3,7-dimethylocta-1,3,6-triene

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Mar 2014 to 26 Mar 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

July, 2011

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

August, 2009

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Version / remarks:

May, 2012

Qualifier:

according to guideline

Guideline:

other: “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23)

Version / remarks:

December, 2000

Principles of method if other than guideline:

The test was terminated after 48 hours instead of 72 hours due to significant loss of the test substance. Therefore, results were based on 48 hour values.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from the control and all test concentrations (from a replicate of each test concentration with and without algae dedicated exclusively to chemical analyses) at the start (t=0h) and every day thereafter until the end of the test.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The standard test procedures required preparation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures. Given the volatility of the test item (Vapour Pressure: 251 Pa at 25°C), the stock and test solutions were prepared under closed conditions and gently stirred to avoid production of a dispersion.

Stock solution was prepared by slow-stirring. The mixing vessel was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the saturated solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and 1 L of the test water was added. This is to use a maximum volume and to minimize headspace whilst maintaining optimum surface contact between test item and the water. Then an excess of the test item (0.85 g) was carefully added directly to the surface of the test water and the vessel was sealed immediately. 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 kept as low as possible to maintain mixing of the water phase without dispersing the test substance in the water phase. After 24 ± 2 hours of gentle stirring and without further standing time (although stirring was stopped before drawing off the aqueous phase), the saturated aqueous phase was extracted via the drain port. The first 100 mL were discarded and samples were taken from the remaining stock solution and chemically analyzed (measured concentration: 3.9 mg/L). Then the stock solution was directly diluted into flasks with test water and a fixed amount of inoculum to obtain the required test concentrations in the flasks and a cell density of 5.10E+03 cells/mL per vessel.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Origin: Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 -75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes under standardised conditions according to the test guidelines.

ACCLIMATION
- Stock Culture: algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2°C.
- Pre-culture: 2 to 4 days before the start of the test, cells from the algal stock culture were inoculated in test water at a cell density of 1.10E+4 cells/mL. The pre-culture was maintained under the same conditions as used in the test (see 'Details on test conditions'). The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.5 - 22.7 °C

pH:

- Start: 8.00 - 8.14
- End: 8.11 - 9.06

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 0.3, 0.5, 0.8, 1.2, 1.9 and 3.0 mg/L (based on a range-finding study)
- Measured concentrations (geometric means): - (control), 0.101, 0.101, 0.217, 0.289, 0.358 and 0.805 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessels: 100 mL, all-glass flasks sealed with a fritted glass stopper.
- Replicates: 6 replicates of the controls and 3 replicates of each test concentration.
- Volume: volume of 50 mL of algal suspension per replicate
- Cell concentration: 5.10+03 cells/mL (initial cell density)
- Test Environment: controlled environment room; vessels were distributed randomly in the incubator and redistributed over the test at t=24h and t=48h. During incubation, the algal cells were kept in suspension by continuous shaking.

GROWTH MEDIUM
- Type of medium: Original medium of OECD TG 201. Since the test was performed under closed conditions, 3 mL of stock solution 4 were added to the sterilised water during test water reconstitution (instead of 1 mL) to obtain a final concentration of 150 mg NaHCO3/L
- Stock solution 1 (macronutrients): 1.5 g/L NH4Cl; 1.2 g/L MgCl2·6H2O; 1.8 g/L CaCl2·2H2O; 1.5 g/L MgSO4·7H2O; 0.16 g/L KH2PO4
- Stock solution 2 (iron): 64 mg/L FeCl3·6H2O; 100 mg/L Na2EDTA·2H2O
- Stock soltuion 3 (trace elements): 185 mg/L H3BO3; 415 mg/L MnCl2·4H2O; 3 mg/L ZnCl2; 1.5 mg/L CoCl2·6H2O; 0.01 mg/L CuCl2·2H2O; 7 mg/L Na2MoO4·2H2O
- Stock solution 4 (bicarbonate): 50 g/L NaHCO3
- Test water preparation: the stock solutions 1 and 3 were sterilised by autoclaving (120 °C, 15 min), then stored in the dark at 4°C. The stock solutions 2 and 4 were sterilised by membrane filtration (mean pore diameter 0.2 μm). The growth medium was prepared by adding an appropriate volume of the stock solutions 1-4 to water. To 500 mL of sterilised water were added:10 mL of stock solution 1, 1 mL of stock solution 2, 1 mL of stock solution 3 and 3 mL of stock solution 4. Then, the mixture was made up to 1000 mL with sterilised water. The pH of this solution was approximately in the range of 8.1 ± 0.2.

OTHER TEST CONDITIONS
- Light regime: continuous illumination
- Light intensity: 4440 - 8880 lux

EFFECT PARAMETERS MEASURED:
- Cell densities: cell numbers were counted daily by microscope using a counting chamber.
- Other: pH, measured at the beginning and at the end of the test in one vessel per concentration and the control (same vessel at t=0h and t=48h); Temperature of medium: measured continuously in the growth chamber, over the study period; Light intensity: Light intensity was measured once (t=0h) during the test at 5 positions distributed over the experimental area at the surface of the test media.

