Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

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:

2019-05-16

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

Specific details on test material used for the study:

- Name: Azuril [Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3ene-1-carbonitrile]
- EC number: 915-371-2, (268-417-2, 244-530-2)
- CAS number: n/a, (68084-04-8, 21690-43-7)
- Old/other identifiers: EC 268-417-2, EC 244-530-2, CAS 68084-04-8, CAS 2169043-7
- Batch/Lot number: A170421E
- Appearance: Clear, almost colorless liquid
- Purity: 99.35%
- Expiry date: 06 June 2020
- Storage conditions: Room temperature (15-25°C, ≤70 RH%), under inert gas, protected from humidity (tightly closed container)
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Analytical monitoring:

yes

Details on sampling:

Based on poor solubility of the test item in water, a given amount test item was weighed directly into each individual flask just before the start of the experiment.As recommended by the OECD 209 Guideline, initial weighed nominal concentration were used for subsequent calculations to avoid time-consuming and expensive analytics. Therefore, no sampling was performed for test concentration validation.

For the measurement of the respiration rate a well-mixed sample of each treatment was poured into a BOD flask after approximately 3-hours incubation, and was not further aerated.

Vehicle:

no

Details on test solutions:

Test concentrations:

Due to the observed destructive effect on the caps of the oxygen electrodes during the preliminary test at concentration level of 1000 mg/L (during the abiotic control measurement), the experiment was terminated and a trial experiment was performed (with the following concentrations: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/L) to find the non-destructive concentration. Based on the results of the trial experiment, further preliminary tests were required with a maximum technical feasible 10 mg/L concentration. The main experiment was performed at 3 concentrations of the test item with 5 replicates per concentration: 1 mg/L (TI11, TI12, TI13, TI14, TI15), 5 mg/L (TI21, TI22, TI23, TI24, TI25) and 10 mg/L (TI31, TI32, TI33, TI34, TI35) were used. Flasks contained deionised water, synthetic sewage, inoculum and the test item.

Controls:

Abiotic Control (A): In parallel to the study, abiotic control flasks (A1, A2, A3, A4 and A5) were tested under identical test conditions. Flasks contained deionised water, synthetic sewage and the highest concentration of the test item (10 mg/L nominal concentration), without the addition of inoculum.

Untreated Control (B): 1-1 member of five replicates (B1, B2, B3, B4 and B5) of the untreated control group was tested in parallel with all of the test series. Flasks contained deionised water, synthetic sewage and inoculum, without addition of the test item.

Reference Control (REF): In parallel with the test item, the reference item 3,5-Dichlorophenol (REF1, REF2, REF3, REF4, REF5) was tested at nominal test concentrations of 1.0, 3.2, 10, 21 and 32 mg/L under identical test conditions. Flasks contained deionised water, synthetic sewage, inoculum and the reference item. A stock solution of 3,5-Dichlorophenol was prepared according to the OECD Guideline No. 209 (1.0 g of powdered 3,5-Dichlorophenol dissolved in 1000 mL of water). Warm deionised water and ultra-sonication was used to help the dissolution of the reference item and the volume of the solution was made up to 1.0 L with deionised water and then cooled to room temperature. The pH of the solution was checked and adjusted with NaOH solution (2 mol/L) to pH 7.38.

Preparations of the test flasks:

Each glass flask had a final volume of 400 mL (ratio of composition of each test mixture referring to 500 mL according to the guideline). A volume of 12.8 mL synthetic sewage (peptone, meat extract, urea and salts), an adequate amount of the test item and deionised water were placed into the test flask before the start of the incubation. At the start of the test, 200 mL activated sludge inoculum with a sludge concentration of 3 g/L of suspended solids was added to all flasks, except of the abiotic control flasks.

Abiotic controls in 5 parallels (A1, A2, A3, A4 and A5), and one untreated blank (B1) control was started as the first step of the test. Then at appropriate time intervals of approximately 30 minutes the further test groups were started. The second series were 5 different concentrations (without replicates) of the reference item (REF1, REF2, REF3, REF4 and REF5) and to the second blank control (B2). The third series was the 5 replicates of the test item concentration of 1 mg/L nominal concentration (TI11, TI12, TI13, TI14 and TI15) and the third blank control (B3). The fourth series was the five replicates of test item concentration 5 mg/L nominal concentration (TI21, TI22, TI23, TI24 and TI25) and the fourth blank control (B4). The final series was the five replicates of test item concentration 10 mg/L nominal concentration (TI31, TI32, TI33, TI34 and TI35) and the fifth blank control (B5).

