Bis(2-ethylhexyl) cyclohexane-1,4-dicarboxylate

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:

2009-04-30 to 2009-09-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Specific details on test material used for the study:

Batch No.: 20090226
Purity: 99.38%

Analytical monitoring:

yes

Details on sampling:

Test concentrations were verified by chemical analysis. Water samples were taken from the controls and each exposure level at the start (0h), before and after renewing of test solution (24 hours) and at the end (48 hours) of the test for analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water accommodated fraction (WAF)
- 100 mg of test substance was added to 1 L of OECD medium to prepare a nominal concentration of 100 mg/L
- This solution was homogenised with ultra-homogeniser for 30 mins and stirred for 48 hrs to ensure that the limit of solubility was reached.
- After 48 hrs the solution was centrifuged (approx. 20,000× g, 20 mins) to remove any undissolved test substance.
- No remarkable phenomenon was observed in the WAF.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: UTEX 1648
- Source: UTEX The Culture Collection of Algae, The University of Texas at Austin, Austin, Texas, United States
- Age of inoculum (at test initiation): Log phase growth
- Method of cultivation: In sterile OECD nutrient medium

ACCLIMATION
- Acclimation period:
- Culturing media and conditions: Same as test
- Any deformed or abnormal cells observed:

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

No data, however the contents of OECD culture medium were specified.

Test temperature:

22.2 ± 0.1 °C

pH:

7.99-8.20

Dissolved oxygen:

No data

Salinity:

No data, however the contents of OECD culture medium were specified.

Nominal and measured concentrations:

Nominal: 100 mg/L
Measured: 0.27 mg/L

Details on test conditions:

RANGE-FINDING STUDY
- Concentrations: 100 mg/L nominal water accommodate fraction (WAF); negative control.
- Cell density: 1.5 × 10³ cells/mL.
- No. of vessles per concentration (replicates): 1
- Exposure period: 72 hrs

TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Aeration: Shaking incubator, 100 cycles/min
- Initial cells density: 5.1 × 10³ cells/mL.
- Control end cells density: 5.1 × 10³ cells/mL.
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, OECD medium
- Detailed composition if non-standard medium was used: See attached supporting information.


OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: 80-87 µE/m²/s

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Direct counting using an optical microscope.
- Observation intervals:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: None; limit concentration used.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: No
- No sign of contamination by foreign algal cells or protozoa.

Reported statistics and error estimates:

No statistical treatment was done because the limit test was carried out with only one test concentration. The EC50 values were expressed as above the test concentration.

Validity

The following four validity criteria were met and therefore the test was considered valid:

Verification items	Criteria	Results
Increase of biomass in control cultures within the 72 hr test period	≥16×	113×
Mean coefficient of variation for section-by-section specific growth rate in the control cultures	≤ 35 %	26.6 %
Coefficient of variation of average specific growth rates in replicate control cultures during the whole test period	≤ 7 %	3.2 %
Change of pH in control medium during the test period	≤ 1.5	0.1

Validity criteria fulfilled:

yes

Conclusions:

The 72 h EC10, EC50 values expressed in terms of the mean measured concentration of the test substance was all determined as > 0.27 mg/L for avergae growth rate and yield, respectively.

Description of key information

A growth inhibition test to alga, Pseudokirchnerilla subcapita was conducted according to OECD 201.

72 hr EC10 > 0.27 mg/L, 72 hr EC50 > 0.27 mg/L (limit of solubility)

Additional information

The toxicity test was performed with a nominal concentration of 100 mg/L Water Accommodated Fractions (WAFs). The mean concentrations (geometric average) of the test substance measured at the nominal concentrations of 100 mg/L WAFs during the test was 0.27 mg/L. The 72 h EC10, EC50 expressed in terms of the mean measured concentration of the test item was all > 0.27 mg/L for average growth rate and yield, respectively.