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 aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-04-23 to 2019-04-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name: Azuril [Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-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 21690-43-7
- Batch/Lot number: A170421E
- Appearance: Clear, almost colourless liquid
- Purity: 99.35%
- Expiry date: 06 June 2019
- Storage conditions: Room temperature (15-25°C, ≤70 RH%), under inert gas, protected from humidity (tightly closed container)

Analytical monitoring:

yes

Details on sampling:

Analytical measurement was performed from the control at the beginning and at the end of the experiment, at the applied test concentration levels at the beginning of the experiment and 24-hour intervals thereafter, in order to better define loss of the test substance during the exposure period. Samples without algae were also taken (from remaining test solutions) for analysis at each test concentration level in order to distinguish degradation and adsorption of the test item to algae.

Duplicate samples were taken (~10 mL) in glass tubes at the applied test concentrations (with and without algae) at the beginning of the test and 24 hour intervals thereafter during the experiment. Sample from the control was only be taken for analysis at the start and at the end of the test. After sampling, samples were frozen and kept approximately at -20°C at the Test Facility. One set of the samples was sent to the Test Site for analysis and one set was retained as a back-up at the Test Facility, if required for any confirmatory analyses (discarded after satisfactory results were obtained for the first set).

Total samples:
- 42 tubes of test solution (~40 mL aliquots, measured with 0.01 g precision) were received at 20 February 2019 from the Test Facility corresponding to the control and each test concentration at the start and at the end of the experiment.
- 135 tubes of test solution (~10 mL aliquots, measured with 0.01 g precision) were received at 30 April 2019 from the Test Facility corresponding to the control and each test concentration with and without the presence of algae at 0 h, 24 h, 48 h and 72 h.

Vehicle:

no

Details on test solutions:

Solution preparation: Because the test item is poorly soluble in water, test solutions were prepared individually using a saturated solution method (water accommodated fraction, WAF) according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.

Saturated test item solutions (0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mg/L nominal loading rates WAFs) were prepared individually by dispersing/dissolving the amount of test item into the test medium (OECD medium) two days before the start of the study. These solutions were shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. The non-dissolved test material was removed by filtration through a fine (0.22 µm) filter to give appropriate WAF solutions.

The test solutions were prepared and distributed into test vessels prior to introduction of algae. During the preparation of WAF solutions and the study the usage of plastic lab wares was omitted.

The preliminary range-finding concentrations were prepared individually as per the OECD No. 23 methods (1, 5, 50, 100 mg/L nominal loading rates WAFs), and by dilution (0.001, 0.01, 0.1 mg/L nominal loading rates WAFs).

Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests. For the untreated control, algal growth medium was inoculated with algal cells (without test item) and was examined in parallel to the test item concentrations.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

- Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
- Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Algal cells were cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of Citoxlab Hungary Ltd.
- Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
- Culture conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines. The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The preculture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of two days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

- Target Temperature: 21-24°C maximum deviation of ± 2°C
- Actual Temperature: Actual Temperature:Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was between 22.4 and 22.5 °C measured in the flask and between 21.7 and 23.2 °C measured within the climate chamber.

pH:

- Target pH: Maximum deviation of ± 1.5 units
- Actual pH: The pH was checked at the beginning and at the end of the test, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.60 –8.94 during the experiment.

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

The following loading concentrations were tested in the definitive test: 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mg/L nominal loading rate WAFs. The test item was found to degrade over the study duration. The corresponding measured geometric mean test item concentrations were: 0.18, 0.39, 0.96, 1.99, 2.29 and 2.96 mg/L. All the results/conclusions in this report are expressed as the nominal loading rate WAFs and as the geometric mean measured concentration.

Details on test conditions:

Three concentration range-finding tests were conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test. Algal cells were exposed to each concentration of the test item plus controls, for 72 hours. The tests were performed with 2 replicates per each test concentration and 3 replicates in the control group.

The definitive test was started (0 hours) by inoculation of a biomass of approximately 10^4 algal cells per mL test medium. The test was performed with three replicates per test concentration and six replicates in the control group. Volumes of 150 mL algal suspension per replicate in 250 mL Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers. The exposure time was 72 hours.

The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 7502 lux (equivalent to ~101 µE/m2 /s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15% and therefore provided equal conditions for each test vessel.

