Dec-9-en-1-ol

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:

14 January 2018 - 10 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd. 22 January 2018

Test material

Test material information

Constituent 1

Reference substance name:

Dec-9-en-1-ol

EC Number:

235-878-6

EC Name:

Dec-9-en-1-ol

Cas Number:

13019-22-2

Molecular formula:

C10H20O

IUPAC Name:

dec-9-en-1-ol

Test material form:

liquid

Details on test material:

- Physical appearance: clear colourless liquid
- Storage conditions: at room temperature, protected from light

Specific details on test material used for the study:

The test item is insoluble in water.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 h, t=24 h and t=72 h
Volume: 6.0 mL
Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration (i.e. 3.2% of SS) but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions were prepared at a loading rate of 100 mg/L applying an overnight period of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for 1 hour- 1 hour and 15 minutes. The aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
- Controls: Test medium without test item or other additives.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis)) 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 21-24°C, until the pre-culture started.

PRE-CULTURE
- 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Pre-culture medium as used in the Final test: Standard M2 medium, according to OECD 201 Guideline.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

21.1-23.5 °C

pH:

At the start of the test: 8.2-8.3
At the end of the test: 7.9-8.2

Nominal and measured concentrations:

Nominal concentrations: 0.32, 1.0, 3.2, 10 and 32% of a saturated solution prepared at 100 mg/L
Measured concentrations (TWA): 0.034, 0.23, 0.82, 0.81, 2.7 and 7.9 mg/L, respectively to nominal concentrations stated above.
The Time Weighted Average was calculated as the concentrations decreased below the limit of detection at the end of the exposure perio at all test concentrations. For measured concentrations per time point, see table 1.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL all glass, capped, vessels; fill volume: 50 mL
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 206 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- In addition: 1 or 2 replicates of each concentration without algae; 1 replicates of each test concentration for sampling purposes after 24 hours of exposure.
- Other: during incubation the algal cells were kept in suspension by continuous shaking.

GROWTH MEDIUM
- Medium used: M2 medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2; according to the OECD 201 Guideline, formulated using Milli-RO water.
- Intervals of water quality measurement: pH measured at beginning and end of test; temperature of medium continuously measured in a temperature control vessel.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuously using TLD-lamps with a light intensity of 88 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length =10 mm). Algal medium was used as blank.
- At the end of the final test microscopic observations were performed on solutions containing 3.2 and 10% of the SS to
observe for any abnormal appearance of the algae compared to the control.

LIMIT/RANGE-FINDING STUDY:
- Test concentrations: 0.10, 1.0, 10 and 100% of the SS prepared at 100 mg/L
- Results used to determine the conditions for the definitive study: yes, at concentrations of 10% and 100% of the SS, growth inhibition was 66% and 100%, respectively, while inhibition of growth was only 0.039% and 0.63% in the 0.10 and 1.0% SS test solutions, respectively. The EC50 for growth rate inhibition was therefore expected to be between a concentration of 1.0% and 10% of the SS.
- Test item analysis of the 1.0 and 10% SS concentrations showed that initial test concentrations were 0.87 and 7.2 mg/L, respectively and that these concentrations decreased to 1.3% and 0.6% of initial at the end of the test.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate (test performed January 2018).

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 3.4-3.8

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 1.6-1.9 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.82 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

Statistically significant

Effect conc.:

0.034 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a concentration of 0.28 and 2.7 mg/L (TWA) when compared to the control.

- All test concentrations were decreased below the limit of detection at the end of the test. The concentrations measured in samples with algae were comparable with the concentrations measured in the samples without algae. Based on these results, TWA concentrations were calculated.

- Growth rate inhibition was statistically significant at TWA concentrations of 0.23 mg/L and above but considered to be biologically not relevant (<10%) at TWA concentrations of 0.23 and 0.82 mg/L.
- The 72h-NOErC was 0.0.34 mg/L, based on statistical significance. Based on biological relevance (inhibition >10%), the 72h-NOErC was determined to be 0.82 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Tested concentrations: 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L
- 72-h ErC50: 0.86 mg/L; 95% CI: 0.84-0.88 mg/L
- Other: the results were within the historical range of the test facility.

Reported statistics and error estimates:

NOEC and EC50: Step-down Jonckheere-Terpstra test procedure, α=0.05, one-sided, smaller.
ECx values: probit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding TWA concentrations of the test item.

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Table 1 Measured concentrations in the final test

Time of sampling [hours]	Percentage of SS^a [%]	Analyzed concentration [mg/L]	Relative to initial [%]
0	0	< 0.01^d
	0.32	0.223
	1.0	0.71
	3.2	2.40
	3.2^b	2.46
	10	7.89
	32	23^c
24	0	< 0.01^d	n.a
	0.32	0.027	12
	1.0	0.506	72
	3.2	2.13	89
	3.2^b	2.33	95
	10	7.41	94
	32	22^c	99
72	0	< 0.01^d	n.a.
	0.32	< 0.01^d	n.a.
	1.0	< 0.01^d	n.a.
	3.2	< 0.01^d	n.a.
	3.2^b	< 0.01^d	n.a.
	10	< 0.01^d	n.a.
	32	< 0.04^d	n.a.

^a     Percentage of a saturated solution (SS) prepared at a loading rate of 100 mg/L; ^b     Without algae.; ^c     Estimated value, calculated by extrapolation of the calibration curve.; ^d    Response below the limit of detection (LOD) of the analytical method which was determined to be at 0.010 mg/L and 0.040 mg/L taking a dilution factor of 0.333 or 1.33 into account, respectively.

n.a. Not applicable.

Table 2 Growth rate and percentage inhibition for the total test period

Test item TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.776	0.0143	6
0.034	1.759	0.0039	3	0.93
0.23	1.739	0.0059	3	2.1^#
0.82	1.663	0.0490	3	6.4^#
2.7	1.254	0.0395	3	29*
7.9	0.129	0.1068	3	93*

* - effect was statistically significant,^#- effect was statistically significant but biologically not relevant (<10%)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of a toxicity study towards algae, performed according to OECD guideline 201 and GLP principles, the 72h-ErC and the 72h-ErC10 of Trepanol were determined to be 3.6 and 1.8 mg/L, respectively (based on TWA concentrations). The statistically significant 72h-NOErC was 0.034 mg/L (based on TWA concentrations). The biologically relevant 72h-NOErC was 0.82 mg/L (based on TWA concentrations).