Reaction mass of (4R)-isopropenyl-1-methylcyclohexene and (4S)-isopropenyl-1-methylcyclohexene and 1-isopropyl-4-methylcyclohexa-1,3-diene and 1-isopropyl-4-methylcyclohexa-1,4-diene and 4-isopropylidene-1-methylcyclohexene

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:

19-26 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline No. 202. All validity criteria were fulfilled

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

11 January 2013

Analytical monitoring:

yes

Details on sampling:

Chemical analyses: Single samples for analysis were taken from the control and all test concentrations from replicates without daphnids (except at the end of the test: replicates with daphnids) at the start of the test (t=0 h), at t=24 h (new and old solutions) and at the end of the test (t=48 h) in all vessels.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The study was carried out using WAFs (Water Accommodated Fractions). The WAFs (for the fresh mediums at t=0 h and t=24 h) were prepared in the dark under closed conditions and by slow stirring to avoid production of a dispersion.
The mixing vessel was a graduated 5 L cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the WAFs. A magnetic stirring bar was placed in each test vessel and test water (2.2.) was added. The loading rates of the test item were weighed on glass slides that afterwards were placed under the surface of the test water contained in the mixing vessels through fishing wire. The mixing vessels were thereafter carefully filled with the remaining volume of test water to obtain a final volume of 5.5 L in order to use a maximum volume and to minimize headspace. Then the mixing vessels were closed immediately. The mixing was carried out at a speed that was slow enough not to cause dispersion or emulsification of the undissolved fraction of the test item. Mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. The stirring speed was as low as possible to maintain mixing of the water phase. After 24 ± 2 h of gentle stirring in the dark, the WAFs were allowed to stand for 1 h before use and were taken out of the drain port. The first 100 mL were discarded. Then the WAFs were directly added into test tubes (without headspace) that were immediately sealed with screwcaps after introduction of daphnids. No small bubble was observed in the test tubes. The test solution was observed to be clear and colourless and at all concentrations.

- Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna (Straus), clone 5
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at study initiation: < 24 h
- Breeding Conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass beakers containing purified drinking water (Composition (mg/L): Calcium 11.5 – Magnesium 8.0 – Sodium 11.6 – Potassium 6.2 – Silica 31.7 – Bicarbonates 71.0 – Sulfates 8.1 – Chlorides 13.5 – Nitrates 6.3; pH = 7). During the week the stock daphnids were fed daily with a suspension of freshwater algae (mix of 3 algae strains: Chlorella vulgaris = 2.5x10^6 cells/mL/day/daphnid, Desmodesmus subspicatus = 2.5x10^6 cells/mL/day/daphnid and Pseudokirchneriella subcapitata = 5x10^6 cells/mL/day/daphnid). The water was changed once per week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Feeding during test: No feeding

ACCLIMATION
- At least 48 h prior to the start of the test, gravid daphnids were transferred to OECD test water and held at similar temperature and light conditions as used in the test. During this period, daphnids were fed in the same manner as that of the stock population. Only daphnids up to 24 h old were used for the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

Total water hardness was approximately 250 mg/L (as CaCO3)

Test temperature:

19.9-20.7 °C throughout the test (average value: 20.4 °C)

pH:

7.64-8.08
pH: 6.0-9.0, not varying by more than 1.5 units

Dissolved oxygen:

8.06-8.70 mg O2/L
oxygen: ≥ 60% of the air-saturation value at the end of the test

Salinity:

None

Nominal and measured concentrations:

Nominal WAFs concentrations: 1.0, 1.4, 2.1, 2.9, 4.2 and 6.0 mg/L (loading rate)
Summed measured concentrations: 0.866, 1.212, 1.820, 2.512, 3.637 and 5.196 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass test tubes of approximately 20 mL capacity sealed with screw caps.
- Aeration: No aeration of the test solutions occurred throughout the test.
- Renewal rate of test solution: A semi-static test was performed with renewal of test solutions after 24 h, in order to maintain test concentrations within 80-120 % of nominal values.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Loading: 5 daphnids per vessel each completely filled with test solution and without headspace.
- Introduction of Daphnids: Daphnids were introduced into the test medium immediately after filling the test tubes with test solutions.
- 4 replicates with daphnids for the control and per WAFs concentration. Moreover, 3 abiotic replicates for the control and per WAFs concentration were prepared:
- two for sampling for analysis of test concentrations from the freshly prepared solutions (t=0 h and t=24 h fresh)
- and another for sampling from the 24 h old solutions (t=24 h old).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water as prescribed by the OECD Guideline 202
- Conductivity: < 10 μS/cm

