4-(1,1-dimethylethyl)cyclohexyl methacrylate

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:

Experimental starting date: 04-04-2017 Experimental completion date: 06-04-2017

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 (Carbon and Ammonium Oxidation))

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch/lot No.: DAR-16030
Appearance: Colorless liquid
Water solubility: Immiscible
Volatile: Expected non-volatile (vapour pressure 0.4 kPa at 40 °C)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

4-(1,1-dimethylethyl)cyclohexyl methacrylate was weighed directly in the incubation vessels. 3,5-Dichlorophenol was added to the incubation vessels using a 1.0 g/L stock solution.
The stock solution of 3,5-dichlorophenol was prepared by dissolving 0.1008 g of 3,5-dichlorophenol (99.7%) in approximately 40 mL of deionized water with the addition of a few drops of 2 M NaOH. Finally the pH was corrected with 2M H2SO4 to approximately pH 7 and the solution was diluted further to 100 mL with deionized water.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Unadapted secondary activated sludge was obtained (04-04-2017) from the WWTP Nieuwgraaf in Duiven. The WWTP Duiven is an activated sludge plant treating predominantly domestic wastewater. Prior to use the activated sludge was homogenized with a syringe. The suspended solids (dry weight) content of the homogenized activated sludge was determined and subsequently concentrated by settlement to the required dry weight concentration. The activated sludge was aerated and fed overnight with OECD synthetic sewage concentrate at 50 mL/L prior to use on the second day following collection. The dry weight of the activated sludge in the incubation vessels was 1.5 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.1-20.5 °C

pH:

6.9-7.0

Conductivity:

<5 μS/cm

Nominal and measured concentrations:

Concentration of test material: 19.8, 37.2, 74.2, 150.6, 295.8, 594.8, 1195.2 mg/L

Details on test conditions:

Test conditions
The test was performed in 300 mL Erlenmeyer flasks with a total working volume of 50 mL. The homogenized activated sludge was incubated in a shaking water bath (100 rpm, 20 °C) for 3 hours with various concentrations of the test compound and synthetic sewage.

Synthetic sewage feed and stocks
The synthetic sewage feed was made by dissolving the following amounts of substances in 1 liter of deionized water: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O, 2.8 g K2HPO4. In the incubation vessels 1.6 mL of the synthetic sewage feed is added in a total volume of 50 mL.

Test procedures
The activated sludge respiration inhibition test was performed according to the study plan.
The study plan was developed from an OECD Test Guideline (OECD, 2010). In this test the inhibition of oxygen uptake by micro-organisms oxidizing organic carbon was not separately expressed from the respiration by micro-organisms oxidizing ammonium. The total respiration rate of activated sludge fed with a standard amount of synthetic sewage was measured. The respiration rate of the same activated sludge in the presence of various concentrations of the test substance under otherwise identical conditions was also measured. The inhibitory effect of the test substance at a particular concentration was expressed as a percentage of the mean respiration rates of the controls. Some aspects of the test are listed below:
Prior to use the activated sludge was homogenized with a syringe.
The activated sludge with test substances was incubated in a shaking bath (100 rpm/min) for 3 hours.
The test substance and the reference compound (3,5-dichlorophenol) concentrations were spaced by a factor two.
An abiotic control was not performed because neither reduction of oxygen by the test substance nor formation of oxygen clue to chemical breakdown of the test substance is expected.
No specific analyses were performed.

Determination of respiration rates, pH, dry weight and temperature
The respiration rates of the activated sludge were measured in a Biological Oxygen Monitor (BOM) YSI 5300. The BOM consisted of a thermostated vessel with a magnetic stirrer and an oxygen electrode. The electrode to measure the oxygen depletion tightly closed the vessel. The volume of the vessel was 10 mL and the temperature in the vessel was 20°C. The pH was measured using an EUTECH cyberscan pH11 pH meter. The temperature was measured with a Testo 925 digital thermometer. The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a pre-weighed 12 µm cellulose nitrate filter. This filter was dried for minimal 1.5 hours at 104 ± 5 °C and weighed after cooling. Dry weight was calculated by subtracting the weight of the filters and dividing the difference by the filtered volume.

Calculation of test results
The respiration rate was calculated from the linear part of the respiration curve as mg O2/L/min. Divided by the activated sludge dry weight concentration in the incubation vessels a respiration rate (activity) in mg O2/g/min was calculated.
In order to calculate the inhibitory effect of a test substance at a particular concentration, the respiration rate was expressed as a percentage of the mean of two controls of the respiration rate:
(1 - 2Rs / (Rc1 + Rc2)) x 100 = percentage inhibition
Rs = oxygen-consumption rate at tested concentration of test substance.
Rc = oxygen-consumption rate of controls.
The EC values were computed from the best-fitted line (linear maximum likelihood regression) through the points given by the probit of the percentage inhibition and the logarithm of the concentration of the substance. All computations were performed with the program ToxRatPro version 2.1.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 280 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.:

850 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

> 1 280 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 280 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Test conditions
The pH of the reaction mixtures after the incubation period ranged from 7.0 to 7.2. Temperature varied from 19.8 to 20.5 °C. These conditions allow respiration of the activated sludge used.

