Reaction products of alkenyl dihydrofuran-2,5-dione and polyisoalkenyl dihydro-2,5-furandione with alkylene glycol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1995-06-23 to 1995-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

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

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
See read-across justification attached in Section 13 of the IUCLID.

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Range finder: 0.1, 1.0, 10 and 100 mg/L
Definitive test: 100 mg/L
- Sampling method: Water samples were taken from the control and 100 mg/L test groups (replicates R1-R3 and R4-R6 pooled) at 0 and 96 hours for quantitative analysis.
- Sample storage conditions before analysis: room temperature and storage in both light and dark conditions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct dispersion in culture medium
100 mg of test material was dissolved in culture medium and the volume adjusted to 500 mL to give a 200 mg/L stock solution. This stock solution was mixed with 500 mL of algal suspension to give a test concentration of 100 mg/L.

Range finder: The stock solution (200 mg/L) was diluted to give 20, 2.0 and 0.20 mg/L stock solutions. Aliquots of each stock solution (100 mL) were mixed with 100 mL of algal solution to give test concentrations of 0.10, 1.0, 10 and 100 mg/L.

Filtration of the test concentrations during analysis resulted in a loss of test material. Test samples were not filtered prior to analysis.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Centre for Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria.
- Method of cultivation: Maintained in house by replenishment of culture medium at 21 ± 1°C and constant illumination (ca. 7000 lux) and constant aeration

ACCLIMATION
- Acclimation period: NO
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

Not stated

Test temperature:

24±1°C

pH:

9.9 - 10.2

Dissolved oxygen:

Not measured

Salinity:

Not applicable

Nominal and measured concentrations:

Range finder:
nominal: 0.10, 1.0, 10 and 100 mg/L.
Definitive:
nominal: 100 mg/L
measured: 63 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flask
- Type: closed
- Material, size, headspace, fill volume: 200 mL fill volume
- Aeration: no
- Initial cells density: nominal 10E04 cells/mL (definitive study mean = 1.07E04 cells/mL)
- Control end cells density: mean = 1.23E04 cells/mL
- No. of vessels per concentration (replicates): 6 (definitive study)
- No. of vessels per control (replicates): 3 (definitive study)

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Direct counting using a haemocytometer at 0, 24, 48, 72 and 96 hours
pH determined ay initiation of study and after 96 hours.
Temperature: every 3.2 mins


TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 63 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

96 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Range-finder
The results showed no effect on growth at all concentrations tested.
Based on this information a single test concentration of six replicates, of 100 mg/L was seleceted for the definitive study. This experimental design conforms to a limit test to confirm at the maximum test concentration given in the OECD/EEC test guidelines no effect on growth was observed.
Definitive study
Neither the growth or the biomass of Selenastrum caprornutum was affected by the presence of 100 mg/L of the test material, over the 96 hour exposure period.
Statistical analysis of the area under the growth curve data was carried out for the control and 100 mg/L test group using a Students t-test. There wer no statistically significant differences (P>= 0.05) between the control and 100 mg/L test groups and therefore the "NoObserved Effect Concentration" (NOEC) is given as >= 100 mg/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.
The cell concentration of the control cultures increased by a factor of 36 after 72 hours in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours, and by a factor of 182 after 96 hours in line with the EPA Guideline that states that the enhancement must be at lest by a factor of 100 after 96 hours.
Control mean cell density:
0 hours = 1.23E04 cells/mL
72 hours = 4.41E05 cells/mL
96 hours = 2.24E06 cells/mL

All test and control cultures were inspected microscopically at 96 hours. There were no abnormalities detected in any of the control or test cultures.
The pH values of the control and test cultures were observed to increase from pH 7.9 - 8.2 at 0 hours to pH 9.9 - 10.2 at 96 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.

Physico-chemical measurements
Temperature was maintained at 24 ± 1°C throughout the study, with the exception of the forst 35 minutes of the study period. During this time the temperature in the incubator was 21.9°C to 22.9°C, this initial low temperature is considered to be due to the incubator being opened to allow the test vessels to be placed inside, and hence reducing the temperature inside the incubator to that of the surrounding room.