In summary, 3 test item concentration series and one abiotic control series were prepared in 5 parallels with one blank control series by series. In parallel with the above test mixtures 5 reference item concentrations without replicates were also prepared with one blank control.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Species: Activated sludge, microorganisms from a domestic waste water treatment plant.
- Source: The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
- Conditioning: The activated sludge was supplied by the sewage plant for domestic sewage 1 day before the start of the experiment.Prior to use, the sludge was fed daily with 50 mL synthetic sewage (see section 3.7.2.) per litre and kept aerated at 20 ± 2°C until use. The activated sludge used for this study was washed and centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in chlorine-free tap water and again centrifuged. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight was determined. Based on this ratio, calculated amounts of wet sludge were suspended in chlorine-free tap water to yield a concentration equivalent to 3 g/L (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.20. The activated sludge was used directly after conditioning.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not reported

Test temperature:

Target Temperature: The test should be performed at a temperature within the range 20± 2°C.
Actual Temperature: The test temperature was in the range of 20.6 - 21.4 ºC.

pH:

Target pH: The test should be performed at a pH within the range 7.5 ± 0.5.
Actual pH: The pH was in the range of 7.20 - 8.12

Dissolved oxygen:

Not applicable

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

The concentrations of test item used in the main experiment were: 1, 5, 10 mg/L nominal concentrations. Concentrations in solution were not measured.

Details on test conditions:

Appropriate glass beakers for 500 mL volume and BOD bottles with 300 mL volume were used. Each test flask was uniquely identified with study code, treatment and replicate codes.

Test was performed in a temperature controlled laboratory an aeration with compressed air (about 1 L/min) was used. Test conditions were measured with suitable instruments and documented in the raw data.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Details on results:

Preliminary tests:

A destructive effect on the caps of the oxygen electrodes was observed during the first preliminary test at concentration level of 1000 mg/L. A trial experiment was performed (with the following concentrations: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/L) and showed that the highest technical feasible test concentration was 10 mg/L.

Definitive test:

Validity: During the experiment the blank controls oxygen uptake rate was <20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour. The specific respiration rate of blank controls (mg O2/L/h/g solid sludge) are: B1=17.68; B2=16.63; B3=17.65; B4=17.87; B5=16.16. The average specific oxygen uptake of controls was 17.20 mg O2/L/h/g solid sludge. Although this is slightly below the OECD recommended level, this deviation was considered not to adversely affect the result or integrity of the study. Taking into account the lack of any significant inhibition of test item and appropriate reference control results, the study is considered to be valid, in agreement with the Sponsor. As required by the guideline, the coefficient of variation of oxygen uptake rate in control replicates lower than 30% at the end of the test (4.40%), and the EC50 of the positive control substance 3,5-Dichlorophenol (3,5-DCP) was in the required range of 2 mg/L to 25 mg/L for total respiration (10.22 mg/L with 95% confidence limits of 8.11 – 12.88 mg/L).

Respiration inhibition: In comparison to the inoculum controls the inhibition of the respiration rate in the case of the activated sludge was between -8.82% and 1.86% in the examined range of 1–10 mg/L (nominal concentration). Under the conditions of this study the observed inhibition of activated sludge respiration by the test item was an 3-hour EC50 >10 mg/L (nominal). The test item had no significant inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 10 mg/L (nominal), but further testing at higher concentrations was not technically feasible due to the chemical reactivity of the test item with the oxygen sensors.

Results with reference substance (positive control):

In comparison to the controls, the inhibition of the respiration rate of the activated sludge was 74.91% at the highest nominal concentration of 32.0 mg/L. At the nominal concentrations of 1.0, 3.2, 10.0 and 21.0 mg/L 11.12%, 30.30%, 46.08% and 63.29% inhibition of the respiration rate was calculated respectively. The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 10.22 mg/L with 95% confidence limits of 8.11 – 12.88 mg/L.

Reported statistics and error estimates:

Calculation of oxygen uptake rates:

The oxygen uptake rates (R) was calculated from the measured values, based on the linear part of the graphs of oxygen concentration (values taken in consideration between 2.0 mg/L and 7.0 mg/L only) versus time.
R = (Q1 – Q2)/Δt × 60
where:
Q1: oxygen concentration at the beginning of the selected section of the linear phase (mg/L).
Q2: oxygen concentration at the end of the selected section of the linear phase (mg/L).
Δt: time interval between the two measurement (min).
The R value was composed for all of the parallels and the mean R values were calculated from them, to determine the inhibition effects.

