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 fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-05-06 to 2019-05-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity 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-3ene-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 R H % ), under inert gas, protected from humidity (tightly closed container)

Analytical monitoring:

yes

Details on sampling:

The concentration of the test item was measured at the test concentrations at the beginning and at the end of each renewal period, from the control at the beginning and end of experiment. Additional samples in absence of fish were also taken for analysis at each test concentration level in order to distinguish adsorption of the test item to the test system. Duplicate samples were taken (~10 mL) in glass tubes at the applied test concentration levels as well as from the test solutions in absence of fish at the beginning and at the end of each 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:
- 12 tubes of test solutions (~50 mL aliquots, measured with 0.01 g precision) were received from the Test Facility, corresponding to control (0 mg/L) and test concentration (1.28, 3.2, 8, 20 and 50 mg/L) samples at the start and the end of the experiment.
- 39 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 (1.28, 3.2, 8, 20 and 50 mg/L) samples at the start and the end of each renew period with and without the presence of animals.

Vehicle:

no

Details on test solutions:

The test item is poorly soluble in water, so test solutions were prepared 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 (1.28, 3.2, 8, 20 and 50 mg/L nominal loading rates WAFs) were prepared individually by dispersing/dissolving the amount of test item into the test medium (aquarium water) two days before the start of the study. This solution was 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.

The same method was used to prepare concentrations of the two preliminary range-finding tests (0.1, 1, 10 or 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.

The test item is not stable over the 96-hour exposure period, so the tests were performed under semi-static conditions. 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. During the preparation of WAF solutions and the study the usage of plastic lab wares was omitted.

For the untreated control, the dilution water (circulated and filtered) was used without of addition of the test item.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

- Species: Zebrafish (Brachydanio rerio)
- Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary
- Justification of species: Zebrafish (Brachydanio rerio) is one of the convenient species for acute fish toxicity test.
- Body length of animals: 2.0 – 2.6 cm
- Acclimatisation: more than 12 days
- Animal health: Fish were bred in a well-known fish farm, under disease- and parasite-controlled conditions. Fish were observed during the acclimatisation and test periods, the fish were healthy. There was no mortality of the population for seven days before the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Target hardness: The hardness of the dilution water should range between 40 and 250 (preferably <180) mg/L CaCO3.
Actual hardness: The hardness of the dilution water was determined as 155 mg/L (as CaCO3).

Test temperature:

Target temperature: The water temperature should range between 21 and 25 ºC.
Actual temperature: The water temperature was measured at the beginning and at the end of the renewal periods during the experiment in each test aquarium (where the fish were alive). The test temperature was between 20.9 and 22.0 °C.

pH:

Target pH: The pH should range between 6 and 8.5.
Actual pH: The pH was measured at the beginning and at the end of the renewal periods during the experiment in each test aquarium (where the fish were alive). The pH was in the range of 7.82 - 8.41

Dissolved oxygen:

Target dissolved oxygen: The dissolved oxygen concentration should be ≥60% of the air saturation value in all test vessels throughout the exposure.
Actual dissolved oxygen: The dissolved oxygen concentration was measured at the beginning and at the end of the renewal periods during the experiment in each test aquarium (where the fish were alive). The dissolved oxygen concentration was in the range of 79 – 100 % of the air saturation value at the temperature used.

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

The nominal concentrations in the main test were: 1.28, 3.2, 8, 20 and 50 mg/L test item nominal loading rates WAFs. The corresponding measured geometric mean test item concentrations were: 0.86; 2.40; 6.25; 16.68 and 17.74 mg/L.

Details on test conditions:

Two preliminary 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. Four fish were exposed to each concentration of the test item plus a control, for 96 hours under semi-static conditions (with 24-hour renewal in absence of stability data).

The definitive test had a duration of 96 hours. One aquarium was used for each test group and for the control group, respectively. Each aquarium comprised 7 fish and 5 L test solution.The loading of the test aquaria was less than 1.0 g fish/L test solution at the start of the experiment. The light-dark cycle during the test was 16 hours light and 8 hours darkness. The animals were not fed during the test. The frequency of the water renewal periods was 24 hours.

The observations of fish were carried out in the following intervals: at 3, 6, 24, 48, 72 and 96 hours. Mortality or any sub-lethal effects were observed during the experiment. The body weights of the fish were recorded before the treatment. The body weight of 7 fish per aquarium was registered. The loading of the aquaria was calculated on the basis of these body weights and it did not exceed the recommended maximum loading of 1.0 g fish/L.

Reference substance (positive control):

no

Remarks:

Reference controls are not tested in the standard regulatory acute fish tests.

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

3.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.86 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Preliminary range-finding tests:

Preliminary test 1: No effects (0/4) were observed in the control (0 mg/L) and 0.1, 1 and 10 mg/L WAFs, while 100% mortality was observed at 100 mg/L.
Preliminary test 2: No effects (0/4) were observed in the control (0 mg/L) and 1 mg/L WAFs, while at 10 mg/L, 1 of the 4 fish tested showed clinical signs (dark colour, localisation at the bottom of aquarium, decreased activity, unbalanced/vertical/diagonal swimming, lateral position, spasmodic twitching) and the animal was euthanized because of the significant clinical signs.

