Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene

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:

2019-05-27 to 2019-05-31

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: Reaction mass of (Z)-3,7-dimethylocta-1,3,6,-triene and dipentene
- Other name/Synonym: Ocimene PQ
- Chemical name: (Z)-3,7-dimethylocta-1,3,6,-triene (CAS 3338-55-4) and dipentene (CAS 138-86-3)
- EC numbers: 222-081, 205-341-0
- CAS numbers: 3338-55-4/138-86-3
- Batch/Lot Number: A170524D
- Description: Colorless Liquid
- Purity: Treated as 100 %
- Expiry date: 06 June 2019
- Storage condition: Room temperature (15-25 °C, ≤70 RH%), under inert gas, protected from humidity (tight closed container)

Analytical monitoring:

yes

Details on sampling:

Based on the analytical method validation (Study Code: 17/180-920AN) samples were taken from each concentration at the beginning and end of the first and last renewal periods. Samples from the control were taken for analysis at the start and at the end of the test only. Additional samples in the absence of fish were taken (from the first and last remaining test solutions) for possible analysis in order to distinguish adsorption of the test item to the fish/test system. Duplicate samples were taken (2 x ~10 mL) in glass tubes from each concentration level as well as from the first and last remaining test solutions (2 x ~10 mL) collected in the absence of fish at the beginning and end of the first and last 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:
- 27 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 (6.25, 12.5, 25, 50 and 100 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:

Because the test item is a multi-constituent substance and poorly soluble in water, 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 (2000).

Saturated test item solutions (nominal loading rates of 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L) were prepared individually by dispersing/dissolving the amount of test item into the test medium (aquarium water) two days before the start of the experiment. 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 the appropriate WAF solutions. The test solutions were prepared just before introduction of the fish (start of the treatments).

During the preparation of WAF solutions and the study the usage of plastic lab wares was omitted. Remaining test solutions from the first renewal period were kept and placed next to test aquariums during the whole experiment.

The same method was used to prepare concentrations of the two preliminary range-finding tests (0.1, 1, 10 or 100 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. In the first preliminary test, plastic lab wares were used during the preparation of WAF solutions, while usage of plastic lab wares was omitted during the second preliminary test.

Based on the analytical method validation (Study Code: 17/180-920AN) the preliminary and definitive 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.

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 – 2.8 cm
- Acclimatisation: more than 12 days
- Observations: 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 149 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 20.5 and 21.9 °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 8.02 – 8.49.

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 72 – 100 % of the air saturation value at the temperature used.

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

The nominal concentrations of test item used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L nominal loading rates WAFs.

Despite renewing the test item every 24 hours the test item could not be detected in the system at the end of each 24 hour period and so the Geometric mean could not be calculated. In order to estimate the geometric mean it was assumed the

Details on test conditions:

Two preliminary concentration range-finding tests were conducted to determine the approximate toxicity of the test item. Four fish were exposed to each concentration of the test item plus a control, for 96 hours under semi-static conditions in both preliminary range-finding tests. The frequency of the water renewal periods was 24 hours. The first preliminary test, for which plastic lab wares were used during the preparation of WAF solutions, had inconclusive regarding the toxicity of the test item. In the second case, usage of plastic lab wares was omitted and mortality was observed in the highest concentration, i.e., 100 mg/L nominal loading rate WAFs.

The definitive test had a duration of 96 hours. One aquarium was used for test groups 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 and any sub-lethal effect were determined in each observation period. Dead fish were removed. The body weights of the fish were recorded before the introduction of fish. The body weight of 7 fish per aquarium was registered. The loading of the aquaria was calculated on the basis of these body weights (1.0 g fish/L testing liquid).

Reference substance (positive control):

no

Remarks:

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

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

35.01 mg/L

Nominal / measured:

nominal

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.785 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC10

Effect conc.:

0.566 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 any of the treatments.
Preliminary test 2: 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.

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 concentrations of test item used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L nominal loading rates WAFs. The test concentrations were analytically determined at the start and at the end of the first and at the last renewal periods. Concentration of the two highest examined test groups of 50.0 and 100.0 mg/L nominal loading rates WAFs were only analysed at the start of the experiment and 24 hours later, as all animals were observed as dead at either 3 or 24 hours after the start of the test. The test concentrations could be measured at the start of the renewal periods but the measured concentrations were mostly below the Limit of Quantification (LOQ = 0.21 mg/L) at the end of the renewal periods at all test levels measured (except at 100.0 mg/L WAF at the end of the first renewal period and at 25.0 mg/L WAF at the end of the last renewal period). Consequently, the geometric mean measured concentrations could not be calculated. The biological results are expressed in the nominal loading rates WAFs (water accommodated fraction) based on OECD TG NO. 23 on poorly soluble and multiconstituent substances.

Mortality: No mortalities were recorded in the control and 6.25, 12.5 and 25.0 mg/L concentrations All fish had died in the 100 and 50 mg/L concentrations after 3 and 24 hours, respectively.

