations positive control); 5 replicates (all concentrations test item);
- Blank control: 2 replicates before and 2 after measuring positive control and test item, respectively
- Water: tap water, see annex 2, page 13
- Feeding: nutrient solution, 16 mL/vessel

TEST PROCEDURE
In the blank control vessels, 16 mL nutrient solution was mixed with 234 mL water. The positive control vessels and the treatments were prepared by putting the appropriate amount of positive control solution or the appropriate amount of test item into the respective test vessel, adding 16 mL nutrient solution and water to reach a total volume of 250 mL. Then, 250 mL inoculum was added in 5 minute intervals and the mixtures were closed with sealed lids. After 3 hours, the content of the first vessel was shaken vigorously for 30 seconds, poured in a 250 mL narrow-neck bottle and the respiration rate was determined by measurement of the O2-concentration over a period of max. 5 minutes1. The following vessels were measured likewise in 5 minute intervals. The test item was added to the test vessels directly.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

0.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

72 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

7.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

A statistically significant effect was first seen at a test concentration of 2.3 mg/L (measured) at which 20.3% inhibition of respiration was noted. At the higher tested concentrations percentages inhibition of respiration plateaued at ca. 47.5%.

Results with reference substance (positive control):

3-h EC50 = 7.9 mg/L (95% C.L. 7.3 - 8.5 mg/L). The test was performed 29 September 2016.

Reported statistics and error estimates:

- EC50 and EC10: For the calculation of the EC10 and EC50, the percentage inhibition was plotted versus concentration in a Gauß-logarithmic diagram. EC10 and
EC50 were determined from the x-values of the regression line at y = 10% and y = 50%.
- NOEC: The t-test was used for treatment 10 mg/L; whereas the WEIR test was used for treatment 3.2 mg/L.

Table: Oxygen consumption and percentages inhibition of respiration

Nominal test conc. (mg/L)	Measured test conc. (mg/L)	Oxygen consumption			Mean % inhibition
		mg/L per min.	mg/L per hour	% Inhibition
0 (control)	0	0.6396	38.377	0	0
		0.6505	39.028	0
		0.6383	38.298	0
		0.6205	37.230	0
		0.7764	46.583	0
		0.6407	38.445	0
3.2	0.9	0.6811	40.867	-3.0	-2.3
		0.6530	39.182	1.2
		0.6921	41.523	-4.7
		0.6969	41.813	-5.4
		0.6577	39.461	0.5
10	2.3	0.5187	31.119	21.5	20.3
		0.5526	33.159	16.4
		0.6208	37.250	6.1
		0.5419	32.511	18.0
		0.3995	23.970	39.6
32	14.8	0.2873	17.237	56.5	45.7
		0.4200	25.200	36.5
		0.3422	20.534	48.2
		0.4026	24.156	39.1
		0.3426	20.556	48.2
100	25.5	0.3813	22.875	42.3	45.4
		0.3663	21.978	44.6
		0.3116	18.695	52.9
		0.3500	20.998	47.1
		0.3960	23.759	40.1
320	55.1	0.3619	21.713	45.3	47.58
		0.4063	24.377	38.5
		0.2033	12.197	69.2
		0.2986	17.915	54.8
		0.4620	27.719	30.1

Note: The difference between treatment 3.2 mg/L and the blank control can be considered as not significant as

the calculated t-value lay below the significance limit and O2 consumption in the treatment was higher than in the

blank control. Therefore, the nominal concentration 3.2 mg/L resp. 0.9 mg/L measured concentration is stated as NOEC.

Validity criteria fulfilled:

yes

Conclusions:

The 3-h EC50, EC10 and NOEC values are 22.0, 2.1 and 0.9 mg/L, respectively in microorganisms in activated sludge.

Executive summary:

The toxicity of the substance to microorganisms was investigated in an activated sludge respiration inhibition study according to OECD TG 209 and in compliance with GLP criteria (LAUS, 2017). In this study non-adapted activated sludge from a predominantly domestic sewage was exposed to nominal test substance concentrations of 0 (control), 3.2, 10, 32, 100 and 320 for 3 hours. As a certain degree of volatilisation could not be excluded, the test was performed in a closed system without aeration and the nominal test concentrations were analytically verified at the start and at the end of exposure. Within the test period of 3 hours, undissolved test item was dissolved, especially in the higher concentrated treatments and measured concentrations at the end of the test were clearly higher than at the beginning. Therefore, the geometric mean measured concentrations were calculated which were 0 (control)*, 0.9, 2.3, 14.8, 25.5 and 55.1 mg/L. Effect values are based on measured concentrations. The effects on microorganisms was determined by measuring total respiration rates and comparing these to an unexposed control. A statistically significant effect was first seen at a test concentration of 2.3 mg/L (measured) at which 20.3% inhibition of respiration was noted. At the higher tested concentrations percentages inhibition of respiration plateaued  at ca. 47.5%. Based on these findings, the 3 -h EC50, EC10 and NOEC values were determined at 22.0, 2.1 and 0.9 mg/L, respectively.

* In the blank solution, a substance was detected at the same retention time as the test item. The concentration was determined at 0.33 mg/L. However, as the shape of the peak in the test solution and the peak in the blank control is completely different, it was concluded that no test item was present in the blank control.

Description of key information

The 3-h EC50, EC10 and NOEC values are 22.0, 2.1 and 0.9 mg/L, respectively in microorganisms in activated sludge. The EC10 value of 2.1 mg/L is used as key value for assessment.

Key value for chemical safety assessment

EC50 for microorganisms:

22 mg/L

EC10 or NOEC for microorganisms:

2.1 mg/L