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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-04-23 to 2019-04-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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-3ene-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)
- 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 measurements were performed at the applied test concentration levels at the beginning and end of each renewal period, from the control at the beginning and end of experiment. Additional samples in absence of daphnia were also taken for analysis at each test concentration level in order to distinguish adsorption of the test item to the daphnia/test system test.

Duplicate samples were taken (~10 mL) in glass tubes at the applied test concentration levels (three replicates) from the stock solution at the beginning of the renewal periods and from the test solutions in absence/presence of daphnia at the end of the renewal periods. 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 on the first set).

Total samples:
- 36 tubes of test solutions (~15 mL aliquots, measured with 0.01 g precision) were received from the Test Facility, corresponding to control (0 mg/L) and test concentration (0.625, 1.25, 2.5, 5 and 10 mg/L) samples at the start and the end of the experiment.
- 90 tubes of test solutions (~10 mL aliquots, measured with 0.01 g precision) were received from the Test Facility, corresponding to control (0 mg/L) and test concentration (0.625, 1.25, 2.5, 5 and 10 mg/L) samples at the start and the end of each renew period.

Vehicle:

no

Details on test solutions:

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 (0.625, 1.25, 2.5, 5 and 10 mg/L nominal loading rate WAFs) were prepared individually by dispersing/dissolving the amount of test item into the test medium (ISO-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 same method was used to prepare concentrations of the two preliminary range-finding tests (0.1, 1, 10 and 100 mg/L; 1 and 10 mg/L), which 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.

All the tests were performed under semi-static conditions because the test item is not stable over the 48-hour exposure period. The frequency of the water renewal periods was 24 hours. Prior to treatment of each renewal period, test item solutions were prepared by the method described above. The test solutions were prepared just before introduction of the Daphnia (start of the treatments). During the preparation of WAF solutions and the study the usage of plastic lab wares was omitted.

Reconstituted (ISO-medium, according to OECD No. 202) was used as dilution water for both the range finding and definitive tests. For the untreated control, diluted water (ISO-medium) was used without addition of the test item and was examined in parallel to the test item concentrations.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Species and strain: Daphnia magna
- Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary
- Breeding: The Daphnia are bred in Ecotoxicological Laboratory of Citoxlab Hungary Ltd. The health of the stock animals is continuously monitored by visual daily checking. Abnormal behaviour or significant decrease of population is recorded.
- Justification of strain: Daphnia magna is the standard species of the acute immobilisation test.
- Age of the animals: Animals were less than 24 hours old at the beginning of the test. They were not the first breeding of the previous generation.
- Acclimatization: There was no acclimatization because the water used was similar to the culture water.
- Holding water: Same water is used as test water.
- Food and Feeding: The Daphnia are fed with concentrated algal suspension of Pseudokirchneriella subcapitata at least three times per week during the holding.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

Target hardness: The reconstituted water (ISO medium) has an approximate theoretical total hardness of 249 mg/L (as CaCO3).
Actual hardness: The reconstituted water (ISO medium) had a total hardness of 247 mg/L (as CaCO3).

Test temperature:

Target Temperature: 18–22°C with a maximum deviation of ± 1°C
Actual Temperature: The test temperature was in the range of 20.2 – 20.8°C measured in the test vessels. The additionally measured temperature in the climate chamber was between 19.8 – 21.0°C.

pH:

Target pH: 6-9 with a maximum deviation of ± 1.5 units
Actual pH: The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was measured at the start and at the end of the renewal periods in all test vessels and was in the range of 7.63 – 7.87.

Dissolved oxygen:

Target dissolved oxygen: The dissolved oxygen concentration at the end of the test in control and test vessels should be ≥ 3 mg/L of the air saturation value at the temperature used.
Actual dissolved oxygen: The dissolved oxygen concentration was measured in all test vessels at the start and at the end of the renewal periods and was in the range of 6.6 – 8.6 mg/L.

