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

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:

2019-05-14 to 2019-05-16

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: Ocimene PQ [Reaction mass of (Z)-3,7-dimethylocta1,3,6,-triene and dipentene]
- 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:

Analytical measurements were performed at the applied test concentration levels at the beginning and end of each renewal period (24 h). Samples from the control were taken for analysis at the start and at the end of the test only. 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:
- 86 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 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 (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 (ISO Medium) two days before the start of the treatments. 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 Daphnia (start of the treatments). During the preparation of WAF solutions and the study the usage of plastic lab wares was omitted.

The same method was used to prepare concentrations of the preliminary range-finding test (0.1, 1, 10 and 100 mg/L), which was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test.

Daphnids in each test concentration and control were exposed for 48 hours 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.

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.
- Number of animals: There were 20 animals in test group and control group respectively, divided into 4 replicates (5 animals / replicate).
- 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 245 mg/L (as CaCO3).

Test temperature:

Target Temperature: Target Temperature: 18–22°C with a maximum deviation of ± 1°C.
Actual Temperature: The water temperature was measured at the start and at the end of the renewal periods in one test vessel at all concentration levels. The test temperature was in the range of 20.0– 20.5°C measured in the test vessels. The additionally measured temperature in the climate chamber was between 19.5 and 20.6°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 one test vessel at all concentration levels and was in the range of 7.69 – 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 one test vessel at all concentration levels at the start and at the end of the renewal periods and was in the range of 6.9 – 8.9 mg/L.

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

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 rate 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:

A concentration range-finding test was 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.

For the definitive test, twenty daphnids, divided into four groups (glass beakers) of five animals each (at least 5 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

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

66.04 mg/L

Nominal / measured:

nominal

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.273 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.315 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Preliminary range-finding test:

No effects (0/10) were observed at 48 hours at 0 mg/L (control), 0.1 mg/L, 1 mg/L and 10 mg/L. All the daphnids were immobilised at 100 mg/L (10/10).

Definitive test:

Validity: There was no immobilization in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L. 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 rate WAFs. Test concentrations were analytically determined at the start and at the end of the renewal periods. However, the test concentrations could only be measured at the start of the renewal periods in most cases and measured concentrations were below the Limit of Quantification (LOQ = 0.21 mg/L) at the end of the renewal periods at all tested levels. Consequently, the geometric mean measured concentrations could not be calculated. The biological results are expressed in the nominal loading rate WAFs (water accommodated fraction) based on OECD TG NO. 23 on poorly soluble and multiconstituent substances.

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), 6.25 mg/L, 12.5 mg/L and 25.0 mg/L. No significant effects were observed at 50.0 mg/L (1/20), while all the daphnids were immobilised at 100 mg/L (20/20). 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 Ocimene PQ were as follows:

The 24h EL50 value: 82.52 mg/L nominal loading rate WAF (95 % conf. limits: 70.40 – 95.37 mg/L nominal loading rate WAF)
The 48h EL50 value: 66.04 mg/L nominal loading rate WAF (95 % conf. limits: 57.70 – 78.19 mg/L nominal loading rate WAF)
The 48h EL100 value: 100.0 mg/L nominal loading rate WAF
The 48h No-Observed Effect Concentration (NOELR): 50.0 mg/L nominal loading rate WAF
The 48h Lowest Observed Effect Concentration (LOELR): 100.0 mg/L nominal loading rate WAF
The 48h EC50 (immobilisation): 0.315 mg/L est.geometric mean
The 48h EC10 (immobilisation): 0.273 mg/L est.geometric mean

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) is: 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. Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Reported statistics and error estimates:

The 24 and 48 hours EL50 values of the test item were calculated using Probit analysis by SPSS PC+ software. The 48 hours EL100 values of the test item were determined directly from the raw data. For the determination of the LOELR and NOELR, the immobilization at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.

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

Results of the Preliminary Range-Finding Test::

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	1	10

Number and percentage of immobilised animals

Test concentration [mg/L nominal lading rate WAF]	Number of treated animals	Immobilised animals
		24 hours		48 hours
		number	percent	number	percent
Control	20	0	0	0	0
6.25	20	0	0	0	0
12.5	20	0	0	0	0
25.0	20	0	0	0	0
50.0	20	0	0	1	5
100.0	20	16*	80	20*	100

Immobilisation of the test animals

Test concentration (mg/L nominal loading rate WAF)	Replicate	Number of treated animals	Number of immobilised animals
			24 h	48 h
Control	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
6.25	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
12.5	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
25.0	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
50.0	1	5	0	0
	2	5	0	1
	3	5	0	0
	4	5	0	0
100.0	1	5	3	5
	2	5	5	5
	3	5	3	5
	4	5	5	5

Estimation of EC50 and EC10 based on geometric mean measured concentrations

		Data from regression statistics
Geometric mean using 1/2 LOD	% inhibition	% inhibition	Log conc	Logit (% inhibiton)	Probit (% inhibiton)
0	0	0	0	-	-
0.257	0.0001	0.000001	-0.595053658	-13.81550956	0.246575691
0.160	0.0001	0.000001	-0.822752458	-13.81550956	0.246575691
0.203	0.0001	0.000001	-0.750609735	-13.81550956	0.246575691
0.335	5	0.05	-0.475173709	-2.944438979	3.355146373
0.324	99.9999	1.00000	-0.498533123	13.81550956	9.753424309

Slope	63.91298438	20.8558565
Intercept	32.78959685	15.46489836
Test values 50% effect	0	5
Log of Er50 concentration	-0.51	-0.50
ErC50 mg/l	0.307	0.315
Test values 10% effect	-2.197224577	3.718448434
Log of Er10 concentration	-0.55	-0.56
ErC10 mg/l	0.284	0.273

Conclusions:

Acute toxicity of Ocimene PQ [Reaction mass of (Z)-3,7dimethylocta-1,3,6,-triene and dipentene] 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 not be analytically determined. Under the conditions of this test, the observed endpoints for the effect of Ocimene PQ were as follows:

The 24h EL50value:       82.52 mg/L nominal loading rate WAF (95 % conf. limits: 70.40 – 95.37 mg/L nominal loading rate WAF)
The 48h EL50value:       66.04 mg/L nominal loading rate WAF (95 % conf. limits: 57.70 – 78.19 mg/L nominal loading rate WAF)
The 48h EL100value:       100.0 mg/L nominal loading rate WAF
The 48h No-Observed Effect Concentration (NOELR): 50.0 mg/L nominal loading rate WAF
The 48h Lowest Observed Effect Concentration (LOELR): 100.0 mg/L nominal loading rate WAF
The 48h EC10 value: 0.273 mg/L Geometric mean measured
The 48h EC50value: 0.315 mg/L Geometric mean measured

Executive summary:

Acute toxicity of Ocimene PQ 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 0.315 mg/L based on the mean measured concentration

Description of key information

Acute toxicity of Ocimene PQ was assessed with Acute immobilisation test onDaphnia 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 0.315 mg/L based on the mean measured concentration

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Remarks:

immobilisation

Effect concentration:

0.315 mg/L