5,6,7-TRIMETHYLOCTA-2,5-DIEN-4-ONE

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 3 July 2020 to 1 December 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(2E,5Z)-5,6,7-trimethylocta-2,5-dien-4-one

Cas Number:

357650-26-1

Molecular formula:

C11H18O

IUPAC Name:

(2E,5Z)-5,6,7-trimethylocta-2,5-dien-4-one

Constituent 2

Reference substance name:

(2E,5E)-5,6,7-trimethylocta-2,5-dien-4-one

Cas Number:

847144-75-6

Molecular formula:

C11H18O

IUPAC Name:

(2E,5E)-5,6,7-trimethylocta-2,5-dien-4-one

Test material form:

liquid

Specific details on test material used for the study:

Purity: 94.0 % (sum of peaks #2 and #3 ; sum of the two main isomers)
Appearance: Yellow liquid
Expiration date: July 17, 2021

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: For the determination of the actual test item concentrations, duplicate samples (10 mL) were taken from each treatment at the start of the test.After 24 hours, 48 hours and at the end of the test stability samples (containing algae) were taken in duplicate from all test concentrations and from the control.
For sampling at the end of the test, the test medium of the treatment replicates was pooled.

- Sample storage conditions before analysis: All samples were stored frozen (at -20 ± 5 °C) immediately after sampling

- concentration (nominal): 0.10; 0.32; 1.0; 3.2; 10 mg/L

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

Method: At the start of the exposure period, the test medium of the highest nominal concentration of 10 mg/L was prepared by completely dissolving 20.0 mg of test item in 2000 mL of test water. For this, stirring for 15 minutes in a closed vessel at room temperature was applied. The mixing vessel was nearly completely filled (including a small headspace) and tightly sealed with glass stoppers. After this treatment, this highest test medium was a clear solution.

The test media of the lower test item concentrations were prepared in a series of further dilution steps. The test media were prepared just before the start of the test.

- control: test water without test item

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum, occasionally also listed as Raphidocelis subcapitata)
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures - SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany
- Method of cultivation: under standardized conditions according to the test guidelines

Study design

Test type:

static

Water media type:

other: Synthetic test water (AAP medium)

Remarks:

The culture media was prepared according to the test guidelines, but modified according to the International Standard ISO 14442 since a closed test system was applied.

Total exposure duration:

72 h

Test conditions

Hardness:

0.15 mmol/L

Test temperature:

23°C

pH:

7.5 - 7.8

Nominal and measured concentrations:

Nominal concentrations: 0.0, 0.10, 0.32, 1.0, 3.2, 10 mg/L
Measured concentrations: 0.0, na, 0.27, 0.84, 2.8, 8.8 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: completely filled (without headspace) with about 60 mL of test medium and tightly sealed with glass stoppers to avoid losses of the volatile substance by evaporation
- Initial cells density: 5000 algal cells/mL
- No. of vessels per concentration (replicates): Triplicates
- No. of vessels per vehicle control (replicates): 6 replicates (test water without test item)

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:
Ingredients Concentration
Macro-nutrients NaHCO3 250.0 mg/L
K2HPO4 1.044 mg/L
MgSO4 × 7 H2O 14.6 mg/L
MgCl2 × 6 H2O 12.16 mg/L
CaCl2 × 2 H2O 4.41 mg/L
NaNO3 25.5 mg/L

Trace elements H3BO3 186.0 µg/L
MnCl2 × 4 H2O 415.0 µg/L
ZnCl2 3.27 µg/L
CoCl2 × 6 H2O 1.43 µg/L
CuCl2 × 2 H2O 0.012 µg/L
Na2MoO4 × 2 H2O 7.26 µg/L
FeCl3 × 6 H2O 160.0 µg/L
Na2EDTA × 2 H2O 300.0 µg/L



