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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:
24 April 2003 to 29 April 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
minors deviation with no impact on the final results
Principles of method if other than guideline:
No positive control substance was detailed within the study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material :
Not applicable.
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
The concentrations of H-25424 in the working stock (nominal 60 mg H-25424/L), blank control, and test concentration (nominal 3.75, 7.5, 15, and 30 mg H-25424/L) solutions were verified at test initiation (day 0). An aliquot from each of the working stock, the blank control, and test concentration solutions was taken for measurement of pH and test substance concentrations prior to the addition of Selenastrum capricornutum.
The pH and concentrations of H-25424 in the blank control, test concentration (nominal 3.75, 7.5, 15, 30, and 60 mg H-25424/L), and abiotic control (nominal 60 mg/L H-25424/L) solutions were also analyzed at test termination (day 3). These samples were prepared by pooling all 3 replicates for each of the control and test concentration solutions and taking an aliquot from each of the pooled samples.

The concentrations of H-25424 in each of the test solutions were determined by high performance liquid chromatography (HPLC).

- Sampling method:
An aliquot from the working stock, blank control, and test concentrations were sampled at the study start (day 0) and submitted for analysis. An aliquot from the blank control, test concentrations, and abiotic control were sampled at the end of the exposure (day 3) and submitted for analysis. Back-up solutions for each sample also were provided. The day 3 exposure samples were centrifuged in an IEC clinical centrifuge for 30 minutes in order to remove algae. Aliquots of all test solutions were transferred to autosampler vials for analysis by HPLC.

- Sample storage conditions before analysis: Not detailed; presumed analysis undertaken at point of sampling.

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
AAP nutrient medium(2) was prepared by adding 1 mL of each of the 6 macronutrient stock solutions and 1 mL of the micronutrient stock solution to approximately 800 mL of Milli-Q® (deionized) water, with mixing after each addition. The volume of the medium was brought to 1 liter with additional Milli-Q® water. Appropriate proportions (1 mL of each stock solution for each 1 liter of medium) were used to prepare the larger volumes of the medium required for the definitive and recovery tests.
The medium pH was adjusted to 7.49 with 0.1N sodium hydroxide. For both the definitive and recovery tests, the medium was filter-sterilized using Corning® pre-sterilized filtration systems each with a 0.22 μm cellulose acetate filter. The containers with the resulting filter-sterilized AAP nutrient medium for use in the definitive and recovery tests were stored in the refrigerator in the dark at approximately 4°C and acclimated to room temperature prior to use. Any remaining unused medium was properly stored.

Prior to the initiation of the definitive test, method development was conducted to determine the appropriate concentrations to be used in the definitive test. Details are attached in "background information" below.

A working stock solution was prepared by dissolving the test substance in 1000 mL filter-sterilized AAP nutrient medium for a nominal concentration of 60 mg H-25424/L (= 60 ppm). Aliquots of the filter-sterilized AAP nutrient medium were used for the blank (normal culture medium) control solution. Test solutions were prepared using aliquots of the nominal 60 mg/L working stock solution and diluting with filter-sterilized AAP nutrient medium to make nominal concentrations of 3.75, 7.5, 15, and 30 mg H-25424/L. Aliquots of the nominal 60 mg/L working stock solution were used for the 60 mg H-25424/L test concentration solution and the abiotic (stability) control solution.

- Eluate: None
- Differential loading:
- Controls: Abiotic Control - nominal 60 mg/L H-25424/liter nutrient medium; (no Selenastrum capricornutum)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None specified.
Test organisms (species):
Scenedesmus capricornutum
Details on test organisms:
TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain: Not applicable.
- Source (laboratory, culture collection): The original culture source was the Department of Botany - Culture Collection of Algae - The University of Texas at Austin - Austin, Texas 78713-7640.
- Age of inoculum (at test initiation): Fresh.
- Method of cultivation: The culture method for Selenastrum capricornutum was based on published literature. Prior to the study, Selenastrum capricornutum cultures were maintained under the same environmental conditions used in the study. The organisms were cultured in sterilized 250 mL Erlenmeyer flasks containing approximately 50 mL of filtered (= filter-sterilized) AAP nutrient medium and were aseptically transferred to fresh medium every 3 to 7 days. The flasks were fitted with foam stoppers to permit gas exchange.