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

The nominal concentrations in the main test were: 0.625, 1.25, 2.5, 5 and 10 mg/L test item nominal loading rates WAFs. The corresponding measured geometric mean test item concentrations were: 0.33; 0.66; 1.27; 3.11 and 6.27 mg/L.

Details on test conditions:

Two 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. Ten daphnids (divided into two replicates) in each test concentration and control were exposed for 48 hours under semi-static conditions (in absence of stability data).

For the definitive test, twenty daphnids, divided into four groups (glass beakers) of five animals each (~100 mL test solution/flask; 20 mL test solution/animal), were used at the test concentrations and for the control group as well for 48 hours under semi-static conditions.

The light-dark cycle during the test was 16 hours light (artificial illumination) and 8 hours darkness. Daphnids were not be fed during the test.

The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile. The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Preliminary range-finding test:

In the first preliminary study, no effects (0/10) were observed at 48 hours at 0 mg/L (control), 0.1 mg/L and 1 mg/L, with some effects at 10 mg/L (3/10) and 100 mg/L (10/10). In the second preliminary test: no effects (0/10) were observed at 48 hours at 0 mg/L (control) and 1 mg/L, while effects were seen at 10 mg/L (10/10).

Definitive test:

Validity: There were no immobilized animals in the control group and the dissolved oxygen concentration at the end of the test in the control and test vessels was more than 3 mg/L.All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Concentrations of the test item: The following nominal loading rates were tested: 0.625, 1.25, 2.5, 5 and 10 mg/L. Test concentrations were analytically determined at the start and at the end of the renewal periods. Additional samples in absence of daphnia were also taken for analysis at each test concentration level in order to distinguish adsorption of the test item to the daphnia/test system. As no significant differences were detected in the absence and presence of the animals, the corresponding measured geometric mean test item concentrations were calculated with the data of the samples in presence of daphnids.The test item was found to degrade over the study duration. The starting concentrations were generally within 20% of the nominal concentrations. The corresponding measured geometric mean test item concentrations were: 0.33, 0.66, 1.27, 3.11 and 6.27 mg/L. All the results/conclusions in this report are expressed as the nominal loading rate WAFs besides the measured values.

Immobilisation: The number of immobilised animals and the percentage of immobility were determined at the 24th and 48th hour. No effects (0/20) were observed at 48 hours at 0 mg/L (control), 0.625 mg/L (0.33 mg/L) and 1.25 mg/L (0.66 mg/L). No statistically significant effects (6/20) were observed at 2.5 mg/L (1.27 mg/L), while all the daphnids were immobilised (20/20) at 5 mg/L (3.11 mg/L) and 10 mg/L (6.27 mg/L). Based on the request of the Sponsor additional statistical calculation was made with data of the 48 hours by SPSS/PC+ validated statistical software with Duncan’s Multiple Range Test. With these new calculations, results at 2.5 mg/L (1.27 mg/L) were considered statistically significantly dfifferent compared to the control values. In addition to immobility, no abnormal behaviour or appearance of test animals was detected.

Under the conditions of this test, the observed endpoints for the effect of Azuril were as follows:

Calculation based on the initial nominal loading rates (WAFs):
The 24h EL50 value: 5.1 mg/L (nominal loading rate WAF) (95 % conf. limits: 4.25 – 6.07 mg/L nominal loading rate WAF)
The 48h EL50 value: 2.8 mg/L (nominal loading rate WAF) (95 % conf. limits: 2.46 – 3.47 mg/L nominal loading rate WAF)
The 48h EL100 value: 5 mg/L (nominal loading rate WAF)
The 48h No-Observed Effect Loading Rate (NOELR):
1.25 mg/L (nominal loading rate WAF)
The 48h Lowest Observed Effect Loading Rate (LOELR):
2.5 mg/L (nominal loading rate WAF)

