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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 23 to June 24, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 201 and the EC Method C.3 with GLP statement. All validity criteria were fulfilled.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on April 12, 2005/ signed on June 01, 2005)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The aqueous samples were diluted with methanol and/or water to bring the expected analyte concentration within the calibration range of 0 – 5.3 mg/L.
- Sampling method: Samples of 100 mL of media were taken from the control and test vessels at 0 and 72 hours for analysis.
- Sample storage conditions before analysis: Sample were analyzed within three days of sampling and /or stored in a refrigerator in case further analysis was required.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test media were prepared from an aqueous preparation in which the test substance (200 mg) was added directly to algal culture medium (2 L) in a volumetric flask. The content of the flask was shaken vigorously and treated with ultrasound for 30 minutes, with periods of intermittent vigorous shaking every five minutes. After ultrasound treatment, the medium was stirred for approximately one hour in darkness before this primary aqueous dilution (100 mg/L) was either used directly, or diluted with sterile culture medium to provide the media at the remaining lower test concentrations. An aliquot (2.21 mL) of the secondary algal inoculum was added to a portion (600 mL) of each test medium. An aliquot (100 mL) of the appropriate inoculated test medium was added to each of the test vessels.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain No: CCAP 278/4.
- Source: Culture Collection of Algae and Protozoa, Institute of Freshwater Ecology, Cumbria, UK
- Method of cultivation: Sterile algal nutrient medium

ACCLIMATION
- Pre-culture: The liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21-25 °C. Subsequently, appropriate volumes of these primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these cultures were incubated, as stated above, for a further three days to provide an inoculum in the log phase of growth, characterized by a cell density of 2.72 x 10^6 cells/mL.
Test type:
other: non-axenic
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
No data
Test temperature:
24.6-25.5 °C
pH:
7.3-9.8
Dissolved oxygen:
No data
Salinity:
See Table 6.1.5/1: Algal nutrient medium (OECD)
Nominal and measured concentrations:
Nominal concentrations: 4.27, 9.39, 20.7, 45.5 and 100 mg/L
Arithmetic mean measured concentrations: 3.97, 8.74, 19.0, 43.6 and 96.7 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flasks (250 mL)
- Initial cells density: Inoculated test medium (100 mL/flask) was added to each flask to provide an initial cell density of 1 x 10^4 cells/mL (nominal).
- Control end cells density: The control cultures were prepared as for the test media except that no test substance was added and a larger volume (900 mL) of medium was made.
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): Three flasks for each test group (two additional flasks were prepared without algae at the highest and lowest test concentrations)
- No. of vessels per control (replicates): Six flasks were established for the control group

GROWTH MEDIUM
- Standard medium used: Yes; See Table 6.1.5/1: Algal nutrient medium (OECD)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: See Table 6.1.5/1: Algal nutrient medium (OECD)

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Environmental conditions: Conical flasks (250 mL) each containing control or test culture (100 mL) were placed in an illuminated orbital incubator according to a random number sequence. The cultures were incubated, without renewal of medium for 72 hours under continuous illumination of between 6080 and 6760 lux provided by 6 x 30 W “cool white” 1 metre fluorescent tubes. The temperature of the incubator was maintained between 22.9 and 24.6 °C.
Temperature and pH of control and test flasks at the start and end of the test were recorded. Gaseous exchange and suspension of the algal cells were ensured by the action of the orbital shaker, oscillating at a nominal 150 cycles per minute. The minimum and maximum temperature and light intensity in the test area were determined each day.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Samples were taken from control and test flasks at 24, 48 and 72 hours and the cell densities measured using a haemacytometer (Improved Neubauer). The estimate of cell numbers in each sample was based on the mean of four or eight consecutive counts depending on the cell density of the cultures. The presence of any abnormal cells was also noted during counting.


TEST CONCENTRATIONS
- Range finding study: The range finding test used nominal test concentrations of 1, 10 and 100 mg/L. The definitive test concentrations, selected based on the results of the range finding test, were: Nominal concentrations: 4.27, 9.39, 20.7, 45.5 and 100 mg/L
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
14.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% confidence limits 13.9 and 15 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
26.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits 25.4 and 27.8 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.97 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: biomass and growth rate
Details on results:
After 48 hours, at nominal test concentrations of 45.5 and 100 mg/L, cells were visibly swollen and/or ruptured; at the remaining test concentrations the cells appeared normal.
Results with reference substance (positive control):
The results of the most recent laboratory reference test using potassium dichromate indicated that its 72-hour EbC50 to Selenastrum capricornutum was 0.92 mg/L; this was within the range typically obtained in this laboratory (0.3 to 1 mg/L).
Reported statistics and error estimates:
When compared to the algal nutrient medium control group, the mean cell density at 3.97 mg/L was reduced by 5%. Although not statistically significant in terms of growth rate (mean, 1.1%), this slight reduction in the cell density at 3.97 mg/L was statistically significant for the area under the growth curve (mean, 4.6%). This is not considered to be of biological significance because growth in two replicate cultures at 3.97 mg/L was comparable to the control and is not thought to have an impact on the results of the study.

Chemical analysis:

At the start of the test, the measured levels of test item in samples of the test cultures ranged between 96 and 99% of their nominal values. After 72 hours, the measured levels were between 88 and 95% of their nominal values (between 90 and 97% of their starting values). The overall arithmetic mean measured levels of test item were 3.97, 8.74, 19.0, 43.6 and 96.7 mg/L. After 72 hours, analysis of samples of media containing test item at 4.27 and 100 mg/L, which had been incubated without algal cells, gave similar results to test media incubated in the presence of algal cells. These results indicate that the presence of algal cells did not affect the stability of the test substance.

