Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene

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:

2019-05-27 to 2019-05-30

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name: Reaction mass of (Z)-3,7-dimethylocta-1,3,6,-triene and dipentene
- Other name/Synonym: Ocimene PQ
- Chemical name: (Z)-3,7-dimethylocta-1,3,6,-triene (CAS 3338-55-4) and dipentene (CAS 138-86-3)
- EC numbers: 222-081, 205-341-0
- CAS numbers: 3338-55-4/138-86-3
- Batch/Lot Number: A170524D
- Description: Colorless Liquid
- Purity: Treated as 100%
- Expiry date: 06 June 2019
- Storage conditions: Room temperature (15-25 °C, ≤70 RH%), under inert gas, protected from humidity (tight closed container)

Analytical monitoring:

yes

Details on sampling:

Analytical measurement was performed at the applied test concentration levels and from the control at the beginning of the experiment and 24-hour intervals thereafter, in order to better define loss of the test substance during the exposure period.

Duplicate samples were taken (2 x ~10 mL) in glass tubes at the applied test concentration levels (each replicate) as well as from the remained test solutions (2 x 2 x ~10 mL) in absence of algae at the beginning of the test and 24 hour intervals thereafter during the experiment. Sample from the control was only be taken for analysis at the start and at the end of the test. After sampling, samples were frozen and kept approximately at -20°C at the Test Facility. One set of the samples was sent to the Test Site for analysis and one set was retained as a back-up at the Test Facility, if required for any confirmatory analyses (discarded after satisfactory results were obtained on the first set of).

Total samples:
- 98 tubes of test solutions (~10 mL aliquots, measured with 0.01 g precision) were received from the Test Facility, corresponding to samples from the control and each test concentration (6.25, 12.5, 25, 50, 100 mg/L) at 0 h, 24 h, 48 h and 72 h, with and without the presence of algae.

Vehicle:

no

Details on test solutions:

Because the test item is a multi-constituent substance and poorly soluble in water, test solutions were prepared individually using a saturated solution method (water accommodated fraction, WAF) according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23 (2000).

Saturated test item solutions (nominal loading rates of 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L) were prepared individually by dispersing/dissolving the amount of test item into the test medium (OECD Medium) two days before the start of the experiment. These solutions were shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. The non-dissolved test material was removed by filtration through a fine (0.22 µm) filter to give the appropriate WAF solutions.

The test solutions were prepared and distributed into test vessels prior to introduction of algae. During the preparation of WAF solutions and the study the usage of plastic lab wares was omitted.

The same method was used to prepare concentrations of the preliminary range-finding test (0.1, 1, 10 or 100 mg/L), which 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.

Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests. For the untreated control, algal growth medium was inoculated with algal cells (without test item) and was examined in parallel to the test item concentrations.

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.
- Culture 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, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

- Target Temperature: 21-24°C maximum deviation of ± 2°C
- Actual 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 between 22.4 and 22.5 °C measured in the flask and between 21.3 and 23.5 °C measured within the climate chamber.

pH:

- Target pH: Maximum deviation of ± 1.5 units
- Actual pH: The pH was checked at the beginning and at the end of the test, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.71 –8.99 during the experiment.

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

The concentrations of test item used in the definitive test: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L nominal loading rates WAFs.
Despite analysing the concentrations of the substance every 24 hours after 24 hours the substance could not be detected by the analytical method and so the Gemetric mean could not be calculated. In order to estimate the geometric mean it was assumed the

Test concentrations were analytically determined at the start and 24-hour intervals thereafter during the experiment in order to better define loss of the test substance during the exposure period. However, the test concentrations could only be measured at the start of the experiment and measured concentrations were below the Limit of Quantification (LOQ = 0.21 mg/L) later during the exposure period at all tested levels. Consequently, the geometric mean measured concentrations could not be calculated.

Details on test conditions:

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. During the formulation procedure was prepared by the similar method described in “Details of test solutions”.

The definitive test was started (0 hours) by inoculation of a biomass of approximately 10^4 algal cells per mL test medium. The test was performed with three replicates per test concentration and six replicates in the control group. Volumes of 150 mL algal suspension per replicate in 250 mL Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers. The exposure time was 72 hours.

The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 7502 lux (equivalent to ~101 µE/m2 /s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

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

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

61.47 mg/L

Nominal / measured:

nominal

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

48.24 mg/L

Nominal / measured:

nominal

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

45.65 mg/L

Nominal / measured:

nominal

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.199 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.225 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Preliminary range-finding test:

The average cell number (x10^4 cells/mL) at 72 hours was 75.00 at 0 mg/L (control), 75.00 at 0.1 mg/L, 75.50 at 1 mg/L, 73.00 at 10 mg/L and 1.0 at 100 mg/L.

