Octadecene, reaction products with hexadecene, hydrogenated

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:

03 December 2019 to 06 December 2019

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:

OECD Guideline for Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted 28th July, 2011.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Annex IV Part C, C.3

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

EPA Ecological Effects Test Guidelines, OCSPP 850.5400, Algal Toxicity, Tiers I and II, EPA 712-C-96-164, April 1996.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

Duplicate samples (from three replicates) were taken (2 x ~15 mL) in glass tubes at the beginning and at the end of the experiment from the control and at the applied test concentration level.

Vehicle:

no

Details on test solutions:

The test solutions were prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23 (2000).
Saturated test item solution with the nominal loading rate of 100.0 mg/L (water accommodated fractions, WAF) was prepared with direct addition of the test item, mixed into the algal growth medium (OECD Medium) and then continuously stirred gently for approximately 48 hours using magnetic stirrer. Then, the emulsion was settled for approximately 1 hour, and the middle layer was collected as the test solution. This test solution (100.0 mg/L WAF) was divided into two parts. The non-dissolved test material was removed by filtration through a fine (0.22 µm) filter to give the appropriate WAF solution in the first case, while the other set was not filtered and used as test solution as it is.

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of Charles River Laboratories Hungary Kft.
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
Breeding conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, after an incubation period of three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Not specified

Test temperature:

Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 22.4 – 22.6 °C measured in the flask and between 21.2 and 23.2 °C measured within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the test in each test vessels. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.01 – 8.52 during the experiment.

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal loading rate WAF

Details on test conditions:

PERFORMANCE OF THE TEST
The exposure time was 72 hours. The test was started (0 hours) by inoculation of a biomass of approximately 10e4 algal cells per mL test medium.
The test was performed with six replicates per test concentration and six replicates in the control group. Volumes of 100 mL test solution per replicate in 250 mL sterile Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers.
All procedures were performed under sterile conditions during the test.

Preliminary Range-Finding Test
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. Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group.
Saturated test item solutions with the nominal loading rates of 0.1, 1.0, 10.0 and 100.0 mg/L (water accommodated fractions, WAFs) were individually prepared with direct addition of the test item, mixed into the algal growth medium (OECD Medium) and then continuously stirred gently for approximately 48 hours using magnetic stirrer. Then, the emulsions were settled for approximately 1 hour, and the middle layer was collected as the test solution in each tested level.
Test solution at the highest test level (100.0 mg/L WAF) was divided into two parts as described above.
The concentration levels used and results (72 h) of the Preliminary Range-Finding Test are summarised in table form - see "Any other information" for details.

Test item concentrations in the Definitive Test
Because significant inhibition was not observed at the highest concentration level during the preliminary range-finding test, only one test concentration (a filtered and a non-filtered one in parallel to demonstrate that any effect observed is due to physical effects and not toxicity) in the test medium (100 mg/L nominal loading rate WAF) and one control group were tested in a Limit Test.

OBSERVATIONS
The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate is (Batch Number: A0345704): 10 – 13 September 2019.

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass, growth rate & yield

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biosmass, growth rate & yield

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

VALIDITY
The cell density in the control cultures increased by the factor of 66.33 within three days.
The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 11.83 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 1.13 %.
All validity criteria were met; therefore, the study can be considered as valid.

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS
There were no any observed morphological deviations during the experiment.

AVERAGE SPECIFIC GROWTH RATES
The results of the statistical evaluation (based on Dunnett’s Test; α=0.05) show that the 0-72 h average specific growth rate was not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 100.0 mg/L nominal loading rate (WAF).

AREAS UNDER THE GROWTH CURVES
The results of the statistical evaluation (based on Dunnett’s Test; α=0.05) show that the 0-72 h areas were not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 100.0 mg/L nominal loading rate (WAF).

YIELD
The results of the statistical evaluation (based on Dunnett’s Test; α=0.05) show that the 0-72 h yield was not statistically significantly different from the untreated control value; accordingly the No Observed Effect Concentration (NOEC) was determined as 100.0 mg/L nominal loading rate (WAF).