The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in each concentration and in the control was performed (at 24, 48 and 72 hours) to detect any abnormal appearance of the algae.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.39 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Preliminary range-finding tests:

Three preliminary experiments were performed with different concentration ranges. The summarised average cell number (x10^4 cells/mL) at 72 hours was 72.5/75.0 at 0 mg/L (control), 70.0 at 0.001 mg/L, 69.5 at 0.01 mg/L, 68.0 at 0.1 mg/L, 66.5 at 1 mg/L, 2.0 at 5 mg/L, 1.0 at 50 mg/L and 1.0 at 100 mg/L. Chuff and/or thin cells were observed at the concentration level of 100 mg/L.

Defnitive test:

Validity: The cell density in the control cultures increased by the factor of 71.83 within three days (OECD guideline requires ≥ 16). The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 10.27% (OECD guideline requires ≤ 35%). The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.75% (OECD guideline requires ≤ 7%). All validity criteria were met; therefore, the study can be considered as valid.

Conentrations of test item: The following nominal loading rates were tested: 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mg/L. Test concentrations were analytically determined at the start and 24-hour intervals thereafter during the experiment in order to better define loss of the test substance during the exposure period. Additional samples in absence of algae were also taken for analysis at each test oncentration level in order to distinguish adsorption of the test item to the test system. As no significant differences were detected in the absence and presence of the algae, the corresponding measured geometric mean test item concentrations were calculated with the data of the samples in presence of algae. The test item was found to degrade over the study duration, a semi-static approach is not feasible with the Algae test, so a minimal WAF method was used to ensure maximal concentrations of test item in aquatic media at time zero. The corresponding measured geometric mean test item concentrations were: 0.18, 0.39, 0.96, 1.99, 2.29 and 2.96 mg/L. Therefore, all the results/conclusions in this report are expressed as the nominal loading rates; the measured values are also shown.

Morphological deviation of the algal cells: In the preliminary range-finding test chuff and/or thin cells were observed at the concentration level of 100 mg/L nominal loading rate WAF. Chuff cells were observed in the main experiment at the concentration levels of 2.0, 3.0, 4.0 and 5.0 mg/L nominal loading rates WAFs.

Growth rate (r): Growth rate is the increase in cell density per time unit. The average specific growth rate for a specific period was calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments, following the equations in OECD Guideline No 201. The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value at the measured concentration range of 2.0 – 5.0 mg/L nominal loading rates WAFs, accordingly the No Observed Effect Loading Rate (NOELR) was determined as 1.0 mg/L nominal loading rate WAF. The 72 h ErL50 was determined [by Probit analysis (TOXSTAT software)] as 2.8 mg/L nominal loading rate WAF (95 % confidence limits: 2.62 – 3.02 mg/L nominal loading rate WAF). The 72 h ErC50 was determined [by Probit analysis (TOXSTAT software)] as 1.6 mg/L (95 % confidence limits: 1.42 – 1.70 mg/L).

Biomass (b): The areas under the growth curve were used to calculate biomass, i.e., the actual number of cells per volume of medium (cells/mL). The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h areas were statistically significantly different from the untreated control value at the measured concentration range of 1.0 – 5.0 mg/L nominal loading rates WAFs, accordingly the No Observed Effect Loading Rate (NOELR) was determined as 0.5 mg/L nominal loading rate WAF. The 72 h EbL50 value was determined [by Probit analysis (TOXSTAT software)] as 1.6 mg/L nominal loading rate WAF (95 % confidence limits: 1.49 – 1.76 mg/L nominal loading rate WAF). The 72 h EbC50 value was determined [by Probit analysis (TOXSTAT software)] as 0.8 mg/L (95 % confidence limits: 0.68 – 0.84 mg/L).

Yield (y) or cell number: Yield is calculated as the biomass at the end of the test minus the starting biomass for each single vessel of controls and treatments. For each test concentration and control, mean yield values were calculated following the equations in OECD Guideline No 201. The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h yield was statistically significantly different from the untreated control value at the tested concentration range of 1.0 – 5.0 mg/L nominal loading rates WAFs, accordingly the No Observed Effect Loading Rate (NOELR) was determined as 0.5 mg/L nominal loading rate WAF. The 72 h EyL50 value was determined [by Probit analysis (TOXSTAT software)] as 1.7 mg/L nominal loading rate WAF (95 % confidence limits: 1.56 – 1.81 mg/L nominal loading rate WAF). The 72 h EyC50 value was determined [by Probit analysis (TOXSTAT software)] as 0.8 mg/L (95 % confidence limits: 0.72 – 0.87 mg/L).