Activated sludge respiration inhibition test
The inhibitory effect of 4-(1,1-dimethylethyl)cyclohexyl methacrylate at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of 4-(1,1-dimethylethyl)cyclohexyl methacrylate for activated sludge after 3 hours contact time is >1280 mg/L, the highest concentration tested. The EC10, EC20 and EC80 values are 850 mg/L, >1280 mg/L and >1280 mg/L, respectively. 4-(1,1-dimethylethyl)cyclohexylmethacrylate is therefore considered not harmful to activated sludge.

The validity of the test is demonstrated by the three validity criteria. First, the prescribed oxygen uptake rate of >20 mg O2/g dry weight/hour for the control measurements was met (26 mg O2/g dry weight/hour). Second, the coefficient of variation of the replicates of the control oxygen uptake rates was 5%, which shows a high precision and fulfills the maximum prescribed variation of <30%. Third, the EC50 of the reference compound for activated sludge after 3 hours contact time is 12 mg/L, which is within the prescribed range of 2 to 25 mg/L.

Results with reference substance (positive control):

The EC50 of the reference compound for activated sludge after 3 hours contact time is 12 mg/L, which is within the prescribed range of 2 to 25 mg/L.

Respiration rates of the activated sludge, inhibition percentages, pH and temperature at various concentration of 3,5-dichlorophenol after 3 hours contact time.

Concentration (mg/L)	Activity (mg O2/g/min)	Inhibition (%)	pH at start test	pH at end test	Temperature at end test (°C)
Control	0.4533	-	6.9	7.0	19.9
Control	0.4000	-	6.9	7.1	19.8
2.5	0.3889	9	6.9	7.2	19.8
5.0	0.3762	12	6.9	7.1	20.0
10.1	0.2500	41	7.0	7.1	19.9
20.1	0.1000	77	7.0	7.1	20.3
40.2	0.0444	90	6.9	7.2	20.4

Concentration stock solution 3,5-dichlorophenol = 1005 mg/L

pH stock solution 3,5-dichlorophenol = 7.0

pH synthetic sewage = 7.1

 

Respiration rates of the activated sludge, inhibition percentages, pH and temperature at various concentrations of 4-(1,1-dimethylethyl)cyclohexyl methacrylate after 3 hours contact time.

Weighed test substance (mg)	Concentration (mg/L)	Activity (mg O2/g/min)	Inhibition (%)	pH at start test	pH at end test	Temperature at end test (°C)
-	Control	0.4000	-	6.9	7.2	20.4
-	Control	0.4556	-	6.9	7.2	20.0
-	Control*	0.4333	-	6.9	7.2	20.3
-	Control*	0.4333	-	6.9	7.1	20.3
0.99	19.8	0.4222	1	6.9	7.1	20.3
1.86	37.2	0.4476	-5	6.9	7.1	20.5
3.71	74.2	0.4278	0	6.9	7.1	20.2
7.53	150.6	0.4095	4	7.0	7.2	20.4
14.79	295.8	0.4167	3	7.0	7.1	20.3
29.74	594.8*	0.3889	10	6.9	7.1	20.4
59.76	1195.2*	0.3857	11	6.9	7.1	20.1

* = corresponding control and reference concentration

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of 4-(1,1-dimethylethyl)cyclohexyl methacrylate to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance.
The inhibitory effect of 4-(1,1-dimethylethyl)cyclohexyl methacrylate at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of 4-(1,1-dimethylethyl)cyclohexyl methacrylate for activated sludge after 3 hours contact time is >1280 mg/L, the highest concentration tested. The EC10, EC20 and EC80 values are 850 mg/L, >1280 mg/L and >1280 mg/L, respectively. 4-(1,1-dimethylethyl)cyclohexyl methacrylate is therefore considered not harmful to activated sludge.

Executive summary:

In order to predict effects of chemicals in the environment and in biological waste water treatment plants, an activated sludge respiration inhibition test was performed. The toxicity to activated sludge was determined in accordance with OECD Test Guideline 209, and in compliance with the OECD principles of Good Laboratory Practice.

The test is valid as shown by the EC₅₀ of the reference compound 3,5-dichlorophenol (12 mg/L), the coefficient of variation of the replicates of the control oxygen uptake rates (5%), and the average oxygen uptake rate for the control replicates (26 mg O₂/g dry weight/hour).

The toxicity of 4-(1,1-dimethylethyl)cyclohexyl methacrylate to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance.

The inhibitory effect of 4-(1,1-dimethylethyl)cyclohexyl methacrylate at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC₅₀ of 4-(1,1-dimethylethyl)cyclohexyl methacrylate for activated sludge after 3 hours contact time is >1280 mg/L, the highest concentration tested. The EC₁₀, EC₂₀ and EC₈₀values are 850 mg/L, >1280 mg/L and >1280 mg/L, respectively. 4-(1,1-dimethylethyl)cyclohexyl methacrylate is therefore considered not harmful to activated sludge.

Description of key information

The toxicity of 4-(1,1-dimethylethyl)cyclohexyl methacrylate to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance.

The inhibitory effect of 4-(1,1-dimethylethyl)cyclohexyl methacrylate at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC₅₀of 4-(1,1-dimethylethyl)cyclohexyl methacrylate for activated sludge after 3 hours contact time is >1280 mg/L, the highest concentration tested. The EC₁₀, EC₂₀and EC₈₀values are 850 mg/L, >1280 mg/L and >1280 mg/L, respectively. 4-(1,1-dimethylethyl)cyclohexyl methacrylate is therefore considered not harmful to activated sludge.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

850 mg/L