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 19/238-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 10 – 13 September 2019.
The 72h ErC 50: 0.82 mg/L, (95 % confidence limits: 0.76 – 0.90 mg/L)
The 72h EbC 50: 0.58 mg/L, (95 % confidence limits: 0.53 – 0.62 mg/L)
The 72h EyC 50: 0.49 mg/L, (95 % confidence limits: 0.46 – 0.54 mg/L)
These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.
The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel 2007 for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
As a Limit test was performed the ErL50, EbL50 and EyL50 values of the test item were not calculated and determined from the raw data.
For the determination of the LOELR and NOELR, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Dunnett’s -Test (α = 0.05) by TOXSTAT software.

Results of the Preliminary Range-Finding Test

Nominal concentrations [mg/L nominal loading rate WAF]	Untreated control	0.1	1.0	10.0	100.0	100.0*
Average of cell number at 72 hours (x 10^4cell/mL)	71.00	70.50	68.00	69.50	60.50	69.00

*Remark: filtered solution

 

Growth Rates (μ) and Percentage Inhibition of μ during the Test Period

Test group	Growth rate μ and % inhibition of μ
	0-24 h		0-48h		0-72h
	μ	%	μ	%	μ	%
Control	0.0553^+	0.0	0.0592	0.0	0.0582^+	0.0
100.0 mg/L nominal loading rate (unfiltered)	0.0518	6.4	0.0584	1.4	0.0574^+	1.4
100.0 mg/L nominal loading rate (filtered)	0.0558	-0.8	0.0590	0.3	0.0576^+	1.0

+: at these values the rounding of the EXCEL and TOXSTAT software was different, The table contains the values calculated with EXCEL.

 

Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Test group	Areas under the Growth Curve (A) and % inhibition of A
	0-24 h		0-48h		0-72h
	μ	%	μ	%	μ	%
Control	34.0	0.0	262.0	0.0	1240.0	0.0
100.0 mg/L nominal loading rate (unfiltered)	30.0	11.8	246.0	6.1	1172.0	5.5
100.0 mg/L nominal loading rate (filtered)	34.0	0.0	260.0	0.8	1202.0	3.1

 

Yield (Y) and Percentage Inhibition of Y during the Test Period

Test group	Yield (Y) and % inhibition of Y (0-72 h)
	Y	%
Control	65.3	0.0
100.0 mg/L nominal loading rate (unfiltered)	61.7	5.6
100.0 mg/L nominal loading rate (filtered)	62.5	-1.4

 

TEST CONDITIONS

pH Values in the Test media at the Start and End of the Test

Test group	pH-values
	Start	End
Control	7.32	8.32
		5.15
		8.43
		8.52
		8.27
		8.31
100.0 mg/L nominal loading rate (unfiltered)	7.01	8.02
		8.09
		8.11
		8.00
		7.99
		7.93
100.0 mg/L nominal loading rate (filtered)	7.02	8.13
		8.08
		8.15
		7.97
		8.01
		7.90

 

Temperature on the Climate Chamber in the Test Media during the Test

Parameter		Exposure Time
		Day 0	Day 1	Day 2	Day 3
Temperature (°C) measured in flask		22.6	22.5	22.4	22.4
Temperature (°C) of the climate chamber	max	23.2	22.9	22.7	22.6
	min	21.4	21.3	21.2	21.4

 

CELL NUMBER

Cell Number (x 10⁴cell/mL) determined in the Main Experiment

Test group		Number of cells
		0 h	24 h	48 h	72 h
Control		1	4	17	62
		1	3	16	65
		1	4	18	70
		1	3	16	64
		1	5	18	68
		1	4	18	69
	Mean	1.00	3.83	17.17	66.33
	SD	0.0	0.8	1.0	3.1
100.0 mg/L nominal loading rate (unfiltered)		1	4	16	62
		1	3	16	60
		1	4	17	70
		1	4	17	61
		1	3	17	59
		1	3	16	64
	Mean	1.00	3.50	16.50	62.67
	SD	0.0	0.5	0.5	4.0
100.0 mg/L nominal loading rate (filtered)		1	3	16	63
		1	4	17	61
		1	4	18	62
		1	4	17	66
		1	4	18	65
		1	4	16	64
	Mean	1.00	3.83	17.00	63.50
	SD	0.0	0.4	0.9	1.9

 

Validity criteria fulfilled:

yes

Conclusions:

The effect of SynNova Base Oil was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), over an exposure period of 72 hours.
Under the conditions of this algal growth inhibition test the calculated endpoints for the effect of SynNova Base Oil were the following:

Based on the nominal loading rate (WAF):
The 72h EbL50 value (biomass): > 100.0 mg/L
The 72h ErL50 value (growth rate): > 100.0 mg/L
The 72h EyL50 value (yield): > 100.0 mg/L

The 72h No-Observed Effect Loading Rate (NOELR): 100.0 mg/L
The 72h Lowest Observed Effect Loading Rate (LOELR): > 100.0 mg/L

Executive summary:

The effect of SynNova Base Oil test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours.

 

Because a significant inhibition was not observed during the preliminary concentration range-finding test, a Limit Test was carried out using only one test concentration at 100.0 mg/L nominal loading rate WAF (a filtered and a non-filtered one in parallel) and one control group.

 

The WAF solution was prepared with direct addition of the test item. During the preparation periods, droplets in the top layers were observed in the test solution, so the solution in the middle layer was collected and siphoned into another test vessel as exposure solution, certainly, there no obvious droplets were observed in the exposure solution. Any remaining non-dissolved test chemical component in the WAF has the possibility to cause physical effects on the test organism unrelated to inherent chemical toxicity. For this reason, a filtered and a non-filtered test item group was tested in parallel to demonstrate that any effect observed is due to physical effects and not toxicity.

 

The test concentration was analytically determined at the start and at the end of the experiment. The measured concentration was below the Limit of Quantification (LOQ = 0.28 mg/L) at the unfiltered sample, while it was below the Limit of Detection (LOD = 0.078 mg/L) at the filtered test item group at the start of the experiment. Test concentration was measured below the Limit of Detection in both test item treated group at the end of the test. The biological results are based on the nominal loading rate.

The test design included six replicates at test concentration groups and six replicates for the untreated control.

 

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using Dunnett’s Test (a= 0.05) by TOXSTAT software.

 

The ErL50, EbL50 and EyL50 values of the test item were determined directly from the raw data.

 

Under the conditions of this algal growth inhibition test the calculated endpoints for the effect of SynNova Base Oil were the following:

 

Based on the nominal loading rate (WAF):

The 72h EbL50 value (biomass): > 100.0 mg/L

The 72h ErL50 value (growth rate): > 100.0 mg/L

The 72h EyC50 value (yield): > 100.0 mg/L

 

The 72h No-Observed Effect Loading Rate (NOELR): 100.0 mg/L

The 72h Lowest Observed Effect Loading Rate (LOELR): > 100.0 mg/L

Description of key information

Based on the nominal loading rate (WAF):

The 72h EbL50 value (biomass): > 100.0 mg/L

The 72h ErL50 value (growth rate): > 100.0 mg/L

The 72h EyC50 value (yield): > 100.0 mg/L

 

The 72h No-Observed Effect Loading Rate (NOELR): 100.0 mg/L

The 72h Lowest Observed Effect Loading Rate (LOELR): > 100.0 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The effect of SynNova Base Oil test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours.

 

The WAF solution was prepared with direct addition of the test item. During the preparation periods, droplets in the top layers were observed in the test solution, so the solution in the middle layer was collected and siphoned into another test vessel as exposure solution, certainly, there no obvious droplets were observed in the exposure solution. Any remaining non-dissolved test chemical component in the WAF has the possibility to cause physical effects on the test organism unrelated to inherent chemical toxicity. For this reason, a filtered and a non-filtered test item group was tested in parallel to demonstrate that any effect observed is due to physical effects and not toxicity.

 

The ErL50, EbL50 and EyL50 values of the test item were determined directly from the raw data.

 

Under the conditions of this algal growth inhibition test the calculated endpoints for the effect of SynNova Base Oil were the following:

 

Based on the nominal loading rate (WAF):

The 72h EbL50 value (biomass): > 100.0 mg/L

The 72h ErL50 value (growth rate): > 100.0 mg/L

The 72h EyC50 value (yield): > 100.0 mg/L

 

The 72h No-Observed Effect Loading Rate (NOELR): 100.0 mg/L

The 72h Lowest Observed Effect Loading Rate (LOELR): > 100.0 mg/L