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED:
- Immobility: Immobility and abnormal behaviour were determined by visual observation after 24 and 48 h. Immobile animals were eliminated from the vessels as soon as they were discovered. The daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of the test vessels.
- pH and dissolved O2: At the start of the test (t=0 h), at t=24 h (new and old solutions) and the end of the test (t=48 h) in all vessels.
- Temperature of Medium: Measured continuously in a vessel next to the test vessels, over the entire study period, beginning at the start of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.43
- Range finding study: Daphnids were exposed to the nominal test concentrations of 0.5, 1, 5, 10 and 15 mg/L and to a control.
- Results used to determine the conditions for the definitive study: The per cent immobility was 0, 0, 0, 30, 100 and 100 (24 h) and 0, 0, 0, 100, 100 and 100 (48 h) at 0, 0.5, 1, 5, 10 and 15 mg/L, respectively. Based on the results of a range-finding test, the following nominal concentrations (spaced by a factor of approximately 1.43): 1.0, 1.4, 2.1, 2.9, 4.2 and 6.0 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

2.217 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 1.353 - 3.506 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.758 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 1.604 - 1.914 mg/L

Details on results:

After 24 h of exposure, immobilisations were 0 % at 1.0 and 1.4 mg/L (loading), 65 % at 2.1 mg/L (loading), 85 % at 2.9 and 4.2 mg/L (loading) and 100 % at 6.0 mg/L (loading).
After 48 h of exposure, immobilisations were 0 % at 1.0 mg/L (loading), 5 % at 1.4 mg/L (loading), 90 % at 2.1 mg/L (loading) and 100 % at 2.9, 4.2 and 6.0 mg/L (loading).

Results with reference substance (positive control):

On July 24, 2013 (most recent test), the 24 h-EC50 was 0.98 mg/L. Hence, the sensitivity of the clone of Daphnia magna was in agreement with the OECD 202 (expected 24 h-EC50: 0.6 to 2.1 mg/L) at this time.

Reported statistics and error estimates:

The evaluation of the effects was based on the nominal WAFs concentrations (nominal loading values). The software ToxRat® Professional was used for the determination of the effective concentrations.

Analytical results

Samples taken from test concentrations were analysed at the start, at t=24 h (new and old solutions) and at the end of the test in order to determine maintenance of actual concentrations of different constituents of the test item.

Overall, the analytical results of this test showed that WAFs were enough stable during the first day (within the ± 20 % of the initial concentration). Although every effort was made to maintain the concentrations of the WAFs (semi-static and closed conditions without headspace), some constituents were lost over the second day, with overall loss of the test item within the 20-30 % of the initial concentration.

It should be noted that chemical analyses were performed only to provide an indication of the stability of the WAFs. A WAF is by definition a complex mixture for which the individual concentration of each constituent differs due to its properties (e.g. solubility, adsorption, volatilisation, bioaccumulation and metabolism). Due to these differences, the ratio of certain constituent may change over time and the composition of the WAFs after 24 h will not be exactly that of the fresh solutions. For information, the results of the nominal WAFs and the summed measured concentrations of the constituents are presented in the table here below. Based on these results some difference between the nominal and measured initial WAF values was found as would be expected. However the differences are not large demonstrating that the WAF was contained efficiently throughout the experiment. Thus, since the study was carried out using WAFs made of several constituents with different stability and behaviours in aqueous solutions during testing, it was considered acceptable to express results based on nominal loading values.

 

Table 6.1.3/1: Acute immobilisation of daphnids after 24 and 48 h in the final test

 

Nominal concentration* (mg/L)	Replicate	Number of daphnids exposed	Response at 24 h		Response at 48 h
			Number	Total %	Number	Total %
Control	1	5	0	0	0	0
	2	5	0		0
	3	5	0		0
	4	5	0		0
1.0	1	5	0	0	0	0
	2	5	0		0
	3	5	0		0
	4	5	0		0
1.4	1	5	0	0	0	5
	2	5	0		1
	3	5	0		0
	4	5	0		0
2.1	1	5	2	65	5	90
	2	5	4		5
	3	5	3		4
	4	5	4		4
2.9	1	5	3	85	5	100
	2	5	5		5
	3	5	4		5
	4	5	5		5
4.2	1	5	4	85	5	100
	2	5	4		5
	3	5	5		5
	4	5	4		5
6.0	1	5	5	100	5	100
	2	5	5		5
	3	5	5		5
	4	5	5		5

* WAF prepared at the given loading rate

 

Water quality parameter values throughout the test:

Test conditions remained within the limits prescribed by the study plan (pH: 6.0-9.0, not varying by more than 1.5 units;oxygen: ≥ 60 % of the air-saturation value at the end of the test), suggesting that pH and oxygen concentrations were not influenced by the test item.

Furthermore, the temperature of the test medium was situated between 19.9 and 20.7 °C throughout the test (average value: 20.4 °C), and complied with the requirements as laid down in the study plan (20 ± 2 °C, constant within 1 °C).

 

Validity criteria of the study:

Controls: In the control, no daphnids became immobilized nor trapped at the surface of the water or showed signs of stress.