OTHER TEST CONDITIONS
- Photoperiod: continuously illuminated by LED light installed above the test flasks
- Light intensity and quality: The mean light intensity at the level of the test solutions was approximately 71 µE.s-1.m-2 (range: 69 to 72 µE s-1 m-2, measured at nine places in the experimental area). The light intensity over the incubation area was within a ±15 %-deviation from the average light intensity as recommended by the guideline.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The test was started using a nominal algal cell density of 5000 cells/mL. The initial cell density was selected according to the recommendations of the OECD test guideline. A small volume (100 µL per sample) of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass, and was not replaced. The algal biomass in the samples was determined by fluorescence measurement (SpectraMax I3x, Molecular Devices Ltd, Wokingham Berkshire/UK). The measurements were performed at least in duplicate at an excitation of 440 nm and emission of 680 nm.
- pH: The pH was measured and recorded in each treatment at the start and end of the test. The pH of the test media was in the range of 7.4 to 7.8 during the test period fulfilling the requirement of the OECD guideline that the pH of the control medium should not increase by more than 1.5 units during the test.
- Temperature: The water temperature during the test was maintained at 23 °C. (The test flasks were incubated in a temperature controlled orbital shaker (Multitron-Pro, Infors HT, Bottmingen/Switzerland))

TEST CONCENTRATIONS
- Range finding study: For determination of the appropriate test concentrations for the main test, a range-finding test with nominal test item concentrations of 0.10, 1.0, 10 and 100 mg/L was performed. For a better understanding of the behavior and fate of the test item under the test conditions, additional test vessels with test medium at a concentration of 10 mg/L were incubated for 72 hours under the following conditions:
- Test medium without algae under light conditions
- Test medium without algae in the dark
- Test medium with algae in a closed vessel

- Test concentrations: Based on the results of the dose range finding test the following nominal concentrations of POMAROSE were selected for the main test: 0.10, 0.32, 1.0, 3.2 and 10 mg/L
- Results used to determine the conditions for the definitive study:
Nominal test item Inhibition of Yield Inhibition of Average
Concentration [mg/L] after 72 Hours [%] Growth Rate after 72 Hours [%]
Control 0.0 0.0
0.10 0.4 0.1
1.0 4.4 1.1
10 97 87
100* 100 100

*: Test medium was a clear solution throughout the test period.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate tested twice a year

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 1.9 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % Confidence Interval : 0.58-2.3 mg / L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 0.86 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % Confidence Interval: 0.81-0.91 mg/L

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 2.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % Confidence Interval: 1.2-2.5 mg / L

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 1.1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % Confidence Interval: 1.0-1.1 mg / L

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 3.1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % Confidence Interval: 3.0-4.4 mg / L

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 1.7 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % Confidence Interval: 1.6-1.7 mg / L

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.27 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

biomass

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.84 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

biomass

Details on results:

The impact of the test item on the growth of the algae is shown in Table 1 to Table 4. A clear concentration-response relationship was observed for both biological endpoints growth rate and yield.

The test item had a statistically significant inhibitory effect growth rate µ and on the yield Y of the algae after the test period of 72 hours at the mean measured concentration of 0.84 mg/L and at all higher test concentrations (results of Williams t-test, one-sided smaller, alpha = 0.05, Table 2 and Table 3).

Therefore, the NOEC values for growth rate and yield was determined to be at the mean measured concentrations of 0.27 mg/L.

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 1.0 mg/L (mean measured 0.84 mg/L) and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.

Results with reference substance (positive control):

For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The 72-hour EC50 for growth rate in the reference test IES Study Number 20200190 was 0.92 mg/L (May 2020) and showed that the sensitivity of the test system was within the range recommended by the guideline (72-hour EC50 for the growth rate 0.92 1.5 mg/L).

Any other information on results incl. tables

The measured concentrations of the test item POMAROSE in the test media are shown in the table below:

Nominal Test Item Concentration [mg/L]	Analytically Measured Concentration of the Test Item [mg/L]				Mean Measured Concentration (Time-weighted Mean) [mg/L]
	0 Hours	24 Hours	48 Hours	72 Hours
0.10	---	---	---	---	---
0.32	0.258	0.328	0.251	0.238	0.27
1.0	0.842	0.933	0.813	0.751	0.84
3.2	2.61	3.39	2.55	2.52	2.8
10	8.80	9.22	8.37	8.93	8.8

