1,5,10-trimethylcyclododeca-1,5,9-triene, epoxidised

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 21st to February 24th 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspection by COFRAC on July 4th, 5th and 6th 2016, signed on January 10th 2017

Specific details on test material used for the study:

No additional information

Analytical monitoring:

yes

Details on sampling:

Chemical analyses : Single samples for analysis were taken from the control and all test concentrations at the start and every day thereafter until the end of the test.
- Centrifugation : Samples were centrifuged before analysis.
- Concentrations: 1.0, 2.2, 4.8, 10.6 and 25 mg test item / L
- Sampling method: The test samples were injected directly after a dilution by a factor of two with acetone (500µL sample + 500 µL of cyclohexanone 10 mg.L-1 in acetone). Cell numbers were counted daily by microscope using a counting chamber.
- Sample storage conditions before analysis: The samples were injected directly after their preparation to avoid any degradation of the test item.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Based on the results of a range-finding test, test solutions used in the definitive test were prepared by direct addition of the required amounts of stock solution to test water and inoculum to obtain the following nominal concentrations: 1.0, 2.2, 4.8, 10.6 and 25 mg test item/L.
- Controls: Test water without test substance but treated in the same way as the test substance solutions.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): The solvent used for the preparation of the spiking solutions was acetone.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): 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.
- Age of inoculum (at test initiation): not specified
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (= test water) 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 1.104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

Not specified

Test temperature:

23 +/- 2°C

pH:

8.06 - 9.44

Dissolved oxygen:

Not specified

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal concentrations : test solutions used in the definitive test were prepared by direct addition of the required amounts of stock solution to test water and inoculum to obtain the following nominal concentrations (spaced by a factor of approximately 2.2) in agreement with the Sponsor and the Study Monitor: 1.0, 2.2, 4.8, 10.6 and 25 mg test item.L-1 [= the highest test concentration (25 mg.L-1, nominal) was expected to be close to/slightly above the solubility of the test item in test water; after two weeks of slow-stirring, the concentration of the stock solution of the test item was approx. 22.0 mg.L-1 (mean of 3 replicates), and since a 1:10 dilution was made by inoculum addition the maximum tested nominal concentration was 20.0 mg.L-1; however concentrations values were calculated considering the test item major constituents included in the main chromatographic peak (representing 70.76% of the test item): 20/70.76% = 28.3 mg.L-1].

Measured concentrations : The highest measured test concentration was slightly lower compared to the expected nominal value (73% of expected nominal concentration in the experimental unit at 28.3 mg.L-1). Since the test item was a UVCB substance, the results were based on the nominal test concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: all-glass closed flasks with ground glass stopper
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Approximately 100 mL (maximum volume capacity: 116 mL), completely filled with test solution with minimum headspace.
- Aeration: The suspensions were continuously aerated

- Initial cells density: 5.103 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: preparation according to OECD 201 TG
- Total organic carbon: Control solutions : 1 and 40 mg Carbon/L


OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Photoperiod: Continuous illumination
- Light intensity and quality: 4 440-8 880 lux , 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.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] : Cell numbers were counted daily by microscope using a counting chamber.
- Other: growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 2.2
- Range finding study : A range-finding test was conducted to determine the range of test loading rates for the definitive test.
- Test concentrations of the range-finding test : nominal concentrations (in triplicate) of 0.10, 0.32 and 1.00 mg/L

Reference substance (positive control):

yes

Remarks:

The sensitivity of the test clone was checked by using potassium dichromate (K2Cr2O7) as reference substance.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

20.457 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits : 18.608 – 22.745 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.51 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits : 2.870 – 4.135 mg/L

Details on results:

- Exponential growth : An initial cell density of 5.103 cells/L using the exponentially growing pre-culture.
- Colour differences: At the start of the test, the solution was observed to be clear and colourless.

Results with reference substance (positive control):

The 72h-EC50 was 1.438 mg/L for the parameter growth rate. The sensitivity of this batch of Pseudokirchneriella subcapitata was consistent with the level proposed by the ISO 8692 (expected 72h-EC50: 0.92 mg/L to 1.46 mg/L).

Reported statistics and error estimates:

The software ToxRat® Professional was used to perform statistical analyses.

Table 1 : Concentrations of the test substance in test water – Final test (main chromatographic peak, representing 70.76% of the test item).

 Biotic system :

Nominal concentration (mg test item.L^-1)	Expected nominal concentration of the main chromatographic peak (mg.L^-1)	Start (t=0h)	t=24h	t=48h	End (t=72h)	Relative loss to initial value (t=0h - t=72h) (%)	Geometric mean measured concentration
							mg.L^-1	%
Control	0	Abs.	Abs.	Abs.	Abs.	N.A.	N.A.	N.A.
1.0	0.71	0.99*	1.03*	0.67*	0.65*	34	0.82	115
2.2	1.56	1.85*	1.81*	1.56*	1.45*	22	1.66	106
4.8	3.40	3.68	3.73	3.41	3.15	14	3.48	102
10.6	7.50	7.31	7.58	7.50	7.30	0	7.42	99
28.3	20.03	14.96	15.11	14.18	14.33	4	14.64	73

*Values below the LOQ but above the LOD

N.A.: not applicable

Absence: concentrations below the LOQ (2.08 mg.L^-1) and the LOD (0.62 mg.L^-1).

Presence: concentrations below the LOQ (2.08 mg.L^-1) and above the LOD (0.62 mg.L^-).

% = Percent of expected nominal concentration of the main chromatographic peak

Samples were centrifuged before analysis.

Table 2 : Concentrations of the test item in test water - Results of the determination of TOC analysis (mg.L^-1) - Final test. 