Table 6.1.5/2: Inhibition of growth

 

Parameter

Mean measured

concentration

(mg/L)

Sample size

Mean

% Inhibition

p

Area under curve to 72 hours

Control

6

30.3

0.0

-

3.97

3

28.9

4.6

0.005**#

8.74

3

24.2

20.1

<0.001***

19.0

3

9.6

68.4

<0.001***

43.6

3

0.5

98.2

<0.001***

96.7

3

-0.2

100.6

<0.001***

Growth rate to 72

Control

6

0.069

0.0

-

3.97

3

0.068

1.1

0.590

8.74

3

0.067

3.6

0.100

19.0

3

0.056

18.4

<0.001***

43.6

3

0.000

99.6

<0.001***

96.7

3

-0.009

114.2

<0.001***

 

* p < 0.05, ** p < 0.01, *** p < 0.001

#: although statistically significant, this is not considered to be of biological significance because growth in the 3.97 mg/L test flasks was greater than or comparable to flasks in the algae nutrient control group.

Validity criteria fulfilled:
yes
Conclusions:
The 72-hour EbC50 and ErC50 were 14.4 and 26.6 mg/L, respectively. The NOEC for both area under the growth curve and growth rate based on the arithmetic mean measured concentrations was 3.97 mg/L.
Executive summary:

The effect of test item on the growth of the unicellular green alga Selenastrum capricornutum (new name: Pseudokirchnerella subcapitata) was assessed under non-axenic conditions according to OECD Guideline 201 and EU Method C.3 with GLP statement.

Triplicate algal cultures, with an initial cell density of 1 x 104/mL, were exposed to test item dissolved in algal nutrient medium at nominal concentrations of 4.27, 9.39, 20.7, 45.5 and 100 mg/L; to aid dissolution, ultrasound treatment and stirring were employed. The cultures were incubated in an orbital incubator under continuous illumination at temperatures ranging from 22.9 and 24.6 °C for 72 hours. The measured levels of test item in samples of the test cultures ranged between 88 and 99% of their nominal values at the start and end of the test, giving overall arithmetic mean measured levels of 3.97, 8.74, 19.0, 43.6 and 96.7 mg/L. Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the area under the growth curve and growth rate.

 

When compared to the algal nutrient medium control group, the mean cell density at 3.97 mg/L was reduced by 5%. Although not statistically significant in terms of growth rate (mean, 1.1%), this slight reduction in the cell density at 3.97 mg/L was statistically significant for the area under the growth curve (mean, 4.6%). This is not considered to be of biological significance because growth in two replicate cultures at 3.97 mg/L was comparable to the control and is not thought to have an impact on the results of the study. After 48 hours, at nominal test concentrations of 45.5 and 100 mg/L, cells were visibly swollen and/or ruptured; at the remaining test concentrations the cells appeared normal.

 

After 72 hours of exposure to test item, the EbC50 and ErC50, respectively based on the arithmetic mean measured concentrations were 14.4 and 26.6 mg/L. The “no observed effect concentration” (NOEC) for both area under the growth curve and growth rate based on the arithmetic mean measured concentrations was 3.97 mg/L.

 

Under the test conditions, the 72-hour EbC50 and ErC50 were 14.4 and 26.6 mg/L, respectively. The NOEC for both area under the growth curve and growth rate based on the arithmetic mean measured concentrations was 3.97 mg/L.

Description of key information

OECD Guideline 201, GLP, key study, validity 1:

72h-ErC50 (Pseudokirchneriella subcapitata) = 26.6 mg/L based on measured concentrations;

72h-NOECr (Pseudokirchneriella subcapitata) = 3.97 mg/L based on measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:
26.6 mg/L
EC10 or NOEC for freshwater algae:
3.97 mg/L

Additional information

The effect of test item on the growth of the unicellular green alga Selenastrum capricornutum (new name: Pseudokirchnerella subcapitata) was assessed undernon-axenic conditionsaccording to OECD Guideline 201 and EU Method C.3 with GLP statement.

Triplicate algal cultures, with an initial cell density of 1 x 104/mL, were exposed to test item dissolved in algal nutrient medium at nominal concentrations of 4.27, 9.39, 20.7, 45.5 and 100 mg/L; to aid dissolution, ultrasound treatment and stirring were employed. The cultures were incubated in an orbital incubator under continuous illumination at temperatures ranging from 22.9 and 24.6 °C for 72 hours. The measured levels of test item in samples of the test cultures ranged between 88 and 99% of their nominal values at the start and end of the test, giving overall arithmetic mean measured levels of 3.97, 8.74, 19.0, 43.6 and 96.7 mg/L. Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the area under the growth curve and growth rate.

 

When compared to the algal nutrient medium control group, the mean cell density at 3.97 mg/L was reduced by 5%. Although not statistically significant in terms of growth rate (mean, 1.1%), this slight reduction in the cell density at 3.97 mg/L was statistically significant for the area under the growth curve (mean, 4.6%). This is not considered to be of biological significance because growth in two replicate cultures at 3.97 mg/L was comparable to the control and is not thought to have an impact on the results of the study. After 48 hours, at nominal test concentrations of 45.5 and 100 mg/L, cells were visibly swollen and/or ruptured; at the remaining test concentrations the cells appeared normal.

 

After 72 hours of exposure to test item, the EbC50 and ErC50, respectively based on the arithmetic mean measured concentrations were 14.4 and 26.6 mg/L. The “no observed effect concentration” (NOEC) for both area under the growth curve and growth rate based on the arithmetic mean measured concentrations was 3.97 mg/L.

 

Under the test conditions, the 72-hour EbC50 and ErC50 were 14.4 and 26.6 mg/L, respectively. The NOEC for both area under the growth curve and growth rate based on the arithmetic mean measured concentrations was 3.97 mg/L.