Table 1: Biomass of Algae

Nominal Test Item Concentration [mg/L]	Mean Measured Concentration	Rep. No.	Biomass of Algae*
	[mg/L]		24 Hours	48 Hours	72 Hours
Control	---	1	3.17	19.5	86.6
		2	3.71	18.1	82.7
		3	3.13	17.7	82.7
		4	3.22	20.0	82.2
		5	2.55	17.4	84.0
		6	3.01	19.3	75.7
		Mean	3.13	18.7	82.3
		SD	0.374	1.07	3.59
0.10	n.a.	1	2.89	19.9	83.9
		2	3.31	20.2	89.1
		3	3.75	19.1	80.4
		Mean	3.32	19.7	84.5
		SD	0.431	0.555	4.40
0.32	0.27	1	3.35	20.1	83.4
		2	3.42	17.7	83.0
		3	3.09	19.0	80.9
		Mean	3.29	18.9	82.4
		SD	0.173	1.20	1.31
1.0	0.84	1	3.25	15.8	76.9
		2	2.14	17.1	71.9
		3	2.20	18.2	75.6
		Mean	2.53	17.0	74.8
		SD	0.625	1.22	2.58
3.2	2.8	1	2.04	6.46	14.4
		2	1.80	6.67	15.5
		3	2.07	7.29	13.1
		Mean	1.93	6.98	14.3
		SD	0.194	0.440	1.71
10	8.8	1	0.661	1.14	0.721
		2	0.622	0.730	0.607
		3	0.695	0.881	0.880
		Mean	0.659	0.916	0.736
		SD	0.036	0.205	0.137

SD:  Standard deviation

n.a.: Not analyzed

*:The biomass was determined by fluorescence measurement (mean of duplicate measurements per replicate) and is given as relative fluorescence units (x 10⁴). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 0.90 x 10⁴relative fluorescence units.

Table 2: Average Growth Rates (µ)

Nominal Test Item Concentration	Mean Measured Concentration	Average Growth Rate µ [day^-1] and Inhibition Ir[%]
		0-24 h		0-48 h		0-72 h
[mg/L]	[mg/L]	µ [day^-1]	Ir[%]	µ [day^-1]	Ir[%]	µ [day^-1]	Ir[%]
Control	--	1.241	0.0	1.515	0.0	1.505	0.0
0.10	n.a.	1.299	-4.7	1.543	-1.9	1.514	-0.6
0.32	0.27	1.295	-4.3	1.523	-0.5	1.506	0.0
1.0	0.84	1.015*	18.2	1.469	3.1	1.473^°	2.1
3.2	2.8	0.781^*	37.1	1.011^*	33.3	0.923^°	38.7
10	8.8	-0.312^*	125.1	0.000^*	100.0	-0.071^°	104.7

n.a.:    not analyzed

°:        Mean value statistically significantly lower than in the control (according to Jonckheere Terpstra test, one-sided smaller, alpha = 0.05).

*:        Mean value statistically significantly lower than in the control (according to Williams t-test, one-sided smaller, alpha = 0.05).

 

Note:  Percentage inhibition values in excess of 100% are obtained when the biomass at the end of the interval is lower than at the start of the interval.

Table 3: Yield (Y)

Nominal Test Item Concentration	Mean Measured Concentration	Yield Y (x 10^4) and Inhibition Iy[%]
		0-24 h		0-48 h		0-72 h
[mg/L]	[mg/L]	Y	Iy[%]	Y	Iy[%]	Y	Iy[%]
Control	--	2.2	0.0	17.8	0.0	81.4	0.0
0.10	n.a.	2.4	-8.3	18.8	-5.9	83.6	-2.6
0.32	0.27	2.4	-7.0	18.0	-1.5	81.5	-0.1
1.0	0.84	1.6*	26.9	16.1*	9.3	73.9°	9.2
3.2	2.8	1.1*	52.1	5.9*	66.8	13.5°	83.5
10	8.8	-0.2*	110.8	0.0*	99.9	-0.2°	100.2

n.a.:    not analyzed

*:        Mean value statistically significantly lower than in the control (according to Williams t-test, one-sided smaller, alpha= 0.05).

°:         Mean value statistically significantly lower than in the control (according to Jonckheere Terpstra test, one-sided smaller, alpha = 0.05).

Note:  Percentage inhibition values in excess of 100% are obtained when the biomass at the end of the interval is lower than at the start of the interval.