ACCLIMATION
- Acclimation period: Not specified.
- Culturing media and conditions (same as test or not): AAP nutrient medium(2) was prepared by adding 1 mL of each of the 6 macronutrient stock solutions and 1 mL of the micronutrient stock solution to approximately 800 mL of Milli-Q® (deionized) water, with mixing after each addition. The volume of the medium was brought to 1 liter with additional Milli-Q® water. Appropriate proportions (1 mL of each stock solution for each 1 liter of medium) were used to prepare the larger volumes of the medium required for the definitive and recovery tests.
The medium pH was adjusted to 7.49 with 0.1N sodium hydroxide. For both the definitive and recovery tests, the medium was filter-sterilized using Corning® pre-sterilized filtration systems each with a 0.22 μm cellulose acetate filter. The containers with the resulting filter-sterilized AAP nutrient medium for use in the definitive and recovery tests were stored in the refrigerator in the dark at approximately 4°C and acclimated to room temperature prior to use. Any remaining unused medium was properly stored.
- Any deformed or abnormal cells observed: None.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
At the end of the 72-hour exposure period, a single randomly selected replicate from the blank control and an aliquot from each of the replicate flasks from each of the test concentrations exhibiting a 50% or greater inhibition of cell counts relative to the blank control (nominal 15, 30, and 60 mg/L) were selected for the recovery test. A blank control was prepared for comparison by diluting an aliquot from a randomly selected single blank control replicate with nutrient medium. An aliquot from each of the replicate flasks for each of the selected test concentrations was removed and combined into a single sterile flask containing enough fresh nutrient medium to dilute the test substance to a concentration (less than the NOEC) that theoretically would not have inhibited algal growth and growth rate. The Selenastrum capricornutum from each test solution replicate selected for the recovery test were exposed to untreated filter-sterilized AAP nutrient medium for 6 days.
Cell counts were made approximately 72 and 144 hours from recovery test initiation.
Hardness:
Not specified.
Test temperature:
24 ± 2 deg C
pH:
pH measurements of the test solutions ranged from 7.55 to 7.79 at the 0-hour and from 7.11 to 7.46 at the 72-hour interval
Dissolved oxygen:
Not specified.
Salinity:
Not applicable.
Nominal and measured concentrations:
Nominal concentrations of 0, 3.75, 7.5, 15, 30, and 60 mg/L, without test medium renewal.
Details on test conditions:
TEST SYSTEM
- Test vessel: sterilized 250 mL Erlenmeyer flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 50ml solution in 250 ml flasks, giving a ratio of 5:1
- Aeration: No. The flasks were fitted with foam stoppers to permit gas exchange
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): Aseptically transferred to fresh medium every 3 to 7 days. The organisms were exposed for 72 hours (3 days), without test medium renewal in the actual test
- Initial cells density: 10,000 cells/mL
- Control end cells density: 10,000 cells/mL
- No. of organisms per vessel: Not specified.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1

