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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Range-finding study: 23 September 1997- 29 September 1997. Definitive study: 17 October 1997- 20 October 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Gel All DX
- Substance type: whote powder
- Physical state: solid
- Lot/batch No.: 4073003
- Storage condition of test material: room tempersture

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Physico-chemical measurements
The pH of each control, solvent control and test flask was determined at initiation of the study and after 72-h exposure. The temperature within the incubator was recorded daily.

Verification of test concentrations
Water samples were taken from the solvent control and the 0.10 mg/L test group (replicates R1 - R3 and R4 - R6 pooled) at 0 and 72 h for quantitative analysis.

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
For the purpose of the definitive study the test material was prepared using a preliminary solution in dimethylformamide.

An amount of test material was dispersed in auxiliary solvent and the volume adjusted to 100 mL to give a 100 mg/100 mL solvent stock solution. An aliquot (200 µl) of this solvent stock solution was dispersed in algal suspension (2 L) to give the test concentration of 0.10 mg/L.

The concentration and stability of the test material in the test solutions were verified by chemical analysis at 0 and 72 h.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Centre for Algae and Protozoa (CCAP), Institute of freshwater Ecology, Ferry House, Ambleside, Cumbria

ACCLIMATION
- Culturing media and conditions (same as test or not): the culture medium used for both the range-finding and definitive studies was the same as that used to maintain the stock culture.
- The culture was maintained in the laboratory at a temperautre of 21 ± 1 °C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Test temperature:
21 ± 1 °C
pH:
8.0- 9.9
Nominal and measured concentrations:
Range-finding study: nominal test concentrations of 0.010, 0.040 and 0.10 mg/L
Definitive test: nominal test concentration of 0.10 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: closed; the flasks were covered with aluminium foil
- No. of organisms per vessel: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 1.54 x 107 cells per mL. This suspension was diluted to a cell density of 1.22 x 104 cells per mL prior to use. At initiation of the study the culture contained a nominal cell density of 104 cells per mL.
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 3
- No. of vessels per solvent control (replicates): 3

OTHER TEST CONDITIONS
- Light intensity and quality: continuous illumination (intensity approximately 7000 lux)
- Flasks were incubated at 24 ± 1 °C and constantly shaken at 100 rpm

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 h and the cell densities determined by direct counting with the aid of a Coulter® Multisizer II particle counter.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: nominal test concentrations of 0.010, 0.040 and 0.10 mg/L
- Test vessel: 250 mL glass conical flask
- Type : closed; loosely covered with aluminium foil to reduce evaporation
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Results used to determine the conditions for the definitive study: The test material was dissolved in dimethylformamide. An amount of test material was dispersed in auxiliary solvent and the volume adjusted to 50 mL to give a 125 mg/50 mL solvent stock solution from which serial dilutions were made to give 1.0, 4.0 and 10 mg/10 mL solvent stock solutions. Aliquots of the solvent stock solutions were separately dispersed in algal suspension to give the test concentrations. The control and the solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 µL/L of dimethylformamide. At the start of the range-finding study a sample of each test and control culture was removed and the cell density using a Coulter® Multisizer II particle counter. The flasks were then covered with aluminium foil and incubated at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at 100 rpm for 72 h. After 72 h the cell density of each flask was determined.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.08 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: results based on the time-weighted mean measured test concentration
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.08 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: results based on the time-weighted mean measured test concentration
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.08 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: results based on the time-weighted mean measured test concentration
Details on results:
RANGE-FINDING STUDY
- Any stimulation of growth found in any treatment: There was no significant effect on growth at all concentrations tested.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: During the preliminary solubility work performed a precipitate of test material was observed (by visual inspection of the prepared test media) at concentrations in excess of 0.10 mg/L thereby indicating this to be the maximum limit of water solubility of the test material under these test conditions. Based on this information a single test concentration of six replicates, of 0.10 mg/L was selected for the definitive study.

DEFINITIVE STUDY
- Growth data: neither the growth or the biomass of Scenedesmus subspicatus (CCAP 276120) were affected by the presence of the test material over the 72-hexposure period.
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 h. There were no abnormalities detected in any of the control or test cultures.
Reported statistics and error estimates:
Statistical analysis of the area under the growth curve data was carried out for the solvent control and 0.10 mg/L test group using a Students t-test. There were no statistically significant decreases in the area under the growth curve data (P ≥ 0.05), between the solvent control and 0.10 mg/L test group.

Any other information on results incl. tables

Growth data

The test concentration of 0.1 0 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guideline. Dimethylformamide was found to give the best testable dispersion of the test material in water. At higher test concentrations there was a marked precipitation of the test material on addition of the solvent stock solution to water.