Table 4 Section by section Growth Rates

Nominal Test Item Concentration	Mean Measured Concentration	Section-by-Section Growth Rates [day^-1] and Inhibition Ir[%]
		0-24 h		24-48 h		48-72 h
[mg/L]	[mg/L]	µ [day^-1]	Ir[%]	µ [day^-1]	Ir[%]	µ [day^-1]	Ir[%]
Control	--	1.241	0.0	1.789	0.0	1.485	0.0
0.10	n.a.	1.299	-4.7	1.788	0.1	1.454	2.1
0.32	0.27	1.295	-4.3	1.750	2.2	1.472	0.8
1.0	0.84	1.015	18.2	1.922	-7.4	1.482	0.2
3.2	2.8	0.781	37.1	1.241	30.6	0.746	49.7
10	8.8	-0.312	125.1	0.313	82.5	-0.214	114.4

n.a.:    not analyzed

 

Note:  Percentage inhibition values in excess of 100 % are obtained when the biomass
at the end of the interval is lower than at the start of the interval.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The impact of the test item POMAROSE on the growth of the freshwater green algal species Pseudokirchneriella subcapitata is summarized in the table below. The results are based on the mean measured time-weighted test item concentrations:

Endpoint after 72 Hours Growth Rate Yield
[mg/L] [mg/L]

EC10 1.9 0.86
(95 % Confidence Interval) (0.58-2.3) (0.81-0.91)
EC20 2.3 1.1
(95 % Confidence Interval) (1.2-2.5) (1.0-1.1)
EC50 3.1 1.7
(95 % Confidence Interval) (3.0-4.4) (1.6-1.7)
NOEC 0.27 0.27
LOEC 0.84 0.84

Executive summary:

The impact of the test item POMAROSE on the growth of the freshwater green algal speciesPseudokirchneriella subcapitatawas investigated in a 72‑hour static test according to the OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU)No 2016/266, C.3.

As the test item is a volatile substance,the test was performedusing glass stoppered Erlenmeyer flasks completely filled (without headspace) with test medium that were tightly sealed with glass stoppers to avoid losses of test item by evaporation (closed system).

 

The nominal test item concentrations tested were 0.10, 0.32, 1.0, 3.2 and 10 mg/L. Additionally, a control (test water without test item) was tested in parallel.

 

The measured concentrations of POMAROSE in the test media of all test concentrations were between 81 and 88 % of the nominal values at the start of the test. Thus, the correct dosage of the test item could be verified. The test item concentrations in the test media aged samples wereranged from 92 % to 106 % at day 1 (24 hours), from 79 to 84 % at day 2 (48 hours) and from 74 % to 89 % at day 3 (72 hours). The test item concentration slightly decreased over the test period. Therefore, themean measured test item concentrations were calculated as thetime-weighted meansover all measurements.

The measured concentrations of the test item POMAROSE in the test media of all test concentrations at the start, after 24 and 48 hours and at the end of the test are shown in the table below.

 

Nominal Test Item Concentration [mg/L]	Analytically Measured Concentration of the Test Item [mg/L]				Mean Measured Concentration (Time-weighted Mean) [mg/L]
	0 Hours	24 Hours	48 Hours	72 Hours
0.10	---	---	---	---	---
0.32	0.258	0.328	0.251	0.238	0.27
1.0	0.842	0.933	0.813	0.751	0.84
3.2	2.61	3.39	2.55	2.52	2.8
10	8.80	9.22	8.37	8.93	8.8

 

---:         not analyzedsince this concentration was below the NOEC determined in this test.

The biological results are based on themean measured time-weighted testitem concentrations.

A clear concentration-response relationship was observed for both biological endpoints growth rate and yield after the exposure period of 72 hours.

 

Endpoint after 72 Hours	Growth Rate [mg/L]	Yield [mg/L]
EC10	1.9	0.86
(95 % Confidence Interval)	(0.58-2.3)	(0.81-0.91)
EC20	2.3	1.1
(95 % Confidence Interval)	(1.2-2.5)	(1.0-1.1)
EC50	3.1	1.7
(95 % Confidence Interval)	(3.0-4.4)	(1.6-1.7)
NOEC	0.27	0.27
LOEC	0.84	0.84

 

 

Validity Criteria:

 

The validity criteria forincrease of biomass,mean coefficient of variation of the daily growth rates andcoefficient of variation of the average specific growth rateswere fulfilled.