GROWTH MEDIUM
- Standard medium used: Yes, AAP nutrient medium(2) was prepared by adding 1 mL of each of the 6 macronutrient stock solutions and 1 mL of the micronutrient stock solution to approximately 800 mL of Milli-Q® (deionized) water, with mixing after each addition. The volume of the medium was brought to 1 liter with additional Milli-Q® water. Appropriate proportions (1 mL of each stock solution for each 1 liter of medium) were used to prepare the larger volumes of the medium required for the definitive and recovery tests
- Detailed composition if non-standard medium was used: N/A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q® (deionized) water
In final stock solution:
- Total organic carbon: 2.143 mg/l
- Particulate matter: Not specified.
- Metals:
Mn 115.374 mg/l
Zn 1.570 mg/l
Co 0.354 mg/l
Cu 0.004 mg/l
Mo 2.878 mg/l
Fe 33.051 mg/l
Na 11.001 mg/l
K 0.469 mg/l
Mg 2.904 mg/l
Ca 1.202 mg/l
- Pesticides: Not specified.
- Chlorine: Not specified.
- Alkalinity: Not specified.
- Ca/mg ratio: 0.41
- Conductivity: Not specified.
- Culture medium different from test medium: No.
- Intervals of water quality measurement: Not specified.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: Medium adjusted to 7.49 with 0.1N sodium hydroxide
- Photoperiod: 24 hours
- Light intensity and quality: 6000 to 10,000 Lux; supplied by cool-white fluorescent tubes
- Shaking speed: 100 rpms
- Temperature: 24 +/- 2 deg C

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Visual, conducted using a hemacytometer and a compound microscope. An aliquot of each sample was loaded into the 2 grid areas of the hemacytometer and 8 squares from each grid area, for a total of 16 squares, were selected for counting. All cells located in the 16 squares were counted and recorded as healthy or unhealthy. The total number of cells counted was multiplied by 10,000 to determine the number of cells per milliliter. Cells outside these 16 squares were not counted nor included in the total number. Observations and counts of healthy and unhealthy cells (e.g., deformed, scenescent, stunted) were recorded in the study records. Counts were made at approximately the same time each counting day. Statistical calculations of the EC50 and NOEC were based on mean healthy cell counts and nominal concentrations.
- Chlorophyll measurement: No.
- Other:

TEST CONCENTRATIONS
Solutions used in the definitive test were as follows:
Blank Control: AAP nutrient medium containing no H-25424
Treatment nominal: 3.75, 7.5, 15, 30, and 60 mg H-25424/liter nutrient medium
Abiotic Control: nominal 60 mg/L H-25424/liter nutrient medium; (no Selenastrum capricornutum)

- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study - None. Prior to the initiation of the definitive test, method development was conducted to determine the appropriate concentrations to be used in the definitive test. Definitive details not specified.
- Test concentrations: 0, 3.75, 7.5, 15, 30, and 60 mg/l
- Results used to determine the conditions for the definitive study: Not applicable.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
7.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
Healthy Cell Count
Remarks on result:
other: 95% C.I. = 6.6 to 9.2 mg/L. NOEC < 3.75 mg/l
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
7.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Area Under the Growth Curve
Remarks on result:
other: 95% C.I. = 6.7 to 8.9 mg/L. NOEC < 3.75 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
21.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.I. = 17.5 to 26.0 mg/L. NOEC < 3.75 mg/L
Details on results:
A. Environmental Conditions - Definitive and Recovery Tests
For the definitive test, the pH measurements of the test solutions ranged from 7.55 to 7.79 at the 0-hour and from 7.11 to 7.46 at the 72-hour interval.

For the definitive test, light intensity for the areas used in the chamber ranged from 6970 to 8010 lumens/m² (= lux). The mean light intensity was 7492 lumens/m².

For the recovery test, light intensity for the areas used in the chamber ranged from 6940 to 8110 lumens/m². The mean light intensity was 7463 lumens/m².

For the definitive and recovery tests, the shaking speed was 96 and 107 revolutions per min (rpm), respectively.

For the definitive and recovery tests, the temperature from the digital readout on the temperature recorder ranged from 24.0 to 24.8°C for the duration of the study.

B. Test Substance Stability and Analytical Verification (Table 3, Figures 1-4, see below)

1. Chromatographic Results
The test substance eluted as a well-resolved peak with a retention time of approximately 3.0 minutes. Figure 1 shows a representative calibration curve obtained for H-25424. A chromatogram of a typical standard is shown in Figure 2. Figures 3 and 4 show chromatograms of a blank control solution and test concentration solution, respectively.
The LOD and LOQ were determined to be 0.002 mg/L and 0.005 mg/L, respectively.

