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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998-05-19 to 1998-05-22 (definitive study) and 1997-12-19 to 1997-12-22 (range-finding study)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1984
Deviations:
yes
Remarks:
pH deviation in control cultures > 1.5 pH units but increase in cell concentration > factor 16
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
1992
Deviations:
yes
Remarks:
see above
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Concentrations: solvent control (0 mg/L) and 4.0 mg/L; stock solution
- Sampling method: Water samples were taken at 0 and 72 hours for quantitative analysis.
- Sample storage conditions before analysis: no (analysis was done immediately after preparation)

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Dissolving of 2 g test material in dimethylformamide; volume adjusted to 50 mL to give a 2 g/50 mL solvent stock solution. An aliquot (100 µL) of this solvent stock solution was dispersed in algal suspension (1 litre) to give the required test concentration of 4.0 mg/L.
- Pre-study recovery analysis showed that the test material was highly unstable in aqueous media. Therefore the test media for replicates R1-R3 and R4-R6 were each prepared separately from the solvent stock solution, a sample of which was extracted for chemcial analysis immediately upon preparation in order to minimise the loss of test material due to hydrolysis.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: CCAP 276/20
- Source: Culture Centre for Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria
- Method of cultivation: Culture maintenance by periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 21 ± 1 "C under continuous illumination (intensity approximately 7000 lux) and constant aeration.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
24 ± 1°C
pH:
pH 7.5 (0 h) to pH 9.0-10.6 (72 h)
Nominal and measured concentrations:
nominal concentration: 4 mg/L
measured concentration (at 0 hours): 2.99 mg/L (replicates R1-R3), 3.14 mg/L (replicates R4-R6)
measured concentration (at 72 hours): 0.648 mg/L (replicates R1-R3), 0.579 mg/L (replicates R4-R6)
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flasks
- Type: closed (covered with alumium foil)
- Material, size, fill volume: glass, 250 mL, 100 mL
- Initial cells density: 10E+4 cells/mL
- Control end cells density: 2.70 x 10E+5 cells/mL (control), 2.37 x 10E+5 cells/mL (solvent control)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: yes

TEST MEDIUM / WATER PARAMETERS
- According to OECD 201 (AAP-medium)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 7000 lux


EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter (at 0, 24, 48 and 72 hours)

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.40 and 4.0 mg/L
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at either concentration tested. During preliminary solubility work precipitation of test material was observed (by visual inspection) at concentrations in excess of 4.0 mg/l indicating this to be the highest test concentration that could be prepared under these test conditions. Based on this information a single test concentration of six replicates, of 4.0 mg/l was selected for the definitive study.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 4 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.:
>= 1.5 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.5 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: yes
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Student t-test was carried out on the area under the growth curve data at 72 h for the solvent control, the 4.0 mg/L test concentration.
There were no statistically significant differences (with P equal to or grater than 0.05), between the solvent control and the 4.0 mg/L test group.
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the growth of Scenedesmus subspicatus has been investigated and gave 72-hour EC50 values of greater than 4.0 mg/L. Correspondingly the No Observed Effect Concentration was greater than or equal to 4.0 mg/L.
The EC50 values based on the time-weighted mean measured test concentrations were greater than 1.5 mg/L, and correspondingly the No Observed Effect Concentration was greater than or equal to 1.5 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 Directive 92/69/EEC.

Procedure:

Following a preliminary range-finging study, Scenedesmus subspicatus was exposed to an aqueous dispersion of the test material at a concentration of 4.0 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 using a Coulter ® Multisizer II Particle Counter.

Results:

Exposure of Scenedesmus subspicatus to the test material gave EC50 values greater than 4.0 mg/L and correspondingly the No Observed Effect Concentration was greater than or equal to 4.0 mg/L.

The test concentration 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.

Pre-study stability analyses performed showed the test material to be highly unstable in aqueous media, with hydrolysis occurring virtually instantaneously on contact with water. The degradation product was found to be insoluble in organic and aqueous solvents, negating the possibility of quantitative analysis of the degradant. Therefore all sovlent stock and test solutions prepared at 0 hours were extracted and analysed immediately after preparation.

Analysis of the test solutions at 0 hours showed the measured test concentrations to be 75% and 79% of nominal. There was a marked decline in measured test concentrations after 72 hours to 16% and 14% nominal.

These results show that despite immediate extraction and analysis of the 0 hour test sample, it was not possible to attain near nominal test concentrations due to the rapid hydrolysis of the test material, and that further hydrolysis occured over the study period. Therefore in order to give a worst case analysis of the data it was considered justifiable to base the results on the time-wighted mean measured test concentrations.

The EC50 values, based on the time-weighted mean measure test concentrations were greater than 1.5 mg/L and correspondingly the No Observed Effect Concentration was greater than or equal to 1.5 mg/L.

So, even at concentrations exceeding the water solubility of the test item, no effects were noted. Consequently, no effects are expected in the aquatic environment.