Observations: Behaviour of fish was found as normal in the control group and at the lowest tested concentration level of 6.25 mg/L nominal loading rate WAF. Localization close to the surface of the water or at the bottom of aquarium and decreased activity was observed at the nominal concentration of 12.5 mg/L nominal loading rate WAF during the first 24 hours after the start of the test. At the test group of 25.0 mg/L WAF dark colour, decreased activity, localization at the bottom of aquarium or close to the surface of the water, swimming problems (unbalanced or vertical/diagonal swimming), immobility and fast motility of operculum was observed during the experiment, while dark colour, decreased activity, localization at close to the surface of the water, unbalanced swimming, fast motility of operculum and gulping was noted during the first 6 hours of the test in the test group of 50.0 mg/L WAF. In the two highest test level of 50.0 and 100.0 mg/L nominal loading rates WAFs all fish were dead 24 hours after the start of the experiment.

Body weight: The body weight of 7 fish was weighed at the start of the test and ranged from 0.21 to 0.26 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 Ocimene PQ were as follows:

The 24, 48, 72 and 96h LL50 value: 35.01 mg/L nominal loading rate WAF (95 % conf. limits: 25.25 – 52.05 mg/L).
The 96h LL100 value: 50.0 mg/L nominal loading rate WAF.
The 96h No-Observed Effect Concentration (NOELR): 12.50 mg/L nominal loading rate WAF.
The 96h Lowest Observed Effect Concentration (LOELR): 25.0 mg/L nominal loading rate WAF.
The 96h LC50 value: 0.785 mg/L geomean measured
The 96h LC10 value: 0.566 mg/L geomean measured

Reported statistics and error estimates:

The 24, 48, 72 and 96 hours LL50 values were calculated by Probit analysis using SPSS PC+ software (based on the measured geometric mean concentrations). The NOELR, LOELR and LL100 were determined directly from the raw data.

The 48 hour EC50 and EC10 based on the geometric mean were estimated using Probit and Logit analysis using Excel.

Sublethal observations / clinical signs:

Results of the first Preliminary Range-Finding Test

Nominal concentrations [mg/L nominal loading rate WAF]	Untreated control	0.1	1	10	100
Number of treated animals	4	4	4	4	4
Number of dead animals	0	0	0	0	0

Results of the second Preliminary Range-Finding Test

Nominal concentrations [mg/L nominal loading rate WAF]	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

Cumulative mortality data in the Definitive Test

Test concentration [mg/L nominal lading rate WAF]	Cumulative mortality[number of fish] (initial population = 7 fish)
	3h	6h	24h	48h	72h	96h
Control	0	0	0	0	0	0
6.25	0	0	0	0	0	0
12.5	0	0	0	0	0	0
25.0	0	0	0	0	0	0
50.0	0	0	7	-	-	-
100.0	7	-	-	-	-	-

Measured and calculated data of bodyweight

Test concentration [mg/L nominal lading rate WAF]	Measured weight of 7 fish (g)	Calculated mean weight of 1 fish (g)	Loading of testing aquarium (g fish/Ltesting liquid)
Control	1.80	0.26	0.36
6.25	1.74	0.25	0.35
12.5	1.84	0.26	0.37
25.0	1.65	0.24	0.33
50.0	1.63	0.23	0.33
100.0	1.50	0.21	0.30

Calculation of EC10 and EC50

		Data from regression statistics
Geometric mean using 1/2 LOD	% inhibition	% inhibition	Log conc	Logit (% inhibiton)	Probit (% inhibiton)
0	0	0	0	-	-
0.290	0.0001	0.000001	-0.537731841	-13.81550956	0.246575691
0.420	0.0001	0.000001	-0.37632424	-13.81550956	0.246575691
0.456	0.0001	0.000001	-0.341011125	-13.81550956	0.246575691
0.475	99.999	0.99999	-0.323085145	11.51291546	9.264890794
2.618	99.999	0.99999	0.418013106	11.51291546	9.264890794
			Slope	25.41870046	9.050458135
			Intercept	2.213706845	5.953860066
			Test values 50% effect	0	5
			Log of Er50 concentration	-0.09	-0.11
			ErC50 mg/l	0.818	0.785
			Test values 10% effect	-2.197224577	3.718448434
			Log of Er10 concentration	-0.17	-0.25
			ErC10 mg/l	0.671	0.566

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of Ocimene PQ [Reaction mass of (Z)-3,7-dimethylocta-1,3,6,- triene and dipentene] 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. Under the conditions of this test, the observed endpoints for the effect of Ocimene PQ were as follows:

The 24, 48, 72 and 96h LL50value:    35.01 mg/L nominal loading rate WAF (95 % conf. limits: 25.25 – 52.05 mg/L)
The 96h LL100 value: 50.0 mg/L nominal loading rate WAF
The 96h No-Observed Effect Concentration (NOELR): 12.50 mg/L nominal loading rate WAF
The 96h Lowest Observed Effect Concentration (LOELR): 25.0 mg/L nominal loading rate WAF
The 96h LC50 value: 0.785 mg/L Geomean measured
The 96h LC10 value: 0.566 mg/L Geomean measured

Executive summary:

The acute toxicity of Ocimene PQ 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 0.785 mg/L (Geomean measured concentration).

Description of key information

The acute toxicity of Ocimene PQ 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 0.785 mg/L (Geomean measured concentration).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

0.785 mg/L