Results with reference substance (positive control):

The experimental period of the last study (Citoxlab Study Code: 19/009-022AL) with the reference item Potassium dichromate was (Batch Number: A0345704) 22 – 25 January 2019. Results were the following:

The 72h ErC 50: 0.87 mg/L, (95 % confidence limits: 0.80 – 0.95 mg/L)
The 72h EbC 50: 0.62 mg/L, (95 % confidence limits: 0.57 – 0.68 mg/L)
The 72h EyC 50: 0.52 mg/L, (95 % confidence limits: 0.48 – 0.57 mg/L)

These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) to demonstrate exponential growth for the entire study period.

The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel 2007 for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).

The ErL50/ErC50, EbL50/EbC50 and EyL50/EyC50 values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software. Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.

For the determination of the LOELR/LOEC and NOELR/NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.

Results of the Preliminary Range-Finding Tests (summarized)

Nominal concentrations	Untreated	0.001	0.01	0.1	1	5	50	100
mg/L nominal loading rates WAFs]	Control	Dilution	Dilution	Dilution	Direct	Direct	Direct	Direct
Average of cell number at 72 hours (x 10^4cell/mL)	72.3/75	70.0	69.5	68.0	66.5	2.0	1.0	1.0

Calculation of exposure concentrations with algae

Loading rate (mg/L)	Mean measured concentrations(mg/L)				Geometric mean
	0h/start	24h	48h	72h/end	mg/L
Control	n.d.	-	-	n.d.	---
0.5	0.313	0.223	0.179	0.090	0.18
1.0	0.637	0.513	0.376	0.182	0.39
2.0	1.787	0.952	1.021	0.493	0.96
3.0	3.186	2.329	1.692	1.255	1.99
4.0	3.434	2.622	2.084	1.473	2.29
5.0	4.018	3.260	2.786	2.107	2.96

n.d. – not detected

Calculation of exposure concentrations without algae

Loading rate (mg/L)	Mean measured concentrations(mg/L)				Geometric mean
	0h/start	24h	48h	72h/end	mg/L
Control	n.d.	-	-	n.d.	---
0.5	0.313	0.226	0.186	0.048	0.16
1.0	0.637	0.604	0.499	0.324	0.50
2.0	1.787	1.654	1.231	0.738	1.28
3.0	3.186	2.728	2.251	1.470	2.32
4.0	3.434	3.135	2.576	1.625	2.59
5.0	4.018	3.933	3.252	2.206	3.26

n.d. – not detected

Growth Rates (r) and Percentage Inhibition of m during the Test Period

Concentration		Growth rate (r) and % inhibition of r
Nominal Loading Rate WAF	Measured	0–24 h		0–48 h		0–72 h
[mg/L]	[mg/L]	r	%	r	%	r	%
Control	-	0.0589	0.0	0.0604	0.0	0.0594	0.0
0.5	0.18	0.0609	-3.4	0.0594	1.6	0.0591	0.5
1.0	0.39	0.0538	8.7	0.0533*	11.7	0.0581	2.1
2.0	0.96	0.0458*	22.2	0.0373*	38.2	0.0458*	22.9
3.0	1.99	0.0289*	50.9	0.0144*	76.1	0.0269*	54.6
4.0	2.29	0.0289*	50.9	0.0144*	76.1	0.0134*	77.5
5.0	2.96	0.0289*	50.9	0.0144*	76.1	0.0096*	83.8

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

Biomass. Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Concentration		Area under the Growth Curves (A) and Percentage Inhibition of A
Nominal Loading Rate WAF	Measured	0–24 h		0–48 h		0–72 h
[mg/L]	[mg/L]	A	%	A	%	A	%
Control	-	38.0	0.0	282.0	0.0	1338.0	0.0
0.5	0.18	40.0	-5.3	276.0	2.1	1304.0	2.5
1.0	0.39	32.0	15.8	208.0*	26.2	1128.0*	15.7
2.0	0.96	24.0*	36.8	108.0*	61.7	480.0*	64.1
3.0	1.99	12.0*	68.4	36.0*	87.2	120.0*	91.0
4.0	2.29	12.0*	68.4	36.0*	87.2	68.0*	94.9
5.0	2.96	12.0*	68.4	36.0*	87.2	60.0*	95.5