The following data show that the cell concentration of the control cultures increased by a factor of 21 and the cell concentration of the solvent control cultures increased by a factor of 18 during the test in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 h:

Mean cell density of control at 0 h: 1.08 x 104cells/mL

Mean cell density of control at 72 h: 2.29 x l05cells/mL

Mean cell density of solvent control at 0 h: 1.03 x l04cells/mL

Mean cell density of solvent control at 72 h: 1.84 x l05cells/mL

Physico-chemical measurements

The pH values of the control cultures were observed to increase from pH 8.0 at 0 h to pH 9.4 - 9.7 at 72 h. 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 slightly higher than that specified in the Test Guideline (1.5 pH unit deviation after 72 h). This is considered not to affect the integrity of the study given that the validation criterion for control cell multiplication was satisfied.

Verification of test concentrations

Chemical analysis of the test preparations at 0 h showed the measured test concentrations to be in excess of the required 80 % of nominal. Analysis of the test preparations at 72 h showed a marked decline in measured test concentrations to 65 % to 66 % of nominal.

Given that pre-study stability analysis showed the test material to be stable in the culture medium used over the study period, the decline in measured test concentrations is considered to be due to adsorption to the algal cells. Adsorption was not a factor in the pre-study stability analyses as no algal cells were present.

In order to give a worst case analysis of the data, it was considered justifiable to base the results on the time-weighted mean measured test concentration.

Cell densities and pH values in the definitive study

Nominal Concentration (mg/L)

pH

Cell densities * (cells/ mL)

pH

0 h

0 h

24 h

48 h

72 h

72 h

Control

R1

8.0

9.59 x 103

1.36 x 104

3.64 x 104

1.81 x 105

9.4

R2

8.0

1.01 x 104

1.35 x 104

3.71 x 104

2.21 x 105

9.5

R3

8.0

1.28 x 104

1.25 x 104

5.25 x 104

2.85 x 105

9.6

Mean

8.0

1.08 x 104

1.32 x 104

4.20 x 104

2.29 x 105

 

Solvent Control

R1

8.0

1.02 x 104

9.51 x 103

3.16 x 104

2.60 x 105

9.6

R2

8.0

1.27 x 104

1.25 x 104

4.07 x 104

1.72 x 105

9.5

R3

8.0

8.11 x 103

1.77 x 104

4.38 x 104

1.19 x 105

9.7

Mean

8.0

1.03 x 104

1.32 x 104

3.87 x 104

1.84 x 105

 

0.10

R1

8.0

9.60 x 103

1.32 x 104

5.20 x 104

2.63 x 105

9.3

R2

8.0

1.10 x 104

9.91 x 104

4.05 x 104

2.24 x 105

9.4

R3

8.0

1.01 x 104

1.26 x 104

3.60 x 104

2.82 x 105

9.5

R4

8.0

1.02 x 104

8.86 x 103

4.14 x 104

2.34 x 105

9.9

R5

8.0

1.09 x 104

1.11 x 104

4.90 x 104

2.48 x 105

9.5

R6

8.0

9.93 x 103

1.08 x 104

4.36 x 104

2.16 x 105

8.7

Mean

8.0

1.03 x 104

1.11 x 104

4.38 x 104

2.44 x 105

 

R1- R6= Replicates 1-6; *cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

Inhibition of growth

Nominal Concentration (mg/L)

Area under curve at 72 h

% inhibition

Growth rate (0- 72 h)

% inhibition

Control

3.42 x 106

-

0.042

-

Solvent

2.83 x 106

-

0.040

-

0.10

3.63 x 106

[28]

0.044

[10]

[increase in growth as compared to solvent control]

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the growth of Scenedesmus subspicatus has been investigated and gave EC50 values of greater than 0.10 mg/l. Correspondingly the No Observed Effect Concentration was greater than or equal to 0.10 mg/l.

The EC50 values based on the time-weighted mean measured test concentration were greater than 0.080 mg/l, and correspondingly the No Observed Effect Concentration was greater than or equal to 0.080 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 No. 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC.

Procedure

Following a preliminary range-finding study, Scenedesmus subspicatus was exposed to an aqueous dispersion of the test material at a concentration of 0.10 mg/l (six replicate flasks) for 72 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

Exposure of Scenedesmus subspicatus to the test material gave EC50 values of greater than 0.1 mg/l and correspondingly the No Observed Effect Concentration was greater than or equal to 0.10 mg/l.

The test concentration of 0.10 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the test under the OECD Guidelines.

Analysis of the test solutions at 0 and 72 h showed a marked decline in test concentrations and so it was considered justifiable to base the results on the time-weighted mean measured test concentration also. The EC50, values, based on the time-weighted mean measured test concentration were > 0.080 mg/l and correspondingly the No Observed Effect Concentration was ≥ 0.080 mg/l.