Cyclohexanethiol

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:

27th July 2009 to 14th August 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline, in compliance with GLP and analytical monitoring was carried out.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexyl Mercaptan

IUPAC Name:

Cyclohexyl Mercaptan

Constituent 2

Reference substance name:

Cyclohexanethiol

EC Number:

216-378-7

EC Name:

Cyclohexanethiol

Cas Number:

1569-69-3

Molecular formula:

C6H12S

IUPAC Name:

cyclohexanethiol

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Cyclohexyl Mercaptan

- Physical state: Clear colourless liquid with no particulate matter

- Analytical purity: 99.85 wt %

- Impurities (identity and concentrations):
Cyclohexene: <0.01 wt %
Heavies: 0.10 wt %
Mercapton sulpher: 27.39 wt %
Dissolved gasses: <0.0500 wt %

- Purity test date: 29/06/2009

- Lot/batch No.: 2009-06-1550

- Expiration date of the lot/batch: Undetermined

- Stability under test conditions: Volatile

- Storage condition of test material: Room temperature in the dark

- Other: Negligable solubility in water

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.33, 1.1, 3.3, 11 and 33 mg/l

- Sampling method: samples were taken at 0 and 72 h.

- Sample storage conditions before analysis: stored in the dark. Duplicate samples were taken at 0 and 72 h and stored at -20 degrees C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 25 mg of test material was dispersed in 500 ml of culture medium in sealed vessels with minimal headspace to give initial test material dispersions of 50 mg/l, which were then shaken at 300 rpm at a temperature of 30°C for 24 hours. Undissolved test material was removed by filtration through a 0.2 µm Gelman Acrocap filter to give a saturated solution with a 0-hour measured concentration of 21 mg/l. This saturated solution was further diluted to give stock solutions of 7.6, 2.6, 0.74 and 0.23 mg/l. An aliquot (1800 ml) of each of the stock solutions was innoculated with 12.5 ml of algal suspension to give the test concentrations of 0.23, 0.74, 2.6, 7.6 and 21 mg/l. All were inverted several times to ensure adequate mixing and homogeneity.

- Controls: Culture medium

- Evidence of undissolved material (e.g. precipitate, surface film, etc): Stock solution was filtered to remove any undissolved test material.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus

- Strain: CCAP 276/20

- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. Master cultures maintained in laboratory by periodic replenishment of culture medium.

- Age of inoculum (at test initiation): Not reported

- Method of cultivation: Not reported

ACCLIMATION
- Acclimation period: Master cultures maintained in laboratory under constant aeration and constant illumination at 21 ± 1°C

- Culturing media and conditions (same as test or not): Same culture medium used during test as that used to maintain the stock culture, except for the addition of 500 mg of sodium bicarbonate.

- Any deformed or abnormal cells observed: Not specified

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ± 1°C

pH:

0 hours: 8
72 hours: Range 8 to 9.5

Dissolved oxygen:

Not measured

Nominal and measured concentrations:

Nominal
0.33, 1.1, 3.3, 11, 33 mg/l

Measured: (Stability Vessel)
0 hour: 0.225, 0.740, 2.58, 7.55, 20.7 mg/l
72 hour: 0.0887, 0.540, 1.95, 6.39, 18.5 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml Conical flask
- Type (delete if not applicable): closed using ground glass stopper
- Material, size, headspace, fill volume: Glass, minimal headspace, completely filled
- Aeration: None
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): No renewal
- Initial cells density: 4 x 10³ cells per ml
- Control end cells density: mean 3.29E+05 cells per 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: reverse osmosis purified deionised water

- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: Yes - addition of 500 mg of sodium bicarbonate
- Photoperiod: Continuous illumination
- Light intensity and quality: 7000 lux, warm white light (380-730 nm)
- Other: constantly shaken at 150 rpm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (Coulter Multisizer Particle Counter), counting cell density, at 0, 24, 48 and 72 hours

- Other: pH measured

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3

- Range finding study
- Test concentrations: 0.0033, 0.033, 0.33, 3.3, 33 mg/l
- Results used to determine the conditions for the definitive study: Range finding results showed no effect on algae growth at test concentrations of 0.0033, 0.033 and 0.33 mg/l. Growth was reduced at concentrations 3.3 and 33 mg/l.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.43 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: Not possible to calculate 95% CL

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

- Exponential growth in the control (for algal test): yes

- Any stimulation of growth found in any treatment: yes, at 0.15 mg/l treatment

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Decline in measured concentrations due to volatility of test material

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: ErC₅₀ (0-72 hrs): 0.79 mg/l

Reported statistics and error estimates:

One way analysis of variance and Dunnett's multiple comparison procedure
SAS computer software package (SAS 1999-2001)

Any other information on results incl. tables

Table 1. Cell Densities in the Definitive Test

Measured Concentration (mg/l)	Cell Densities (cells per ml)
	0 h	24 h	48 h	72 h
Control	4.49E+03	1.47E+04	4.44E+04	3.29E+05
0.15	4.51E+03	1.61E+04	5.88E+04	5.89E+05
0.59	4.26E+03	6.00E+03	1.24E+04	1.44E+04
2.3	4.16E+03	5.62E+03	6.50E+03	6.91E+03
6.6	4.34E+03	4.14E+03	6.15E+03	5.50E+03
18	4.03E+03	4.08E+03	5.36E+03	6.41E+03

  Table 2. Inhibition of Growth Rate and Yield in the Definitive Test

Measured Concentration (mg/l)	Growth Rate (cells/ml/hour)		Yield (cells/ml)
	0 -72 h	% Inhibition	0 -72 h	% Inhibition
Control	0.061		3.25E+05
0.15	0.069	[14]	5.85E+05	[80]
0.59	0.018	71	1.02E+04	97
2.3	0.007	88	2.76E+03	99
6.6	0.004	93	1.17E+03	100
18	0.006	90	2.39E+03	99

[ ] increase in growth as compared to controls

Table 3. Verification of Test Conditions - Measured Concentrations

Sample	Nominal Concentration (mg/l)	Concentration Found (mg/l)	Expressed as a Percent of the Nominal Concentration (%)
0 hours	Control	< LOQ	-
	0.33	0.255	68
	1.1	0.740	67
	3.3	2.58	78
	11	7.55	69
	33	20.7	63
72 hours Open Vessel	Control	< LOQ	-
	0.33	0.0954	29
	1.1	0.475	43
	3.3	2.03	62
	11	5.80	53
	33	16.3	49
72 hours Unopened Vessel	Control	0.0308*	-
	0.33	0.0887	27
	1.1	0.540	49
	3.3	1.95	59
	11	6.39	58
	33	18.5	56

* Possible post sampling contamination

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

A 72-hour EC50 value of 0.43 mg/L and NOEC of 0.15 mg/L have been determined for the effects of the test substance on growth rate of Pseudokirchnerella subcapitata. The results are expressed relative to mean measured concentration.