butyl 4-hydroxycyclohexanecarboxylate

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:

2015-01-07 to 2015-06-15

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:

(2011)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Tightly closed, protected from moisture at room tempereature
- Stability under test conditions: stable (see analytical verification)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 9.38, 18.8, 37.5, 75.0 and 150 mg/L
- Sampling method: Samples were taken at 0 h and 72 h of exposure. All test concentrations and the control were diluted (factor 2) with methanol containing 0.2% formic acid after sampling.
- Sample storage conditions before analysis: Samples were stored at room temperature.

Vehicle:

no

Details on test solutions:

Stock solutions (150 and 75.0 mg/L) were freshly prepared with dilution water. The stock solutions were stirred for 24 hours (approx. 1110 rpm) with a magnetic stirrer. Thereafter, the stock solutions were used to prepare 5 test itejm concentrations (9.38 - 18.8 - 37.5 - 75.0 - 150 mg/L), which have been used for the algae toxicity test.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Göttingen
- Age of inoculum (at test initiation): 4-day old preculture, prepared in dilution water, was used as binoculum

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca + Mg / L

Test temperature:

22.0 - 23.0°C

pH:

7.70 - 8.24

Nominal and measured concentrations:

The measured concentrations of the test item in the fresh media (0 h) were in the range of 105 to 109% of the nominal values at all tested concentration levels. The measured concentrations of the test item in the old media (72 h) were in the range of 95 to 106% of the nominal values of all concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks (250 mL) with cotton wool plugs
- Type: open
- Fill volume: 100 mL
- Initial cells density: 7085 cells/mL
- Control end cells density: 2307104 cells/mL
- 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: yes
- Adjustment of pH: no
- Photoperiod: 24 hours
- Light intensity and quality: 60 - 120 µE x m-2 x s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell density was measured daily via Chlorophyll-a-fluorescence excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found in the definitive test at the test concentration of 150 mg/L at the start of exposure


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: A range finding study was performed with nominal test item concentrations of 1.00 - 10.0 and 100 mg/L. In this non-GLP study, a hrowth rate inhibition of 49% was observed in the highest dose.
- Test concentrations: 9.38 - 18.8 - 37.5 - 75.0 - 150 mg/L

Reference substance (positive control):

yes

Remarks:

tested in a separate study

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 150 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

37.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

71.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no morphological abnormalities at the start of incubation
- Unusual cell shape: At the end of the incubation period, cells in the 2 highest concentrations were rounder.
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No

Results with reference substance (positive control):

The toxicity of potassium dichromate to Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2014-10-14 to 2014-10-17. The reference item toxicity was in the valid range following SOPs from the test facility: ErC50 = 0.749 mg/L

Reported statistics and error estimates:

EC50-values of growth rate after 72 hours was calculated by sigmoidal dose-response regression.
NOEC/LOEC were determined by calculation of statistical significance of growth rate. As standard One Way Analysis of Variance (ANOVA) and DUNNETT's test were used for NOEC/ÖLOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05.

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to principles of OECD 201 and Council Regulation (EC) 440/2008/Method C.3.

The study was conducted under static conditions with an initial cell density of 7085 cells/mL. Five concentrations were tested in a geometrical series with a dilution factor of 2 (nominal): 9.38 - 18.8 - 37.5 - 75.0 - 150 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within acceptable limits.

The concentrations of the test item were analytically verified by LC-MS/MSat the start and at the end of the exposure of all test concentrations and the control. The measured concentrations of the test item at the start of exposure (0 hours) were in the range of 105 to 109% of the nominal values. The measured concentrations of the test item at the end of exposure (72 hours) were in the range of 95 to 106% of the nominal value. Therefore, all effect values are based on the nominal test item concentrations.

The results are provided in the following:

ErC50: > 150 mg/L

NOEC: 37.5 mg/L

LOEC: 75.0 mg/L

Cell densities and growth rate inhibition are summarized in the follwoing table.

Nom. Conc.	Cell Density 0 hours [cells /mL]	Cell Density 72 hours [cells /mL]	Inhibition of Growth rate
150 mg/L	7085	427678	29 %
75.0 mg/L	7085	1101257	13%
37.5 mg/L	7085	2312925	0%
18.8 mg/L	7085	2391365	-1%
9.38 mg/L	7085	2267020	0%
Control	7085	2307104	--

Validity criteria fulfilled:

yes

Conclusions:

ErC50 > 150 mg/L

Executive summary:

see above

Description of key information

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to principles of OECD 201 and Council Regulation (EC) 440/2008/Method C.3.

The study was conducted under static conditions with an initial cell density of 7085 cells/mL. Five concentrations were tested in a geometrical series with a dilution factor of 2 (nominal): 9.38 - 18.8 - 37.5 - 75.0 - 150 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within acceptable limits.

The concentrations of the test item were analytically verified by LC-MS/MS at the start and at the end of the exposure of all test concentrations and the control. The measured concentrations of the test item at the start of exposure (0 hours) were in the range of 105 to 109% of the nominal values. The measured concentrations of the test item at the end of exposure (72 hours) were in the range of 95 to 106% of the nominal value. Therefore, all effect values are based on the nominal test item concentrations.

In this study, the ErC50 value exceeded the highest concentration tested, i.e. 150 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

150 mg/L

EC10 or NOEC for freshwater algae:

71.4 mg/L