diethyl 1,4-cyclohexanedicarboxylate

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:

(Range-finding study) 13 August 1999 to 16 August 1999 (Definitive study) 4th October 1999 to 7th October 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: March 23rd 1998 Date of signature: July 21st 1998

Specific details on test material used for the study:

No additional information

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 6.25, 12.5, 25, 50 and 100 mg/l (nominal concentration).

- Sampling method: Replicates pooled at 0 and 72 hours.

- Sample storage conditions before analysis: Dupl icate samples were taken at each occasion and stored frozen (approximately -20ºC) for further
analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Test material was prepared by direct solution in culture medium.
An amount of test material (100 mg) was dissolved in culture medium with the aid of ultrasonication (approximately 15 minutes) and the volume adjusted to 500 ml to give a 200 mgll stock solution from which dilutions were made to give 100, 50, 25 and 12.5 mg/l stock solutions. An aliquot (250 ml) of each stock solution was separately mixed with algal suspension (250 ml) to give the test concentrations of 6.25, 12.5, 25, 50 and 100 mg/l.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green Algae
- Strain: CCAP 276/20
- Source (laboratory, culture collection):
Liquid cultures of Scenedesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Institute of Freshwater Ecology, The Ferry House, Far Sawrey, Ambleside, Cumbria, United Kingdom.
- Method of cultivation:
Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and continuous illumination at 21 ± 1ºC.
- Culturing media and conditions (same as test or not):
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Hardness:

Not reported.

Test temperature:

24 ± 1ºC.

pH:

7.5 (0-h), 7.5 - 8.4 (72-h)

Dissolved oxygen:

Not reporded.

Salinity:

Not applicable.

Nominal and measured concentrations:

(nominal concentrations)
Range-finding test:
1.0, 10 and 100 mg/l.

Definitive test:
6.25, 12.5, 25, 50 and 100 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flasks
- Type (delete if not applicable): closed.
- Material, size, headspace, fill volume: 250 ml glass conical flasks.
- Aeration: No.
- No. of organisms per vessel: 1E04 cells per ml prior to use.
- No. of vessels per concentration (replicates): 3.
- No. of vessels per control (replicates): 3.

GROWTH MEDIUM/TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis purified deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP)
Stock Solutions
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water. The prepared media was sterilised by 0.2 μm membrane filtration and stored in darkness.


OTHER TEST CONDITIONS
- Adjustment of pH: The pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Photoperiod: Continuous illumination for 72 hours.
- Light intensity and quality: intensity approximately 7000 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisiser II Particle Counter.


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: (nominal concentration) 1.0, 10 and 100 mg/l.
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentrations of 1.0 and
10 mg/l. However, growth was observed to be reduced at 100 mg/l.
Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100 mg/I were selected for the definitive study.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

58 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL of 50 - 68 mg/l

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

86 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

not specified

Remarks:

growth rate and biomass

Details on results:

All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 6.25, 12.5, 25 and 50 mg/l. However cell debris was observed in the 100 mg/l test cultures.

It is clear that both the growth and the biomass of Scenedesmus subspicatus (CCAP 276/20) were affected by the presence of the test material over the 72 hour exposure period.

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

- Observation of abnormalities (for algal test): There were no abnormalities detected.

- Any observations that might cause a difference between measured and nominal values: The pH values of the controls were observed to increase from pH 7.5 at 0 hours to pH 7.7 - 7.9 at 72 hours. This effect is considered to be due to the large number of cells in the log phase of growth respiring oxygen and producing carbonates and bicarbonates as part of photosynthesis/respiration which in solution give rise to alkaline conditions. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours therefore was within the limits given in the Test Guidelines.

- Verification of test concentrations: Analysis of the test solutions at 0 and 72 hours showed the measured test concentrations to range from 90% to 111% of nominal test concentration and so it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations only.

Results with reference substance (positive control):

No applicable.

Reported statistics and error estimates:

There were no statistically significant differences between the control and 6.25, 12.5 and 25 mg/l test concentrations (P>= 0.05), however all other test concentrations were significantly different (P<0.05).

Please refer to Attachment for tables and associated graphs of cell densities and percentage inhibition

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Scenedesmus subspicatus has been investigated over a 72 hour period and gave an EbC50 (72 h) value of 58 mg/I and an ErC50 (0 - 72 h) value of 86 mg/l. The No Observed Effect Concentration at 72 hours was 25 mg/l.

Executive summary:

Methods

A study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus.The method followed that described in the OECD

Guidelines for Testing of Chemicals (1984) No 201, "Alga, Growth Inhibition Test"

referenced as Method C.3 of Commission Directive92/69/EEC (which constitutes Annex

V of Council Directive 67/548/EEC).

 

Procedure

Following a preliminary range-finding study, Scenedesmus subspicatus was exposed to an aqueous solution of the test material at concentrations of 6.25, 12.5, 25, 50 and 100 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24±1ºC.

Samples of the algal populations were removed daily and cell concentrations determined

for each control and treatment group, using a Coulter®Multisizer II Particle Counter.

 

Results

Exposure of Scenedesmus subspicatus to the test material gave an EbC50(72 h) value of

58 mg/land an ErC50 (0 - 72 h) value of 86 mg/l. The No Observed Effect Concentration was 25 mg/l.

Analysis of the test solutions at 0 and 72 hours showed the measured test concentrations to range from 90% to 111% of nominal test concentrations; therefore, the results are based on nominal test concentrations alone.

Description of key information

OECD Guideline 201, EU Method C3, GLP, key study, validity 1:

72h-ErC50 (Scenedesmus subspicatus) = 86 mg/L based on analytically confirmed nominal test concentrations.

72h-NOEC for specific growth (Scenedesmus subspicatus) = 25 mg/L based on analytically confirmed nominal test concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

86 mg/L

EC10 or NOEC for freshwater algae:

25 mg/L

Additional information

Based on the results of one key study (SPL, 1999), performed according to OECD Guideline 201 and EU Method C3 with GLP statement, exposure of Scenedesmus subspicatus to the test material gave an ErC50 (0 - 72 h) value of 86 mg/L. The No Observed Effect Concentration (NOEC) was 25 mg/L.

Analysis of the test solutions at 0 and 72 hours showed the measured test concentrations ranged from 90% to 111% of nominal test concentrations and so the results are based on nominal test concentrations.