Definitive test:

Validity: Constant conditions were maintained in this procedure. There was no mortality in the controls and the dissolved oxygen concentration throughout the test was higher than 60%. All validity criteria were within acceptable limits and therefore the study is considered as valid.

Concentrations of the test item: The following nominal loading rates were tested: 1.28, 3.2, 8, 20 and 50 mg/L. Additional samples in absence of fish were also taken for analysis at each test concentration level in order to distinguish adsorption of the test item to the fish/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 fish. The test concentrations were analytically determined at the start and at the end of each renewal periods. The test item was found to degrade over the study duration. The starting concentrations were generally within 20% of the nominal concentrations, except of the 50 mg/L nominal loading rate WAF (Solubility limit = 20 mg/L) and the start of the third renewal period at nominal loading rate of 1.28 mg/L. The corresponding measured geometric mean test item concentrations were: 0.86; 2.40; 6.25; 16.68 and 17.74 mg/L. All the results/conclusions in this report are expressed as the nominal loading rates besides the measured values.

Mortality: No mortalities were recorded in the control and bottom two doses, while all fish had died in concentrations higher than a nominal loading rate of 8 mg/L by 48 hours. All fish were dead or euthanized after 48 hours. At the nominal loading rate of 20 and 50 mg/L all animals were dead at 3 hours of the experiment.

Observations: Behaviour of fish was found as normal in the control group and at the tested nominal loading rates of 1.28 mg/L. Dark colour, localisation at the bottom of aquarium/close to the surface of the water, swimming problems (decreased activity, lateral position, in one place), fast/slow motility of operculum were observed at the nominal loading rate of 3.2 mg/L nominal loading rate WAF during the experiment. Dark colour, localisation at the bottom of aquarium, swimming problems (decreased activity, lateral position, in one place, swimming/lying on back) were observed at the nominal loading rate of 8 mg/L during the first 48 hours of the experiment.

Body weight: The body weight of 7 fish was weighed at the start of the test and ranged from 0.16 to 0.20 g each. There was no considerable difference observed concerning body weights between the groups.

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 LL50 value: 8.4 mg/L (nominal loading rate WAF)
The 48h, 72h and 96h LL50 value: 5.1 mg/L (nominal loading rate WAF) (95 % conf. limits: 3.03 – 7.90 mg/L nominal loading rate WAF)
The 96h LL100 value: 8 mg/L (nominal loading rate WAF)
The 96h No-Observed Effect Loading Rate (NOELR): 1.28 mg/L nominal loading rate WAF
The 96h Lowest Observed Effect Loading Rate (LOELR): 3.2 mg/L nominal loading rate WAF

Calculation based on the measured test item concentrations:
The 24h LC50 value: 6.6 mg/L (measured)
The 48h, 72h and 96h LC50 value: 3.9 mg/L (measured) (95 % conf. limits: 2.00 – 6.76 mg/L (measured))
The 96h LC100 value: 6.25 mg/L (measured)
The 96h No-Observed Effect Concentration (NOEC): 0.86 mg/L (measured)
The 96h Lowest Observed Effect Concentration (LOEC): 2.40 mg/L (measured)

Reported statistics and error estimates:

The 24, 48, 72 and 96 hours LC50/LL50 values were calculated by Probit analysis using SPSS PC+ software. The NOEC/NOELR, LOEC/LOELR and LC100/LL100 were determined directly from the raw data.

Sublethal observations / clinical signs:

Results of the Preliminary Range-Finding Test I

Nominal concentrations [mg/L nominal loading rates WAFs]	Untreated Control	0.1	1	10	100
Number of treated animals	4	4	4	4	4
Number of dead animals	0	0	0	0	4

Results of the Preliminary Range-Finding Test II

Nominal concentrations [mg/L nominal loading rates WAFs]	Untreated Control	1	10
Number of treated animals	4	4	4
Number of dead animals	0	0	1*

Calculation of exposure concentrations with animals

Nominal Loading rate WAFs (mg/L)	Measured concentrations 1st renewal period (mg/L)		Measured concentrations 2nd renewal period (mg/L)		Measured concentrations 3rd renewal period (mg/L)		Measured concentrations 4th renewal period (mg/L)		Geometric mean
	start	end	start	end	start	end	start	end	mg/L
Control	n.d.	-	-	-	-	-	-	n.d.	-
1.28	1.25	0.67	1.06	0.70	0.85	0.58	1.18	0.78	0.86
3.2	2.79	1.94	3.12	2.20	2.91	1.89	2.95	1.80	2.40
8	8.33	4.68	7.74	5.07	-	-	-	-	6.25
20	18.46	15.07	-	-	-	-	-	-	16.68
50	19.2	16.38	-	-	-	-	-	-	17.74

