Dimethyl cyclohexane-1,4-dicarboxylate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted according to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl cyclohexane-1,4-dicarboxylate

EC Number:

202-347-5

EC Name:

Dimethyl cyclohexane-1,4-dicarboxylate

Cas Number:

94-60-0

Molecular formula:

C10H16O4

IUPAC Name:

1,4-dimethyl cyclohexane-1,4-dicarboxylate

Details on test material:

- Name of test material (as cited in study report): Dimethyl 1 ,4-cyclohexanedicarboxylate (DMCD)
- Physical state: liquid
- Analytical purity: 92.9%
- Isomers composition: mixed
- Lot/batch No.: 05-02

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Concentration determinations were made by analytical personnel at 0, 24, 48, and 72 hours of exposure to evaluate the test substance stability under test conditions.

Test solutions

Vehicle:

no

Details on test solutions:

The test substance (0.13 g) was weighed into a 1000-mL acid-washed, sterile glass Erlenmeyer flask and diluted to 1001.2 g with sterile algal growth media. The mixture was heated at approximately 50°C and sonicated for approximately 15 minutes to fully dissolve the test substance. The solution was then filtered through a sterile 0.45 um Nalgene membrane filter. The resulting nominal concentration of the solution was 129.8 mg/L. An aliquot (1.0 mL) of the test solutions was removed for analysis. The remaining solution was used to initiate the test. All test and control solutions were prepared under aseptic conditions (sterile hood) to avoid contamination of the exposure solutions.

Test organisms

Test organisms (species):

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

Details on test organisms:

The test alga, Selenastrum capricomutum SF-3148, was obtained from the American Type Culture Collection, Rockville, MD and was subsequently cultured in the testing facility. A 4-day culture in liquid algal medium was used as the source of test inoculum. Several previous passages were performed in order to confirm exponential growth under the conditions of the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

A mean temperature of 24C was maintained

pH:

The pH values for the test and control solutions ranged from 7.56 at test start to 7.88 at test end. The pH values of the flasks containing the test solutions did not deviate by more than 1.5 units.

Nominal and measured concentrations:

The nominal concentration of the initial solution was 129.8 mg/L. The exposure concentration was based on the geometric mean of the analyzed solutions at times 0-, 24-, 48-, and 72-hours, averaged over the three replicates. The mean analyzed value for the test substance exposure concentration was determined to be 124.6 mg/L.

Details on test conditions:

Sterile 250-mL Erlenmeyer flasks were conditioned by adding 1-2 mL of the appropriate test solution, swirling to coat the flask, and then removing the solution by inverting the flask into a waste beaker. A total of 8 flasks were prepared. Five flasks were conditioned for the test substance, of which 3 had algae, one served as the chemical control exposed to light, and one served as the chemical control protected from light. Three flasks were conditioned for the negative control using algal growth medium. A hemacytometer count was made to detennine the number of algal cells in the culture flask serving as the inoculum source for the test. Sterile 100-mL volumetric flasks were used to dispense the test substance solutions into the appropriate sets of conditioned flasks. Enough cells were inoculated into each flask containing the test substance solution and into the negative control flasks to give a cell concentration of 10e4 cells/mL. The flasks were secured with foam stoppers and then transferred to the incubator in random order where they were maintained at a temperature of approximately 24°C and shaken at approximately 100 rpm. A mean illumination of 741 ± 3.8 foot-candles was maintained during the test. Flasks were rotated randomly at 24-hour intervals following cell counts. Cell concentrations were measured for each flask at 24-hour intervals from test start. Flasks were swirled to achieve a uniform cell suspension and 4.0 mL were removed for counting. A hemacytometer count was performed to calibrate the Coulter Counter which was then used to make the cell counts.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 124.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 124.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 124.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 124.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The cell count results indicate that algae in the negative control flasks exhibited normal log growth, increasing from l x l04 cells/mL at test start to a mean of 1.29xl0e6 after 72 hours (test end).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

The inhibition of growth of the alga, Selenastrum capricomutum, by solutions of the test substance was determined in a 72-hour growth test. During the test, the algae were exposed in a limit test to one concentration of the test substance prepared in algal growth media over several algal generations. A geometric mean of 124.6 mg/L was calculated from analyzed values of the exposure solutions sampled throughout the test. Cell counts were made at 24-hour intervals and growth curves and graphs of concentration versus percent inhibition were plotted. The EbC50 (0-72hr) value was estimated to be >124.6 mg/L. The ErC50 (0-72hr) value was estimated to be >124.6 mg/L. The 72-hour NOEC value was estimated to be 124.6 mg/L.