Cyclohexanemethanol, 4-(1-methylethyl)-

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:

August 2, 2011 to October 16, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on July 19-21, 2011 / Signed on August 31, 2011

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Purity test date: March 21, 2012

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal - 0.625, 1.25, 2.5, 5.0 and 10 mg/L.
- Sampling method: Samples were taken from the control (replicates R1 – R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis.
- Sample storage conditions before analysis: -20 °C

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An amount of test item (550 mg) was dissolved in culture medium (11 litres) with the aid of prolonged stirring at approximately 1500 rpm for 24 hours to give a 50 mg/L stock solution.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): At the start of the test all control and test cultures were observed to be clear colourless solutions

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland - Master cultures were maintained in the laboratory by the periodic replenishment of culture medium.
- Method of cultivation: Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 1000 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10000-100000 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Hardness:

For the purposes of the definitive test, additional sodium bicarbonate (500 mg/L) was added to the prepared culture medium prior to use.

Test temperature:

24 ± 1 °C

pH:

The pH value of the control cultures was observed to increase from pH 8.0 at 0 hours to pH 9.2 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Dissolved oxygen:

not applicable

Salinity:

not applicable

Nominal and measured concentrations:

Nominal: 0 (control), 0.625, 1.25, 2.5, 5.0 and 10 mg/L.
Measured (0 hours): Measured (72 hours): Measured (72 hours, unopened vessel): Not Reported, 0.594, 1.23, 2.42, 5.14, 10.8

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, completely filled
- Aeration: no
- Initial cells density: 5000 cells/mL (nominal)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: NR
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter® Multisizer Particle Counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: X10
- Range finding study: yes - 0.10, 1.0, 10 and 100 mg/L
- Test concentrations: 0.625, 1.25, 2.5, 5.0 and 10 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

6.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL could not be calculated as the data did not fit model

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

4.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL = 5.4-6.7 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): Yes, factor of 94 by 72 hours
- Observation of abnormalities (for algal test): no abnormalities detected in control or test cultures
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start of the test all control and test cultures were observed to be clear colourless solutions
- Effect concentrations exceeding solubility of substance in test medium: At the start of the test all control and test cultures were observed to be clear colourless solutions

Results with reference substance (positive control):

- Results with reference substance valid: considered to be within normal range.
- EC50: 1.4 mg/L

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups.

Validity criteria fulfilled:

yes

Conclusions:

The 72 hour EC50 and EC10 for growth rate were determined to be 10 mg/L and 5.2 mg/L, respectively, based on nominal concentrations (measured concentrations were >80% to nominal).

Executive summary:

A study was performed, according to OECD Guideline 201 and EU Method C.3, to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter. The test item was suspected to be volatile and hence testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. Following the recommendations of published data (Herman et al 1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.

The 72 hour EC50 and EC10 for growth rate were determined to be 10 mg/L and 5.2 mg/L, respectively, based on nominal concentrations. Also, the 72h-NOEC for growth rate was determined at 2.5 mg/L, based on nominal concentrations. Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 95% to 117% of nominal. Given the potentially volatile nature of the test item, additional test vessels were prepared at the start of the test and incubated alongside the study remaining unopened for the duration of the test. Analysis of these samples at 72 hours showed measured test concentrations to range from 95% to 108% of nominal and as such the results are based on nominal test concentrations only.

Description of key information

The substance was toxic to Pseudokirchnerella subcapitata when tested according to OECD Guidelines for Testing of Chemicals (March 2006) No 201. The ErC50 and ErC10 observed after 72 hours were 10 mg/L and 5.2 mg/L (based on nominal concentrations, measured concentrations were >80% to nominal).

Key value for chemical safety assessment

EC50 for freshwater algae:

10 mg/L

EC10 or NOEC for freshwater algae:

5.2 mg/L

Additional information

The ability of the substance to inhibit the growth of Pseudokirchnerella subcapitata was determined (Harlan Laboratories, 2012). The GLP compliant study followed the OECD Guidelines for Testing of Chemicals No. 201. Potassium dichromate was used as the reference substance and reported a 72-hour EC50 based on growth rate of 1.4 mg/L. The test was performed in a closed system.  The validity criteria as outlined in OECD 201 were meet, the biomass in the control cultures increased exponentially by a factor of at least 16 within the 72-hour test period; the mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35%; and the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%. Based on the growth rate of P. subcapitata, the 72-hour ErC50 is 10 mg/L, the 72 -hour ErC10 is 5.2 mg/L, and the NOEC is 2.5 mg/L; based on nominal concentrations, measured concentrations were >80% to nominal.