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 0.1, 1.0, 2.0 and a concentration close to the water solubility limit

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.342 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.L.: 0.327 - 0.363 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.274 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.L.: 0.249 - 0.290 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% C.L.: 0.270 - 0.368 mg/L

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.199 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% C.L.: 0.133 - 0.236 mg/L

Details on results:

The cell numbers recorded during the final test indicate a clear concentration-response relationship. The test was terminated after 48 hours due to significant loss in the flasks. However, as the test substance still remained in critical test concentrations at 48 hours and all validity criteria were met, following the OECD Guideline it was considered acceptable that the test results were based on the 48 h values rather than continuing the study to 72 hours. See 'Any other information on results inc. tables' for algal densities measured during the test.

Results with reference substance (positive control):

The most recent test performed in July 2013, determined the 72h-ErC50 was 0.92 mg/L.

Reported statistics and error estimates:

The evaluation of the effects was based on the average of measured exposure concentrations. The software ToxRat® Professional was used to for the determination of the 48-hour ErCx and EyCx.

Table: algal densities during the final test, expressed as density of algal cells/mL*E+4

		Measured concentration (mg/L) (geometric means of measured exposure concentrations in the biotic test)
		Control	0.101	0.101	0.217	0.289	0.358	0.805
T = 24h	Mean	3.7	4.5	4.0	5.1	2.7	2.4	0.1
	St. Dev	0.48	0.46	0.69	1.01	0.23	0.40	0.23
T = 48h	Mean	23.1	22.0	21.3	19.5	20.4	2.0	0.4
	St. Dev	2.47	2.88	2.05	2.01	1.83	1.39	0.40
T = 72h	Mean	59.0	49.7	41.1	41.2	34.8	0.7	0.1
	St. Dev	1.51	1.97	1.62	5.67	1.74	0.23	0.23

Validity criteria fulfilled:

yes

Conclusions:

The 48-h ErC50 and 48-h ErC10 are 0.342 mg/L and 0.274 mg/L, respectively in green algae.

Executive summary:

The toxicity towards freshwater algae was determined in a study according to OECD TG 201 and in compliance with GLP criteria. In this study, exponentially growing freshwater algae (Pseudokirchneriella subcapitata) were exposed to nominal test substance concentrations of 0 (control), 0.3, 0.5, 0.8, 1.2, 1.9 and 3.0 mg/L which were based on a range-finding study, under static conditions. Test concentrations were analytically verified at the start and the end of exposure, but showed deviations greater than ± 20% of the initial concentrations in both abiotic and biotic flasks. Therefore, the results were based on the geometric means of measured exposure concentrations in the biotic test. The test was terminated after 48 hours due to significant loss in the flasks. However, as test substance still remained in the flasks at 48 hours and all validity criteria were met, following the OECD Guideline, it was considered acceptable to base the test results on the 48 h values. The cell densities in each flask was determined daily. At the end of the test the percentage inhibition of biomass and average specific growth rate were calculated. The 48-h values for growth rate (ErC50) and biomass (EbC50) were determined at 0.342 and 0.310 mg/L, respectively. The 48-h EC10 values for growth rate and biomass were 0.274 and 0.199 mg/L, respectively.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across from a guideline study

Justification for type of information:

The algae toxicity of Ocimene is based on read-across from Myrcene. The documentation is presented in the Endpoint summary of Aquatic toxicity.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.342 mg/L

Basis for effect:

growth rate

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.274 mg/L

Basis for effect:

growth rate

Description of key information

No data is available for the substance itself. Therefore read-across is performed to the close structural analogue Myrcene (CAS# 123-35-3). The algae toxicity of Myrcene is summarised below. The read across justification is presented in the Aquatic Endpoint summary.

Myrcene and Algae toxicity

Its toxicity towards freshwater algae was determined in a study according to OECD TG 201 and in compliance with GLP criteria. In this study, exponentially growing freshwater algae (Pseudokirchneriella subcapitata) were exposed to nominal test substance concentrations of 0 (control), 0.3, 0.5, 0.8, 1.2, 1.9 and 3.0 mg/L, which were based on a range-finding study, for 48 hours under static conditions. Test concentrations were analytically verified at the start and the end of exposure, but showed deviations greater than ± 20% of the initial concentrations in both abiotic and biotic flasks. Therefore, the results were based on the geometric means of measured exposure concentrations in the biotic test. The test was terminated after 48 hours due to significant loss in the flasks. However, as test substance still remained in the flasks at 48 hours and all validity criteria were met, following the OECD Guideline, it was considered acceptable to base the test results on the 48 h values. The cell densities in each flask was determined daily. At the end of the test the percentage inhibition of biomass and average specific growth rate were calculated. The 48-h values for growth rate (ErC50) and biomass (EbC50) were determined at 0.342 and 0.310 mg/L, respectively. The 48-h EC10 values for growth rate and biomass were 0.274 and 0.199 mg/L, respectively. There is no need to convert the Myrcene toxicity data towards Ocimene, because of the similarities in molecular weight and log Kows.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.342 mg/L

EC10 or NOEC for freshwater algae:

0.274 mg/L