2. Test Solution Results
The mean, measured concentration of the test substance in the day 0 working stock (= nominal 60 mg/L, = highest test concentration, = abiotic control) was 54.6 mg/L. This represents 91% recovery of the test substance. The mean, measured concentrations of the test substance in the day 0 nominal 3.75, 7.5, 15, and 30 mg/L test concentrations were 3.56, 6.95, 13.7, and 27.4 mg/L, respectively. This represents a 95, 93, 91, and 91% recovery of the test substance, respectively. These data indicated the test concentration solution was prepared at the desired concentration. After 3 days, the mean, measured concentrations of the test substance in the day 3 nominal 3.75, 7.5, 15, 30, and 60 mg/L test concentration, and abiotic control solutions were 3.41, 6.31, 12.0, 23.7, and 48.3 mg/L and 47.1 mg/L, respectively. This represents 91, 84, 80, 79, 81, and 79% recovery of the test substance, respectively. The blank control solutions contained no detectable concentrations of the test substance on both day 0 and day 3.
The test substance was determined to be stable over the course of the definitive test as evidenced by the analytical recoveries obtained from the day 0 and day 3 test solutions.

C. Selenastrum capricornutum Growth (Tables 4-8, below)

1. Definitive Test
The organisms were exposed for 72 hours without test medium renewal. The effects were expressed in terms of percent inhibition in growth based on healthy cell count, area under the growth curve, and growth rate relative to the blank control for the 72-hour interval of the test. The 72-hour results are as follows:

Healthy Cell Count
EC50 7.9 mg H-25424/L (95% C.I. = 6.6 to 9.2 mg H-25424/L)
NOEC < 3.75 mg H-25424/L

Area Under the Growth Curve
EC50 7.8 mg H-25424/L (95% C.I. = 6.7 to 8.9 mg H-25424/L)
NOEC < 3.75 mg H-25424/L

Growth Rate
EC5021.7 mg H-25424/L (95% C.I. = 17.5 to 26.0 mg H-25424/L)
NOEC < 3.75 mg H-25424/L

The reductions in healthy cell count, area under the growth curve, and growth rate indicate a dose-dependent response with increasing concentrations of the test substance.

2. Recovery Test
Recovery was assessed for the nominal 15, 30, and 60 mg/L concentrations. The algae from each test solution were exposed to untreated filter-sterilized AAP nutrient medium for 144 hours. The effects upon growth and growth rate of Selenastrum capricornutum were found to be algistatic within 144 hours at nominal concentrations less than or equal to 60 mg/L.
Results with reference substance (positive control):
Not conducted.
Reported statistics and error estimates:
Statistical determination data is listed above.

The tabulated results cannot be pasted into the free text table here, due to the size. These are therefore attached below under "Attached Background Material".

Validity criteria fulfilled:
yes
Conclusions:
Selenastrum capricornutum at 72 hours (3 days) indicate a dose-dependent response for increasing concentrations of the test substance.
The most sensitive parameter was area under the growth curve with an EC50 of 7.8 mg/L and a NOEC of < 3.75 mg/L. The ability to recover was assessed at nominal concentrations of 15, 30, and 60 mg/L. The test substance was determined to be algistatic at nominal concentrations less than or equal to 60 mg/L.
Executive summary:

Selenastrum capricornutum at 72 hours (3 days) indicate a dose-dependent response for increasing concentrations of the test substance.

The results for the growth rate parameter to be taken into account as key values are: EC50 of 21.7mg/L and a NOEC of < 3.75 mg/L. The ability to recover was assessed at nominal concentrations of 15, 30, and 60 mg/L. The test substance was determined to be algistatic at nominal concentrations less than or equal to 60 mg/L.

Description of key information

The substance was tested on Selenastrum capricornutum for 72 hours.

The results for the growth rate parameter to be taken into account as key values are: EC50 of 21.7mg/L and a NOEC of < 3.75 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
7.8 mg/L
EC10 or NOEC for freshwater algae:
3.75 mg/L

Additional information