Definitive test:

Validity: The cell density in the control cultures increased by the factor of 70.67 within three days (OECD guideline requires ≥ 16). The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 11.46 % (OECD guideline requires ≤ 35%). The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 2.13 % (OECD guideline requires ≤ 7%). All validity criteria were met. Therefore, the study can be considered as valid.

Concentrations of the test item: The concentrations of test item used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L nominal loading rate WAFs. Test concentrations were analytically determined at the start and 24-hour intervals thereafter during the experiment in order to better define loss of the test substance during the exposure period. However, the test concentrations could only be measured at the start of the experiment and measured concentrations were below the Limit of Quantification (LOQ = 0.21 mg/L) later during the exposure period at all tested levels so the Gemetric mean could not be accurately calculated. In order to estimate the geometric mean it was assumed the
Morphological deviations of the algal cells: Thin cells were observed at the highest examined concentration level of 100.0 mg/L nominal loading rate WAF 48 and 72 hours after the start of the experiment.

Growth rate (r): Growth rate is the increase in cell density per time unit. The average specific growth rate for a specific period was calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments, following the equations in OECD Guideline No 201. The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value at the tested concentration range of 50.0 – 100.0 mg/L nominal loading rates WAFs, accordingly the No Observed Effect Loading Rate (NOELR) was determined as 25.0 mg/L nominal loading rate WAF. The 72 h ErL50 value was determined [by Probit analysis (TOXSTAT software)] as 61.47 mg/L nominal loading rate WAF (95 % confidence limits: 56.42 – 66.97 mg/L nominal loading rate WAF).

Biomass (b): The areas under the growth curve were used to calculate biomass, i.e., the actual number of cells per volume of medium (cells/mL). The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h areas were statistically significantly different from the untreated control value at the tested concentration range of 50.0 – 100.0 mg/L nominal loading rates WAFs, accordingly the No Observed Effect Loading Rate (NOELR) was determined as 25.0 mg/L nominal loading rate WAF. The 72 h EbL50 value was determined [by Probit analysis (TOXSTAT software)] as 45.65 mg/L nominal loading rate WAF (95 % confidence limits: 41.41 – 50.32 mg/L nominal loading rate WAF).

Yield (y) or cell number: Yield is calculated as the biomass at the end of the test minus the starting biomass for each single vessel of controls and treatments. For each test concentration and control, mean yield values were calculated following the equations in OECD Guideline No 201. The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h yield was statistically significantly different from the untreated control value at the tested concentration range of 50.0 – 100.0 mg/L nominal loading rates WAFs, accordingly the No Observed Effect Loading Rate (NOELR) was determined as 25.0 mg/L nominal loading rate WAF. The 72 h EyL50 value was determined [by Probit analysis (TOXSTAT software)] as 48.24 mg/L nominal loading rate WAF (95 % confidence limits: 43.66 – 53.32 mg/L nominal loading rate WAF).

Results with reference substance (positive control):

The date of the last study (Study Code: 19/009-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 22 – 25 January 2019. Results were the following:

The 72h ErC 50: 0.87 mg/L, (95 % confidence limits: 0.80 – 0.95 mg/L).
The 72h EbC 50: 0.62 mg/L, (95 % confidence limits: 0.57 – 0.68 mg/L).
The 72h EyC 50: 0.52 mg/L, (95 % confidence limits: 0.48 – 0.57 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).

The ErL50, EbL50 and EyL50 values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software.
Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.

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 Bonferroni t-Test.

Probit and Logit analysis was used to estimate the ErC50 usign the estimated Geometric mean. The ErC50 using the geometric mean concentration was 0.19 mg/l

Results of the Preliminary Range/Finding Test:

Nominal concentrations [mg/L nominal loading rate WAF]	Untreated control	0.1	1	10	100
Average of cell number at 72 hours (x 10^4cell/mL)	75.00	75.00	75.50	73.00	1.00

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

Test concentration [mg/L nominal lading rate WAF]	Growth rate (r) and % inhibition ofm
	0–24 h		0–48 h		0–72 h
	r	%	r	%	r	%
Control	0.0553^+	0.0	0.0590	0.0	0.0591	0.0
6.25	0.0538	2.8	0.0590	0.0	0.0588	0.5
12.5	0.0538	2.8	0.0582	1.4	0.0587	0.6
25.0	0.0538	2.8	0.0573	2.9	0.0578	2.2
50.0	0.0289*	47.8	0.0412*	30.1	0.0528*	10.6
100.0	0.0000*	100.0	0.0000*	100.0	0.0000*	100.0

