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:
29 September 1997 - 3 October 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with guideline; restriction due to lack of analytical analyses of test solutions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: US. EPA (TSC) 797.1050
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
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:
- Concentrations: 0, WAF (0.3, 3.3, 33, 330, and 3,300) mg/L

- Sampling method:

Cell counts were made on replicated samples and recorded at 72 and 96 hours.
At the conclusion of the test, a 0.5ml sample of test media from each 3,300 mg/L vessel was combined in a flask with 100 rnl of fresh media to determine whether toxic effects were algicidal or algistatic. The flask was incubated under test conditions for 216 hours and examined for the presence of algal cells.

- Sample storage conditions before analysis: not applicable.

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 25% of the way to the bottom of the mixing vessel) and stirring these mixtures for approximately 20 hours, settling the mixtures for approximately four hours, and siphoning the water phase containing the WAF.

- Eluate: Not applicable.

- Differential loading: Not applicable.

- Controls: Negative control

- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): the 3300 mg/L test solution was cloudy throughout the test.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

- Common name: Algae

- Strain: Scenedesmus subspicatus strain UTEX 1648.

- Source (laboratory, culture collection): from a culture originally procured from the Culture Collection of Algae at the hversity of Texas at Austin and delivered to T.R. Wilbury Laboratories on January 30, 1997.

- Age of inoculum (at test initiation): seven 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:
No applicable

Test conditions

Hardness:
No data available
Test temperature:
23.5 - 23.9 deg. C
pH:
7.0 - 7.5
Dissolved oxygen:
No data
Salinity:
Not applicable since used freshwater
Nominal and measured concentrations:
Nominal: 0.3, 3.3, 33, 330, and 3,300 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: 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: 1.9x106 cells per ml.

- No. of organisms per vessel: no data.

- No. of vessels per concentration (replicates): 2

- No. of vessels per control (replicates): 2

- 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 41 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 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.


TEST CONCENTRATIONS

- Spacing factor for test concentrations: 10x

- Justification for using less concentration than requested by guideline: not applicable.

- Range finding study: same as described above.

- Test concentrations: same as described above.

- Results used to determine the conditions for the definitive study: the study report was entitled as “range –finding study”, but it was a very comprehensive study and was regarded as final main study. Results are present in the following sections.









Reference substance (positive control):
not specified

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
27 mg/L
Nominal / measured:
nominal
Conc. based on:
not specified
Remarks:
cells/mL
Basis for effect:
cell number
Remarks on result:
other: 3.3 - 330 mg/L
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
120 mg/L
Nominal / measured:
nominal
Conc. based on:
other: specific growth rate
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
3.3 mg/L
Nominal / measured:
nominal
Conc. based on:
other: cell/mL
Basis for effect:
cell number
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
3.3 mg/L
Nominal / measured:
nominal
Conc. based on:
other: growth rate
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes

- 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.

- 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: None.

- Effect concentrations exceeding solubility of substance in test medium: None.
Results with reference substance (positive control):
No data available
Reported statistics and error estimates:
None

Any other information on results incl. tables

Table 1: Test Results After 96 Hours

Concentration (mg/L)

Average cells /ml

Percent of Control

0 (control)

1,901,000

0.3

1,875,000

99

3.3

1,825,000

96

33

831,000

44

330

42,000

2

3300

<10,000

<1

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Exposure of freshwater alga to test material at concentrations of 0.3, 3.3, 33, 330, and 3,300 mg/L, plus an untreated control, was conducted in accordance with OECD Guideline 201 and US EPA (TSC) 797.1050. The study report was entitled as “range –finding study”, but the report was comprehensive and well documented. Therefore this study was regarded equivalent to definitive study with Klimisch code of 2.
From this study, a 96 hour EC50 of 27 mg/L (95% confidence interval = 3.3 to 330 mg/L) when calculated using cells/ml, and 120 mg/L (95% confidence interval =96 to 160 mg/L) when determined using the average specific growth rate were obtained. The 96 hour NOEC is 3.3 mg/L when determined using either the number of cells/ml or the average specific growth rate.
Executive summary:

The acute toxicity of the water accommodated fraction (WAF) of test material to the freshwater alga,Selenastrum capricornutum,was investigated during a range-finding study conducted at T.R. Wilbury Laboratories, Inc., in,. The test, which was designed to establish the approximate toxicity of the test substance, was performed for 96 hours from September 29 to October 3, 1997.

The test was performed at 24 ± 1°C under static conditions with a control (0 mg/L) and five concentrations of WAF (0.3, 3.3, 33, 330, and 3,300 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 20 hours, settling the mixtures for approximately four hours, and siphoning the water phase containing the WAF. During the mixing period, the vortex extended approximately 25% of the distance from the water surface to the bottom of the mixing vessels. 

The 3,300 mg/L test solution was cloudy throughout the test. No other 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)

1,901,000

0.3

1,875,000

99

3.3

1,825,000

96

33

831,000

44

330

42,000

2

3300

<10,000

<1

Exposure of freshwater alga to the test item resulted in a 96 hour EC50 of 27 mg/L (95% confidence interval = 3.3 to 330 mg/L) when calculated using cells/ml, and 120 mg/L (95% confidence interval =96 to 160 mg/L) when determined using the average specific growth rate. The 96 hour NOEC is 3.3 mg/L when determined using either the number of cells/ml or the average specific growth rate.