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

Yield (Y) and Percentage Inhibition of Y during the Test Period

Concentration			Concentration
Nominal Loading rate WAF		Measured		0–72 h
[mg/L]		[mg/L]		Y		%
Control		-		70.8		0.0
0.5		0.18		69.3		2.1
1.0		0.39		64.7*		8.7
2.0		0.96		26.0*		63.3
3.0		1.99		6.0*		91.5
4.0		2.29		1.7*		97.6
5.0		2.96		1.0*		98.6

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

Influence of the test item on the Growth ofPseudokirchneriella subcapitatacalculation based on the initial nominal loading rates (WAFs)

Parameter (0-72 h)	Growth rate (r) Loading Rate WAF [mg/L]	Yield (y) Loading Rate WAF [mg/L]	Biomass (b) Loading Rate WAF [mg/L]
Calculation based on the nominal loading rates (WAFs)
EL50	2.8	1.7	1.6
95 % conf. limits	2.62 – 3.02	1.56 – 1.81	1.49 – 1.76
NOELR	1.0	0.5	0.5
LOELR	2.0	1.0	1.0

Influence of the test item on the Growth ofPseudokirchneriella subcapitatacalculationbased on the measured test item concentrations

 

Parameter (0-72 h)	Growth rate (r) [mg/L]	Yield (y) [mg/L]	Biomass (b) [mg/L]
Calculation based on the measured concentrations
EC50	1.6	0.8	0.8
95 % conf. limits	1.42 – 1.70	0.72 – 0.87	0.68 – 0.84
NOEC	0.39	0.18	0.18
LOEC	0.96	0.39	0.39

 

Conclusions:

The effect of test item on algal growth was assessed using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours. All validity criteria were met during this study. The following endpoints have been determined:

72-hour EC50 Growth rate (r) = 1.6 mg/L (95 % conf. limits 1.42 – 1.70 mg/L) (mean measured concentration)
72-hour EC50 Yield (y) = 0.8 mg/L (95 % conf. limits 0.72 – 0.87 mg/L) (mean measured concentration)
72-hour EC50 Biomass (b) = 0.8 mg/L (95 % conf. limits 0.68 – 0.84 mg/L) (mean measured concentration)
72-hour NOEC Growth rate (r) = 0.39 mg/L (mean measured concentration)
72-hour NOEC Yield (y) = 0.18 mg/L (mean measured concentration)
72-hour NOEC Biomass (b) = 0.18 mg/L (mean measured concentration)

Executive summary:

Acute toxicity of Azuril was assessed with a Growth inhibition test on Algae (Pseudokirchneriella subcapitata) over an exposure period of 72 hours according to OECD Guideline No. 201. The test item was found to degrade over the study duration. The 72-hour EC50 Growth rate (r) was reported to be 1.6 mg/L (95 % conf. limits 1.42 – 1.70 mg/L) nominal loading rate WAF, and the 72-hour NOEC Growth rate was reported to be 0.39 mg/L nominal loading rate WAF.

Description of key information

Acute toxicity of Azuril was assessed with a Growth inhibition test on Algae (Pseudokirchneriella subcapitata) over an exposure period of 72 hours according to OECD Guideline No. 201. The test item was found to degrade over the study duration. The 72-hour EC50 Growth rate (r) was reported to be 1.6 mg/L (95 % conf. limits 1.42 – 1.70 mg/L) nominal loading rate WAF, and the 72-hour NOEC Growth rate was reported to be 0.39 mg/L nominal loading rate WAF.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.6 mg/L

EC10 or NOEC for freshwater algae:

0.39 mg/L

Additional information

The effect of test item on algal growth was assessed using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours. All validity criteria were met during this study. The following endpoints have been determined:

72-hour EC50 Growth rate (r) = 1.6 mg/L (95 % conf. limits 1.42 – 1.70 mg/L) (mean measured concentration)

72-hour EC50 Yield (y) = 0.8 mg/L (95 % conf. limits 0.72 – 0.87 mg/L) (mean measured concentration)

72-hour EC50 Biomass (b) = 0.8 mg/L (95 % conf. limits 0.68 – 0.84 mg/L) (mean measured concentration)

72-hour NOEC Growth rate (r) = 0.39 mg/L (mean measured concentration)

72-hour NOEC Yield (y) = 0.18 mg/L (mean measured concentration)

72-hour NOEC Biomass (b) = 0.18 mg/L (mean measured concentration)