Biotic system : 

Nominal concentration (mg test item.L^-1)	Start (t=0h)	t=24h	t=48h	End (t=72h)
Control	0.71	1.49	1.18	1.34
1.0	1.28	2.01	1.69	1.69
2.2	2.29	2.65	2.61	2.56
4.8	3.52	4.24	4.34	4.12
10.6	7.08	7.76	7.44	7.03
28.3	12.70	13.3	12.3	11.70

Samples were centrifuged before analysis; the increase in TOC values was probably due to algal development (despite the centrifugation).

  

Table 3. Algal cell densities during the final test (expressed as density of algal cells.mL^-1x10⁴).

	Replicate	Nominal concentration
		Control	1.0	2.2	4.8	10.6	28.3
t=24h	1	4.0	3.6	2.4	2.0	1.6	1.6
	2	3.2	3.2	2.0	2.8	2.0	1.6
	3	2.8	2.8	2.4	2.8	1.6	2.0
	4	4.0
	5	3.6
	6	5.2
	Mean	3.8	3.2	2.3	2.5	1.7	1.7
	Std. Dev.	0.83	0.40	0.23	0.46	0.23	0.23
	% Inh.	-	7.9	24.9	19.8	38.4	38.4
t=48h	1	12.0	10.4	8.8	8.0	6.0	2.4
	2	10.8	12.4	10.0	7.2	5.6	3.6
	3	9.6	11.6	12.0	12.0	4.0	3.2
	4	12.4
	5	13.2
	6	10.8
	Mean	11.5	11.5	10.3	9.1	5.2	3.1
	Std. Dev.	1.31	1.01	1.62	2.57	1.06	0.61
	% Inh.	-	-0.1	3.6	8.1	25.6	42.4
t=72h	1	50.8	33.6	33.2	28.8	12.0	2.8
	2	44.0	40.0	36.0	20.0	11.6	3.2
	3	42.0	36.0	32.8	26.0	8.8	3.2
	4	45.6
	5	43.6
	6	41.2
	Mean	44.5	36.5	34.0	24.9	10.8	3.1
	Std. Dev.	3.44	3.23	1.74	4.50	1.74	0.23
	% Inh.	-	4.4	6.0	13.1	31.7	59.6

At test start 5000algal cells.mL^-1were incubated; 6 replicates of the controls and 3 replicates of each test loading rate.

Std. Dev.: standard deviation.

% Inh.: %Inhibition of growth rate relative to the control determined by the computer program ToxRat (8).

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based on the nominal test concentrations, the 72-hour EC50 and EC10 based on growth rate were determined at 20.457 and 3.51 mg/L, respectively.

Executive summary:

The influence of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72h static test according to the OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and the EU Method C.3 of Commission Regulation No. 440/2008 amended by Commission Regulation (EU) 2016/266 and the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

 

The nominal tested concentrations of the test item were : 1.0, 2.2, 4.8, 10.6 and 28.3mg/L.

 

Specific analyses of samples at the start and every day thereafter until the end of the test revealed that test concentrations found were stable, with losses of test item < 20%.

TOC analysis confirmed that test concentrations were well maintained throughout the test. However, the highest measured test concentration was slightly lower compared to the expected nominal value (73% of expected nominal concentration in the test unit at 28.3 mg.L^-1). Since the test item was a UVCB substance, the results were based on the nominal test concentrations.

Under the experimental conditions and based on the nominal test concentrations, the 72-hour EC50 and EC10 based on growth rate were determined at 20.457 and 3.51 mg/L, respectively.

All validity criteria were fulfilled. Cell density in controls showed a 89 -fold increase within 72 hours. The specific growth rate was determined at 1.50 day-1, so greater than 0.92 day-1.

The mean coefficient of variation for section-by-section specific growth rates in the control cultures was of 31.4%. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 1.7%.

Description of key information

EU Method C.3, OECD Guideline 201, GLP, key study, validity 1 :

72h-ErC50 (Pseudokirchneriella subcapitata) = 20.457 mg/L (95% CI: 18.608 - 22.745 mg/L), based on nominal concentrations (as UVCB substance)

72h-ErC10 (Pseudokirchneriella subcapitata) = 3.510 mg/L (95% CI: 2.870 - 4.135 mg/L), based on nominal concentrations (as UVCB substance)

Key value for chemical safety assessment

EC50 for freshwater algae:

20.457 mg/L

EC10 or NOEC for freshwater algae:

3.51 mg/L

Additional information

One key study is available to assess the influence of the registered substance on the growth of the freshwater green algal species Pseudokirchneriella subcapitata in a 72h static condition, according to the EU Method C.3 and the OECD Guideline 201 with GLP statement.

The nominal concentrations of the test item of : 1.0, 2.2, 4.8, 10.6 and 28.3mg/L were tested in parallel with controls.

Specific analyses of samples at the start and every day thereafter until the end of the test revealed that test concentrations found were stable, with losses of test item < 20%.

TOC analysis confirmed that test concentrations were well maintained throughout the test. However, the highest measured test concentration was slightly lower compared to the expected nominal value (73% of expected nominal concentration in the test unit at 28.3 mg.L^-1). Since the test item was a UVCB substance, the results were based on the nominal test concentrations.

Under the experimental conditions and based on the nominal test concentrations, the 72-hour EC50 and EC10 based on growth rate were determined at 20.457 and 3.51 mg/L, respectively.

All validity criteria were fulfilled. Cell density in controls showed a 89 -fold increase within 72 hours. The specific growth rate was determined at 1.50 day-1, so greater than 0.92 day-1.

The mean coefficient of variation for section-by-section specific growth rates in the control cultures was of 31.4%. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 1.7%.