Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 6 to 10, 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with agreed test protocols following OECD Guidelines. Restrictions due to lack of test material analyses, but are not expected to have a major impact on the test result.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
The EClO values were calculated and reported. This deviation had no effect on the outcome of the study and no other deviations from the protocol occurred.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: yellow liquid at room temperature
- Analytical purity: not provided.
- Stability under test conditions: stable
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
No data available.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Each of the five WAFS were prepared by combining the appropriate amount of test substance and dilution water in a mixing vessel equipped with a magnetic stirrer (the vortex extended form the surface approximately 5% of the way to the bottom of the mixing vessel) and stirring these mixtures for approximately 24 hours, settling the mixtures for approximately four hours, and siphoning the water phase containing the WAF through glass wool. The control was prepared by passing dilution water through glass wool.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: Negative control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no insoluble material was observed.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Algae
- Strain: Selenastrum capricornutum, UTEX 1648. .
- Source (laboratory, culture collection): from a culture originally procured from the Culture Collection of Algae at the University of Texas at Austin and delivered to T.R. Wilbury Laboratories on May 27, 1999.
- Age of inoculum (at test initiation): nine day- old culture
- Method of cultivation: The culture was transferred to sterile enriched media identical to media used for this test and maintained at test conditions for at least 14 days before the definitive test. The subsample of algae used to inoculate media at the start of the definitive test came from a seven day- old culture. Identification of the culture organisms was verified using an appropriate taxonomic key.
ACCLIMATION
- Acclimation period: 14 days
- 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.

- Any deformed or abnormal cells observed: The number of algal cells/ml in each test vessel and the occurrence of relative size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells ' was determined visually by means of direct microscopic examination with a hemocytometer.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
Not applicable.

Test conditions

Hardness:
No data available.
Test temperature:
23.5~23.9 oC
pH:
7.4~10.0
Dissolved oxygen:
No data available.
Salinity:
Not applicable.
Nominal and measured concentrations:
Nominal concentrations: 0, 1.7, 3.3, 6.5, 13, 25 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: The test vessels were 250 ml glass Erlenmeyer flasks that contained 100 ml of test solution (water depth was approximately 3 cm) and were capped with inverted glass beakers.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 ml glass conical flasks filled with 100 ml of test preparation
- Aeration: no data.
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable.
- Renewal rate of test solution (frequency/flow rate): not applicable.
- Initial cells density: 104 cells per ml.
- Control end cells density: 1.6x106 cells per ml.
- No. of organisms per vessel: no data.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable.

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: not applicable.

TEST MEDIUM / WATER PARAMETERS:
- Source/preparation of dilution water: water used for acclimation of test organisms and for all toxicity testing was sterile enriched media (U.S. EPA, 1978; T.R. Wilbury Standard Operating Procedure number 6) adjusted to a pH of 7.5 ± 0.1 prior to use with 0.1 N hydrochloric acid. Media used for the definitive toxicity test contained <10 mg/L particulate matter at the start of the toxicity test and 29 mg/L particulate matter at the end of the test (the final value resulted, at least in part, from the presence of algal cells).

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: with 0.1N HCl.
- Photoperiod: continuous illumination
- Light intensity and quality: cool-white fluorescent lights that provided a light intensity approximately 390 ~ 400 footcandles.
- Salinity (for marine algae): not applicable.
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: determined visually by means of direct microscopic examination with a hemocytometer.
- Chlorophyll measurement: No.
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2x
- Justification for using less concentration than requested by guideline: not applicable.
- Range finding study: a range finding test was conducted for 96 h with the WAF of five concentrations nominal of test substance.
- Test concentrations: 0.1, 1.0, 10, 100 and 1000 mg/L test material.
- Results used to determine the conditions for the definitive study: a definitive test was conducted for 96 h with the WAF of five concentrations nominal of test substance: 3.3, 6.5, 13, 25 and 50 mg/L test material.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
6.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 5.7 – 7.3 mg/L
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
15 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 14 – 16 mg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
3.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: there were no abnormalities detected in any of the control or test cultures
- Colour differences: no effects.
- Flocculation: no effects
- Adherence to test vessels: no effects.
- Aggregation of algal cells: no effects.
- Other:
- Any stimulation of growth found in any treatment: no effects.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no observations.
- Effect concentrations exceeding solubility of substance in test medium: no.
Results with reference substance (positive control):
No data available.
Reported statistics and error estimates:
STATISTICAL METHODS:
The average specific growth rate was calculated as the natural log of the number of cells/ml at 72 and 96 hours of exposure, minus the natural log of the number of cells/ml at 0 hours of exposure, divided by the hour of exposure. Results of the toxicity test were interpreted by standard statistical techniques (Bruce and Versteeg, 1991).
The weighted least square nonlinear interpolation method was used to calculate the 72 and 96 hour EC50 and EC10 values using the nominal concentrations of test substance and both the cells per mL and the average specific growth reate. The no observed effect concentration (NOEC) is the highest concentration of test substance that allowed at least 90% of control growth. This definition of the NOEC differs slightly from the TSCA test guidelines.