Verification of test concentrations
Analysis of the test concentrations at 0 hours showed the mean measured concentration to be 80% of nominal. At 96 hours analysis of the test preparations showed a marked decline in the mean measured test concentration to 63% of nominal.
Since the pre-study stability analysis showed the test material to be stable over the study period the decline in measured concentrations is considered to be due to absorption by algal cells and/or glassware and deficiencies in the analytical method employed. The stability analysis did not indicate adsorption to glassware since the vessels were rinsed with extraction solvent. This was not performed on test vessels used in the study and may account for the decline in measured concentration.
The test preparations were not filtered prior to analysis due to losses of test material during the filtration process.
The low and variable results obtained during the recovery, stability and test solution analyses are also considered to be due to deficiencies in the analytical method and not due to losses during dosing. The method of analysis was modified for the purposes of quantifying the test material in aqueous media, however, method validation showed that this method gave low and variable recoveries. Attempts to improve the precision and accuracy of the method proved unsuccessful. Alternative methods were assessed, however, one could not be developed in-house.
The above factors must be taken into consideration when interpreting the results of the study.
In order to give a worst case analysis of the data it was considered justifiable to base the EC values on the 96-hour mean measured test concentration.

Results with reference substance (positive control):

Not performed

See tables and cell density plot attached

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test material to aquatic algae was assessed in accordance with OECD Guideline 201. The effect of the test material on the growth of Selenastrum capricornutum gave EC50 values > 100 mg/L. Correspondingly the No Observed Effect Concentration (NOEC) was greater than or equal to 100 mg/L. Based on mean measured test concentrations, the EC50 values were greater than 63 mg/L and the NOEC was greater than or equal to 63 mg/L.

Executive summary:

Introduction

A study was performed to assess the effect of the test material on the growth of the green alga Seelenastrum capricornutum. 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 Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC) and US Code of Federal Regulations, Title 40, Part 797, Section 1050.

Method

Following a preliminary range-finding study, Selenaastrum capricornutum was exposed to an aqueous dispersion of the test material at a concentration of 100 mg/L (six replicate flasks) for 96 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.

Results

The effect of the test material on the growth of Selenastrum capricornutum gave EC50 values > 100 mg/L. Correspondingly the No Observed Effect Concentration (NOEC) was greater than or equal to 100 mg/L.

It was considered unecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Analysis of test solutions at 0 and 96 hours showed a marked decline in test concentrations and so it was considered justifiable to base the results on the measured test concentrations also. Based on mean measured test concentrations, the EC50 values were greater than 63 mg/L and the NOEC was greater than or equal to 63 mg/L.

 

Description of key information

The toxicity of the test material to aquatic algae was assessed in accordance with OECD Guideline 201.  The effect of the test material on the growth of Selenastrum capricornutum gave EC50 values > 100 mg/L. Correspondingly the No Observed Effect Concentration (NOEC) was greater than or equal to 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The key study was performed on the read-across substance Ethylene glycol, reaction products with polyisobutenyl succinic anhydride and hexadecenyl succinic anhydride, salts with dimethylethanolamine. The justification for read-across is attached in Section 13 of the IUCLID.

A study was performed to assess the effect of the test material on the growth of the green alga Seelenastrum capricornutum. 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 Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC) and US Code of Federal Regulations, Title 40, Part 797, Section 1050.

Following a preliminary range-finding study, Selenaastrum capricornutum was exposed to an aqueous dispersion of the test material at a concentration of 100 mg/L (six replicate flasks) for 96 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.

The effect of the test material on the growth of Selenastrum capricornutum gave EC50 values > 100 mg/L. Correspondingly the No Observed Effect Concentration (NOEC) was greater than or equal to 100 mg/L.

It was considered unecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Analysis of test solutions at 0 and 96 hours showed a marked decline in test concentrations and so it was considered justifiable to base the results on the measured test concentrations also. Based on mean measured test concentrations, the EC50 values were greater than 63 mg/L and the NOEC was greater than or equal to 63 mg/L.