Dissolved [O2]: Dissolved oxygen concentration at the end of the test was ≥ 60 % of the air-saturation value in controls and test vessels.

Thus the validity criteria have been fulfilled in the present study.

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal loading rates of the test item and under the experimental conditions, the 48 h EC50 value estimated was 1.76 mg/L (loading).
Therefore, the substance is classified in category 2 (toxic to aquatic life with long-lasting effects) according to CLP regulation (EC) No. 1272/2008.
The substance is classified for acute toxicity in category 2 (toxic to aquatic life) and for chronic toxicity in category 2 (toxic to aquatic life with long-lasting effects) according to GHS regulation.

Executive summary:

A study was performed to assess the acute toxicity of the test item, REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE, to freshwater invertebrate Daphnia magna. It was investigated in a semi-static test using Water Accommodated Fractions according to OECD Guideline 202 with GLP compliance. 

 

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommdated Fractions (WAFs) of the test item over a range of nominal loading rate of 1.0, 1.4, 2.1, 2.9, 4.2 and 6.0 mg/L. The immobility of the daphnids was determined in a semi-static 48 h test by visual observation after 24 h and 48 h. Samples taken from test concentrations were analysed at the start, at t=24 h (new and old solutions) and at the end of the test (t=48 h) in order to determine if the concentrations of different constituents of the test item are maintained. These quantifications were performed only to provide an indication of the stability of the WAFs.

 

The overall loss of the substance displayed by the quantification of different key elements of the test item was within the 20% of the initial concentration on the first day. The second day, the overall loss of the test item was not greater than 30% of the initial concentration. A WAF is by definition a complex mixture for which the individual concentration of each constituent differs due to its properties. Due to these differences, the ratio of certain constituents may change over time and the composition of the WAFs of the 24h old solutions will not be exactly that of the fresh solutions. Thus, since the study was carried out using WAFs made up of several constituents with different stabilities during testing, it was considered acceptable to base the results on nominal loading rate.

 

After 24 h of exposure, immobilisations were 0% at 1.0 and 1.4 mg/L (loading), 65% at 2.1 mg/L (loading), 85% at 2.9 and 4.2 mg/L (loading) and 100% at 6.0 mg/L (loading). After 48 h of exposure, immobilisations were 0% at 1.0 mg/L (loading), 5% at 1.4 mg/L (loading), 90% at 2.1 mg/L (loading) and 100% at 2.9, 4.2 and 6.0 mg/L (loading).

 

Therefore, based on the nominal loading rates of the test item, the 48 h EC₅₀ value estimated was 1.76 mg/L (loading - 95% Cl 1.604-1.914 mg/L) and the 24 h EC₅₀ value estimated was 2.22 mg/L (loading - 95% Cl 1.353-3.506 mg/L).

Therefore, the substance is classified in category 2 (toxic to aquatic life with long-lasting effects) according to CLP regulation (EC) No. 1272/2008.

The substance is classified for acute toxicity in category 2 (toxic to aquatic life) and for chronic toxicity in category 2 (toxic to aquatic life with long-lasting effects) according to GHS regulation.

Description of key information

Based on the nominal loading rates of the test item, the 48 h EC50 of REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA-TERPINENES, TERPINOLENE to freshwater invertebrate Daphnia magna was estimated to be 1.76 mg/L (loading).

Therefore, the substance is classified in category 2 (toxic to aquatic life with long-lasting effects) according to CLP regulation (EC) No. 1272/2008.

The substance is classified for acute toxicity in category 2 (toxic to aquatic life) and for chronic toxicity in category 2 (toxic to aquatic life with long-lasting effects) according to GHS regulation.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.76 mg/L

Additional information

A reliable study was available to assess the acute toxicity of test item REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA-TERPINENES, TERPINOLENE to freshwater invertebrate Daphnia magna. It was investigated in a semi-static test during 48 hours, using Water Accommodated Fractions according to OECD Guideline 202 with GLP compliance. 

Since the study was carried out using WAFs made up of several constituents with different stabilities during testing, it was considered acceptable to base the results on nominal loading rate.

After 24 h of exposure, immobilisations were 0% at 1.0 and 1.4 mg/L (loading), 65% at 2.1 mg/L (loading), 85% at 2.9 and 4.2 mg/L (loading) and 100% at 6.0 mg/L (loading). After 48 h of exposure, immobilisations were 0% at 1.0 mg/L (loading), 5% at 1.4 mg/L (loading), 90% at 2.1 mg/L (loading) and 100% at 2.9, 4.2 and 6.0 mg/L (loading).

Therefore, based on the nominal loading rates of the test item, the 24 h EC50 was 2.217 mg/L (95% Cl 1.353-3.506 mg/L) and the 48 h EC50 value estimated was 1.758 mg/L (95% Cl 1.604-1.914 mg/L).