n.d. – not detected

Calculation of exposure concentrations without animals

Nominal Loading rate WAFs (mg/L)	Measured concentrations 1st renewal period (mg/L)		Measured concentrations 2nd renewal period (mg/L)		Measured concentrations 3rd renewal period (mg/L)		Measured concentrations 4th renewal period (mg/L)		Geometric mean
	start	end	start	end	start	end	start	end	mg/L
Control	n.d.	-	-	-	-	-	-	n.d.	-
1.28	1.25	0.71	1.06	0.21	0.85	0.53	1.18	0.69	0.75
3.2	2.79	1.77	3.12	1.09	2.91	1.81	2.95	1.52	2.12
8	8.33	3.94	7.74	2.82	-	-	-	-	5.20
20	18.46	14.12	-	-	-	-	-	-	16.14
50	19.2	18.24	-	-	-	-	-	-	18.72

Cumulative mortality/morbidity data in the Definitive Test

Concentration		Cumulative mortality[number of fish] (initial population = 7 fish)
Nominal loading rates WAFs(mg/L)	Measured (mg/L)	3h	6h	24h	48h	72h	96h
Control	-	0	0	0	0	0	0
1.28	0.86	0	0	0	0	0	0
3.2	2.40	0	0	0	0	0	0
8	6.25	0	0	3	7	7	7
20	16.68	7	7	7	7	7	7
50	17.74	7	7	7	7	7	7

Measured and calculated data of bodyweight

Concentration		Measured weight of 7 fish (g)	Calculated mean weight of 1 fish (g)	Loading of testing aquarium (g fish/Ltesting liquid)
Nominal loading rates WAFs(mg/L)	Measured (mg/L)
Control	-	1.15	0.16	0.23
1.28	0.86	1.37	0.20	0.27
3.2	2.40	1.15	0.16	0.23
8	6.25	1.17	0.17	0.23
20	16.68	1.15	0.16	0.23
50	17.74	1.20	0.17	0.24

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of Azuril [Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile] was assessed with acute fish toxicity test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a semi-static system. All validity criteria were met during this study. Test concentrations could be analytically determined. 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 LL50 value: 8.4 mg/L (nominal loading rate WAF)
The 48h, 72h and 96h LL50 value: 5.1 mg/L (nominal loading rate WAF) (95 % conf. limits: 3.03 – 7.90 mg/L nominal loading rate WAF)
The 96h LL100 value: 8 mg/L (nominal loading rate WAF)
The 96h No-Observed Effect Loading Rate (NOELR): 1.28 mg/L nominal loading rate WAF
The 96h Lowest Observed Effect Loading Rate (LOELR): 3.2 mg/L nominal loading rate WAF

Calculation based on the measured test item concentrations:

The 24h LC50 value: 6.6 mg/L (measured)
The 48h, 72h and 96h LC50 value: 3.9 mg/L (measured) (95 % conf. limits: 2.00 – 6.76 mg/L (measured))
The 96h LC100 value: 6.25 mg/L (measured)
The 96h No-Observed Effect Concentration (NOEC): 0.86 mg/L (measured)
The 96h Lowest Observed Effect Concentration (LOEC): 2.40 mg/L (measured)

Executive summary:

The acute toxicity of Azuril was assessed with acute fish toxicity test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a semi-static system according to OECD TG 203. The 96 h LC50 was reported to be 3.9 mg/L (measured concentration).

Description of key information

The acute toxicity of Azuril was assessed with acute fish toxicity test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a semi-static system according to OECD TG 203. The 96 h LC50 was reported to be 3.9 mg/L (measured concentration).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

3.9 mg/L

Additional information

The acute toxicity of Azuril [Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile] was assessed with acute fish toxicity test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a semi-static system. All validity criteria were met during this study. Test concentrations could be analytically determined. 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 LL50 value: 8.4 mg/L (nominal loading rate WAF)

The 48h, 72h and 96h LL50 value: 5.1 mg/L (nominal loading rate WAF) (95 % conf. limits: 3.03 – 7.90 mg/L nominal loading rate WAF)

The 96h LL100 value: 8 mg/L (nominal loading rate WAF)

The 96h No-Observed Effect Loading Rate (NOELR): 1.28 mg/L nominal loading rate WAF

The 96h Lowest Observed Effect Loading Rate (LOELR): 3.2 mg/L nominal loading rate WAF

Calculation based on the measured test item concentrations:

The 24h LC50 value: 6.6 mg/L (measured)

The 48h, 72h and 96h LC50 value: 3.9 mg/L (measured) (95 % conf. limits: 2.00 – 6.76 mg/L (measured))

The 96h LC100 value: 6.25 mg/L (measured)

The 96h No-Observed Effect Concentration (NOEC): 0.86 mg/L (measured)

The 96h Lowest Observed Effect Concentration (LOEC): 2.40 mg/L (measured)