Calculation based on the measured test item concentrations:
The 24h EC50 value: 3.1 mg/L (measured) (95 % conf. limits: 2.46 – 3.74 mg/L nominal loading rate WAF)
The 48h EC50 value: 1.5 mg/L (measured) (95 % conf. limits: 1.26 – 2.12 mg/L nominal loading rate WAF)
The 48h EC100 value: 2.7 mg/L (measured) (95 % conf. limits: 1.94 – 14.26 mg/L nominal loading rate WAF)
The 48h No-Observed Effect Concentration (NOEC): 1.27 mg/L (measured)
The 48h Lowest Observed Effect Concentration (LOEC): 3.11 mg/L (measured)

Results with reference substance (positive control):

The experimental period of the last positive control study (Study Code: 19/009-023DA) with reference item Potassium dichromate (Batch no.: A0345704) was 23 - 24 January 2019. The observed 24h EC50 value of the reference item was 0.71 mg/L, (95 % confidence limits: 0.66 – 0.75 mg/L) in that study.

Reported statistics and error estimates:

The 24 and 48 hours EC50/EL50 values of the test item were calculated using Probit analysis by TOXSTAT software. The 48 hours EC100/EL100 values of the test item were determined directly from the raw data. For the determination of the LOEC/LOELR and NOEC/NOELR, the immobilization at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software. Based on the request of the Sponsor additional statistical calculation was made with data of the 48 hours by SPSS/PC+ validated statistical software with Duncan’s Multiple Range Test.

Results of the PreliminaryRange-Finding Test I

Nominal concentrations [mg/L nominal loading rates WAFs]	Untreated Control	0.1	1	10	100
Number of treated animals	10	10	10	10	10
Number of immobilised animals	0	0	0	3	10

Results of the PreliminaryRange-Finding Test II

Nominal concentrations [mg/L nominal loading rates WAFs]	Untreated Control	1	10
Number of treated animals	10	10	10
Number of immobilised animals	0	0	10

Calculation of exposure concentrations with animals

Loading rate WAFs (mg/L)	Mean measured concentrations(mg/L)				Geometric mean
	First renewal period		Second renewal period
	0h/start	24h	0h/start	24h	mg/L
Control	n.d.	-	-	n.d.	---
0.625	0.650	0.312	0.571	0.080	0.33
1.25	1.241	0.554	1.092	0.212	0.66
2.5	2.128	0.738	1.905	0.882	1.27
5	5.129	1.766	4.023	2.552	3.11
10	10.394	3.777	-	-	6.27

Calculation of exposure concentrations without animals

Loading rate WAFs (mg/L)	Mean measured concentrations(mg/L)				Geometric mean
	First renewal period		Second renewal period
	0h/start	24h	0h/start	24h	mg/L
Control	n.d.	-	-	n.d.	---
0.625	0.650	0.295	0.571	0.399	0.46
1.25	1.241	1.016	1.092	0.855	1.04
2.5	2.128	0.733	1.905	0.917	1.29
5	5.129	3.852	4.023	1.667	3.52
10	10.394	4.471	-	-	6.82

Number, percentage of immobilised animals and statistical results with TOXSTAT

Nominal Loading rate WAF	Measured	Number of treated animals	Immobilised animals
			24 hours		48 hours
mg/L	mg/L		number	percentage (%)	number	percentage (%)
Control	-	20	0	0	0	0
0.625	0.33	20	0	0	0	0
1.25	0.66	20	0	0	0	0
2.5	1.27	20	1	5	6	30
5	3.11	20	8*	40	20*	100
10	6.27	20	20*	100	20*	100

*: statistically significantly different compared to the control values (Dunnett’s Test;a= 0.05)

Number, percentage of immobilised animals and statistical results with SPSS

Nominal Loading rate WAF	Measured	Number of treated animals	Immobilised animals
			48 hours
mg/L	mg/L		number	percentage (%)
Control	-	20	0	0
0.625	0.33	20	0	0
1.25	0.66	20	0	0
2.5	1.27	20	6*	30
5	3.11	20	20**	100
10	6.27	20	20**	100