Any other information on results incl. tables

Microscopic examination of the inoculum culture during testing showed no abnormalities with respect to size, shape, color, flocculation or aggregation of cells caused by exposure to the test substance. 

At 96 hours, the cell growth data as % of the control was:

WAF Concentration (mg/L)

Mean cells/mL (% of Control)

Mean Specific Growth Rate  (% of Control)

1.7

90

98

3.3

84

96

6.5

52

87

13

9

53

25

3

30

EC10 and EC50 values calculated using number of cells/mL

72 hr EC10 = 1.9 mg/L (95% confidence interval =1.7 – 2.5 mg/L)

72 hr EC50 = 6.3 mg/L (95% confidence interval =  5.5 – 7.1 mg/L)

96 hr EC10 = 2.8 mg/L (95% confidence interval =  2.3 – 3.5 mg/L)

96 hr EC50 = 6.4 mg/L (95% confidence interval =  5.7 – 7.3 mg/L)

EC10 and EC50 values calculated using average specific growth rate

72 hr EC10 = 6.1 mg/L (95% confidence interval =  4.9 – 7.6 mg/L)

72 hr EC50 = 16 mg/L  (95% confidence interval =  14  – 17 mg/L) 

96 hr EC10 = 4.7 mg/L (95% confidence interval =  3.9 – 5.8 mg/L)

96 hr EC50 = 15 mg/L  (95% confidence interval =  14    16 mg/L)

NOEC using number of cells/mL = 1.7 mg/L

NOEC using average specific growth rate = 3.3 mg/L 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Exposure of freshwater alga to the test material resulted in a 96 hour EC50 of 6.4 mg/L (95% confidence interval = 5.7 to 7.3 mg/L) when calculated using cells/ml, and 15 mg/L (95% confidence interval = 14 to 16 mg/L) when determined using the average specific growth rate. The 96 hour NOEC is 1.7 mg/L when determined using either the number of cells/ml and 3.3 mg/L using the average specific growth rate. The toxic effects were determined to be algistatic rather than algicidal.
Executive summary:

The acute toxicity of the water accommodated fraction (WAF) of test material to the freshwater alga, Selenastrum capricornutum was investigated according to OECD method 201 to establish the approximate toxicity of the test substance.

The test was performed at 24 ± 1°C under static conditions with a control (0 mg/L) and five concentrations of WAF (1.7, 3.3, 6.5, 13, 25, and 50 mg/L). The dilution water was sterile enriched media adjusted to a pH of 7.5 ± 0.1. The five WAFS were prepared by formulating individual concentrations of the test substance and dilution water in mixing vessels equipped with a magnetic stirrer, stirring the mixtures for approximately 24 hours, settling the mixtures for approximately four hours, and siphoning the water phase containing the WAF. During the mixing period, the vortex extended approximately 5% of the distance from the water surface to the bottom of the mixing vessels. 

No insoluble material was noted during the test. Exposure of the algae to the test substance resulted in the following growth after 96 hours:

Concentration (mg/L)    Average cells /ml          Percent of Control

0 (control)                   1,620,000                                

1.7                              1,460,000                                90

3.3                              1,367,000                                84

6.5                              840,000                       52

13                               151,000                         9

25                              48,000                                   3

Exposure of freshwater alga to the test material resulted in a 96 hour EC50 of 6.4 mg/L (95% confidence interval = 5.7 to 7.3 mg/L) when calculated using cells/ml, and 15 mg/L (95% confidence interval = 14 to 16 mg/L) when determined using the average specific growth rate. The 96 hour NOEC is 1.7 mg/L when determined using either the number of cells/ml and 3.3 mg/L using the average specific growth rate.

The determination of whether toxic effects were algistatic or algicida was performed at the conclusion of the test. A 0.5 mL aliquot of test media from each 100 mg/L vessel was transferred into a flask containing 100 mL of fresh media and incubated under test condition for 96 h. Algae increased form the initial density of 720 to 90, 000 cells/mL indicating the test substance was algistatic rather than algicidal.