* : statistically significantly different compared to the control values (Bonferroni t-Test; α = 0.05)

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

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

Test concentration [mg/L nominal lading rate WAF]	Area under the Growth Curves (A) and Percentage Inhibition of A
	0–24 h		0–48 h		0–72 h
	A	%	A	%	A	%
Control	34.0	0.0	260.0	0.0	1288.0	0.0
6.25	32.0	5.9	256.0	1.5	1264.0	1.9
12.5	32.0	5.9	248.0	4.6	1244.0	3.4
25.0	32.0	5.9	240.0	7.7	1176.0	8.7
50.0	12.0*	64.7	100.0*	61.5	704.0*	45.3
100.0	0.0*	100.0	0.0*	100.0	0.0*	100.0

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

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

Test concentration [mg/L nominal lading rate WAF]	Yield (Y)% inhibition of Y
	0–72 h
	Y	%
Control	69.7	0.0
6.25	68.0	2.4
12.5	67.7	2.9
25.0	63.3	9.1
50.0	44.0*	36.8
100.0	0.0*	100.0

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

Calulation of ErC50 using estimated Geometric mean concentrations.

		Data from regression statistics
Geometric mean using 1/2 LOD	% inhibition	% inhibition	Log conc	Logit (% inhibiton)	Probit (% inhibiton)
0	0	0	0	-	-
0.171	0.5	0.005	-0.767833648	-5.29	2.424170696
0.162	0.6	0.006	-0.790411553	-5.11	2.487855672
0.207	2.2	0.022	-0.684805308	-3.79	2.985909188
0.242	10.6	0.106	-0.616683656	-2.13	3.751915189
0.280	99.999	1.0	-0.552722694	11.51	9.264890794
			Slope	58.98592272	24.10897557
			Intercept	39.29396311	20.63711612
			Test values 50% effect	0	5
			Log of Er50 concentration	-0.67	-0.65
			ErC50 mg/l	0.216	0.225
			Test values 10% effect	-2.197224577	3.718448434
			Log of Er10 concentration	-0.70	-0.70
			ErC10 mg/l	0.189	0.199

Validity criteria fulfilled:

yes

Conclusions:

The effect of Ocimene PQ item on algal growth was assessed using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours. All validity criteria were met during this study. Test concentrations could not be analytically determined. Under the conditions of this test, the observed endpoints for the effect of Ocimene PQ were as follows:

72-hour EL50 Growth rate (r) = 61.47 mg/L (95 % conf. limits 56.42 – 66.97 mg/L) nominal loading rate WAF
72-hour EL50 Yield (y) = 48.24 mg/L (95 % conf. limits 43.66 – 53.32 mg/L) nominal loading rate WAF
72-hour EL50 Biomass (b) = 45.65 mg/L (95 % conf. limits 41.41 – 50.32 mg/L) nominal loading rate WAF
72-hour NOELR Growth rate (r) = 25.0 mg/L nominal loading rate WAF
72-hour NOELR Yield (y) = 25.0 mg/L nominal loading rate WAF
72-hour NOELR Biomass (b) = 25.0 mg/L nominal loading rate WAF
72-hour ErC10 Growth = 0.199 mg/l estimated Geometric mean
72-hour ErC50 Growth = 0.225 mg/l estimated Geometric mean

Executive summary:

Acute toxicity of Ocimene PQ was assessed with a Growth inhibition test on Algae (Pseudokirchneriella subcapitata) over an exposure period of 72 hours according to OECD Guideline No. 201. Test concentrations could not be analytically determined. The 72-hour EC50 Growth rate (r) was reported to be 0.225 mg/L geonmetric mean measured concentration, and the 72-hour EC10 Growth rate (r) was reported to be 0.199 mg/l geonmetric mean measured concentration.

Description of key information

Acute toxicity of Ocimene PQ was assessed with a Growth inhibition test on Algae (Pseudokirchneriella subcapitata) over an exposure period of 72 hours according to OECD Guideline No. 201. Test concentrations could not be analytically determined. The 72-hour EC50 Growth rate (r) was reported to be 0.225 mg/L geonmetric mean measured concentration, and the 72-hour EC10 Growth rate (r) was reported to be 0.199 mg/l geonmetric mean measured concentration.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.225 mg/L

EC10 or NOEC for freshwater algae:

0.199 mg/L