*/**: statistically significantly different compared to the control values (Duncan’s Multiple Range Test;a= 0.05), *: p< 0.05, **: p<0.01

Conclusions:

Acute toxicity of 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] was assessed with Acute immobilisation test on Daphnia magna, over an exposure period of 48 hours in a semi-static system. All validity criteria were met during this study. Test concentrations could be analytically quantified. Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of Azuril were the following:

Calculation based on the initial nominal loading rates (WAFs):

The 24h EL50 value: 5.1 mg/L (nominal loading rate WAF) (95 % conf. limits: 4.25 – 6.07 mg/L nominal loading rate WAF)
The 48h EL50 value: 2.8 mg/L (nominal loading rate WAF) (95 % conf. limits: 2.46 – 3.47 mg/L nominal loading rate WAF)
The 48h EL100 value: 5 mg/L (nominal loading rate WAF)
The 48h No-Observed Effect Loading Rate (NOELR): 1.25 mg/L (nominal loading rate WAF)
The 48h Lowest Observed Effect Loading Rate (LOELR): 2.5 mg/L (nominal loading rate WAF)

Calculation based on the measured test item concentrations:
The 24h EC50 value: 3.1 mg/L (measured) (95 % conf. limits: 2.46 – 3.74 mg/L nominal loading rate WAF)
The 48h EC50 value: 1.5 mg/L (measured) (95 % conf. limits: 1.26 – 2.12 mg/L nominal loading rate WAF)
The 48h EC100 value: 2.7 mg/L (measured) (95 % conf. limits: 1.94 – 14.26 mg/L nominal loading rate WAF)
The 48h No-Observed Effect Concentration (NOEC): 1.27 mg/L (measured)
The 48h Lowest Observed Effect Concentration (LOEC): 3.11 mg/L (measured)

Executive summary:

Acute toxicity of Azuril was assessed with Acute immobilisation test on Daphnia magna, over an exposure period of 48 hours in a semi-static system according to OECD TG 202. The 48 h EC50 (immobilisation) was reported to be 1.5 mg/L.

Description of key information

Acute toxicity of Azuril was assessed with Acute immobilisation test on Daphnia magna, over an exposure period of 48 hours in a semi-static system according to OECD TG 202. The 48 h EC50 (immobilisation) was reported to be 1.5 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.5 mg/L

Additional information

Acute toxicity of 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] was assessed with Acute immobilisation test on Daphnia magna, over an exposure period of 48 hours in a semi-static system. All validity criteria were met during this study. Test concentrations could be analytically quantified. Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of Azuril were the following:

Calculation based on the initial nominal loading rates (WAFs):

The 24h EL50 value: 5.1 mg/L (nominal loading rate WAF) (95 % conf. limits: 4.25 – 6.07 mg/L nominal loading rate WAF)

The 48h EL50 value: 2.8 mg/L (nominal loading rate WAF) (95 % conf. limits: 2.46 – 3.47 mg/L nominal loading rate WAF)

The 48h EL100 value: 5 mg/L (nominal loading rate WAF)

The 48h No-Observed Effect Loading Rate (NOELR): 1.25 mg/L (nominal loading rate WAF)

The 48h Lowest Observed Effect Loading Rate (LOELR): 2.5 mg/L (nominal loading rate WAF)

Calculation based on the measured test item concentrations:

The 24h EC50 value: 3.1 mg/L (measured) (95 % conf. limits: 2.46 – 3.74 mg/L nominal loading rate WAF)

The 48h EC50 value: 1.5 mg/L (measured) (95 % conf. limits: 1.26 – 2.12 mg/L nominal loading rate WAF)

The 48h EC100 value: 2.7 mg/L (measured) (95 % conf. limits: 1.94 – 14.26 mg/L nominal loading rate WAF)

The 48h No-Observed Effect Concentration (NOEC): 1.27 mg/L (measured)

The 48h Lowest Observed Effect Concentration (LOEC): 3.11 mg/L (measured)