Finally, this acute toxicity test in algae represents a worst case as the solubility of TODI (and its degradation / hydrolysis product) in water was increased by an auxiliary solvent and the study result is based on time-weighted-mean measured concentrations which can also be regarded as a further overestimation as EC50 refers to a substance concentration which was reduced due to hydrolysis.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
GLP compliance:
not specified
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
Each water sample of 100 mL was removed from each test vessel at 0 hour. Equal volume of samples were removed from replicate test vessels and mixed for each exposure or control group to 100 mL at 72 hours.
Vehicle:
not specified
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 +/- 2°C
Nominal and measured concentrations:
nominal concentrations: 0.32, 0.56, 1.0, 1.8, 3.2, 5.6, 10, 18, 32 mg/L
measured concentrations: 0.267, 0.545, 0.903, 1.75, 3.13, 5,73, 9.55 and 17.6 mg/L (0 hour); 0.205, 0.371, 0.794, 1.39, 2.67, 5.12, 8.85 and 15.5 mg/L (72 hours)
Details on test conditions:
TEST SYSTEM
- Type: closed
- Fill volume: 100 mL
- Initial cells density: 1 x 10E+4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: 4000 - 5000 lx

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
Reference substance (positive control):
not specified
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4.6 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.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
4.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Reported statistics and error estimates:
Doudoroff method
Validity criteria fulfilled:
yes
Conclusions:
In this acute toxicity test with algae the 72-hour EC50 value was determined to be 4.6 mg/L, and the NOEC was determined as 1.0 mg/L based on growth rate.
Executive summary:

A study was performed to assess the acute toxicity of the degradation product ( 4,4'-bi-o-toluidine) to algae (Selenastrum capricornutum) as requested by ECHA. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 201.

Algae (10E+4 cells/mL) were exposed to the test material over a range of nominal concentrations of 0.32, 0.56, 1.0, 1.8, 3.2, 5.6, 10, 18 and 32 mg/L for a period of 72 hours under semi-static test conditions.

 

For analytical monitoring, each water sample of 100 mL was removed from each test vessel at 0 hour. Equal volume of samples were removed from replicate test vessels and mixed for each exposure or control group to 100 mL at 72 hours. Immediately the sample was analyzed by HPLC method. The recovery rate was determined as followed:103.0, 97.5, 99.5% (average: 100.0%) at 0.2 mg/L in media; 98.3, 99.0, 92.5% (average: 96.6%) at 40 mg/L in media.Temperatures of the test solution and pH values were measured at 24 hour intervals.

 

In this acute toxicity test with algae the 72-hour EC50 value was determined to be 4.6 mg/L, and the NOEC was determined as 1.0 mg/L based on growth rate.

 

Description of key information

TODI was assessed in aquatic toxicity test to algae according EU-method C.3 and OECD guideline 201. The 72-hour EC50 value (nominal) was greater than 4.0 mg/L and correspondingly the No Observed Effect Concentration was greater than or equal to 4.0 mg/L. The 72-hour EC50 value based on the time-weighted mean measure test concentrations was greater than 1.5 mg/L and correspondingly the No Observed Effect Concentration was greater than or equal to 1.5 mg/L.
So, even at concentrations exceeding the water solubility of the test item, no effects were noted. Consequently, no effects are expected in the aquatic environment.

 

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
1.5 mg/L
EC10, LC10 or NOEC for freshwater algae:
1.5 mg/L

Additional information

Key study

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 Directive 92/69/EEC.

Procedure:

Following a preliminary range-finging study, Scenedesmus subspicatus was exposed to an aqueous dispersion of the test material at a concentration of 4.0 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 using a Coulter® Multisizer II Particle Counter.

Results:

Exposure of Scenedesmus subspicatus to the test material gave EC50 values greater than 4.0 mg/L and correspondingly the No Observed Effect Concentration was greater than or equal to 4.0 mg/L.

The test concentration 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.

Pre-study stability analyses performed showed the test material to be highly unstable in aqueous media, with hydrolysis occurring virtually instantaneously on contact with water. The degradation product was found to be insoluble in organic and aqueous solvents, negating the possibility of quantitative analysis of the degradant. Therefore all sovlent stock and test solutions prepared at 0 hours were extracted and analysed immediately after preparation.

Analysis of the test solutions at 0 hours showed the measured test concentrations to be 75% and 79% of nominal. There was a marked decline in measured test concentrations after 72 hours to 16% and 14% nominal.

These results show that despite immediate extraction and analysis of the 0 hour test sample, it was not possible to attain near nominal test concentrations due to the rapid hydrolysis of the test material, and that further hydrolysis occured over the study period. Therefore in order to give a worst case analysis of the data it was considered justifiable to base the results on the time-wighted mean measured test concentrations.

The EC50 values, based on the time-weighted mean measure test concentrations were greater than 1.5 mg/L and correspondingly the No Observed Effect Concentration was greater than or equal to 1.5 mg/L.

So, even at concentrations exceeding the water solubility of the test item, no effects were noted. Consequently, no effects are expected in the aquatic environment.

Finally, this acute toxicity test in algae represents a worst case as the solubility of TODI (and its degradation / hydrolysis product) in water was increased by an auxiliary solvent and the study result is based on time-weighted-mean measured concentrations which can also be regarded as a further overestimation as EC50 refers to a substance concentration which was reduced due to hydrolysis.

Supporting study

A study was performed to assess the acute toxicity of TODI's degradation product ( 4,4'-bi-o-toluidine) to algae (Selenastrum capricornutum). The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 201.

Algae (10E+4 cells/mL) were exposed to the test material over a range of nominal concentrations of 0.32, 0.56, 1.0, 1.8, 3.2, 5.6, 10, 18 and 32 mg/L for a period of 72 hours under static test conditions.

 

For analytical monitoring, each water sample of 100 mL was removed from each test vessel at 0 hour. Equal volume of samples were removed from replicate test vessels and mixed for each exposure or control group to 100 mL at 72 hours. Immediately the sample was analyzed by HPLC method. The recovery rate was determined as followed:103.0, 97.5, 99.5% (average: 100.0%) at 0.2 mg/L in media; 98.3, 99.0, 92.5% (average: 96.6%) at 40 mg/L in media.Temperatures of the test solution and pH values were measured at 24 hour intervals.

 

In this acute toxicity test with algae the 72-hour EC50 value was determined to be 4.6 mg/L, and the NOEC was determined as 1.0 mg/L based on growth rate.