Calculation of percentage of inhibition:

The percentage inhibition of the total oxygen consumption (IT) was calculated:
IT = [1-(RT – RTA)/RTB)] × 100%
where:
RT: rate of total oxygen consumption (mg/L/hour).
RTA: rate of total oxygen consumption (mg/L/hour) due to abiotic processes.
RTB: rate of total oxygen consumption (mg/L/hour) due to untreated (blank) controls.

Calculation of specific respiration rate:

The specific respiration rate (Rs) is the ratio of the consumed oxygen and the dry weight (g) of sludge per hour (mg/g/h)
Rs = R/SS
where:
SS: concentration of suspended solids in the test mixture (g/L).
R: oxygen uptake rate (see above)
The oxygen uptake rates, percentage of inhibition and specific respiration rates were calculated by using Excel 2016 for Windows Software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).

Determination of percentage inhibition of oxygen uptake by 50% (EC50):

The 3-hour EC50 value of the reference item and its 95 %-confidence limits were calculated by Probit analysis using TOXSTAT software. In case of the test item the EC50 was determined directly from the values of percentage of inhibition.

Influence of test item on oxygen consumption of activated sludge

Test group	Conc. of test item in test mix.	Total oxygen consumption rate(RT)	Specific respiration rate(RS)	Inhibition	pH values*
Name, ID	mg/L (nominal)	mg O2/L/h	mg O2/L/h/g solid	%	start	end
Abiotic control
A1	10	-0.29	---	---	7.24	7.71
A2		-0.41	---	---	7.20	7.73
A3		-0.54	---	---	7.26	7.75
A4		-0.74	---	---	7.26	7.73
A5		-0.37	---	---	7.27	7.72
Blank control
B1	0.0	26.53	17.68	---	7.21	7.51
B2		24.95	16.63	---	7.36	7.85
B3		26.48	17.65	---	7.47	7.98
B4		26.80	17.87	---	7.51	8.06
B5		24.24	16.16	---	7.55	8.11
Reference item
REF1	1.0	22.46	14.97	11.12	7.35	7.82
REF2	3.2	17.51	11.67	30.30	7.34	7.84
REF3	10.0	13.44	8.96	46.08	7.35	7.88
REF4	21.0	9.00	6.00	63.29	7.35	7.90
REF5	32.0	6.00	4.00	74.91	7.35	7.90
Test item
TI1	1	27.60	18.40	-8.82	7.47	7.98
TI2	5	26.89	17.93	-6.07	7.51	8.06
TI3	10	24.85	16.56	1.86	7.55	8.12

*Note: Individual pH values of the BOD flasks, except TI1-2-3, where the average values are reported.

---: not applicable

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study the observed endpoint for the effect of the test item was the 3-hour EC50 value which was considered to be > 10 mg/L (nominal). The test item had no significant inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 10 mg/L (nominal) i.e., NOEC =10 mg/L (nominal), but further testing at higher concentrations was not technically feasible due to the chemical reactivity of the test item with the oxygen sensors.

Executive summary:

Aquatic toxicity of Azuril towards microorganisms was assessed with an Activated Sludge, Respiration Inhibition Test described by the OECD TG 209 Guideline. Due to the observed destructive effect on the caps of the oxygen electrodes during the abiotic control measurement, the highest technical feasible test concentration was 10 mg/L. Under the conditions of this study the observed inhibition of activated sludge respiration by the test item was a 3-hour EC50 >10 mg/L (nominal), with a corresponding 3-hour NOEC = 10 mg/L (nominal).

Description of key information

Aquatic toxicity of Azuril towards microorganisms was assessed with an Activated Sludge, Respiration Inhibition Test described by the OECD TG 209 Guideline. Due to the observed destructive effect on the caps of the oxygen electrodes during the abiotic control measurement, the highest technical feasible test concentration was 10 mg/L. Under the conditions of this study the observed inhibition of activated sludge respiration by the test item was a 3-hour EC50 >10 mg/L (nominal), with a corresponding 3-hour NOEC = 10 mg/L (nominal).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

10 mg/L

Additional information

The average specific oxygen uptake of controls was 17.20 mg O2/L/h/g solid sludge. Although this is slightly below the OECD recommended level, this deviation was considered not to adversely affect the result or integrity of the study. Taking into account the lack of any significant inhibition of test item and appropriate reference control results, the study is considered to be valid, in agreement with the Sponsor.

In comparison to the inoculum controls, the inhibition of the respiration rate in the presence of the test item was between -8.82% and 1.86% in the examined range of 1–10 mg/L (nominal concentration). Thus, no significant inhibition was observed.