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Ecotoxicological information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Remarks:
Conducted according to guideline in effect at the time of study conduct
Qualifier:
according to
Guideline:
other: Commission Directive 92/69/EEC EEC Method C3 (1992)
Deviations:
no
Remarks:
Conducted according to guideline in effect at the time of study conduct
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: An aliquot from the blank control, working stock, and test concentrations was collected at the study start (day 0) and submitted for analysis. An aliquot from the blank control, test concentrations, and abiotic control was collected at the end of the exposure (day 3). The day 3 exposure samples were centrifuged in the IEC clinical centrifuge for approximately 30 minutes in order to remove algae. Prior to HPLC analysis, all 50 mg/L and 100 mg/L samples were diluted 1:1 and 1:3 with AAP nutrient medium, respectively.
- Sampling method: HPLC with UV absorbance detector
- Sample storage conditions before analysis: Not reported
Vehicle:
no
Details on test solutions:
PREPARATION OF TEST SOLUTION: Prior to the initiation of the definitive test, a range-finding test was conducted to determine the appropriate concentrations to be used in the definitive test. A working stock solution was prepared by dissolving the test substance in 1000 mL filter sterilized AAP nutrient medium for a nominal concentration of 100 mg/L (= 100 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 100 mg/L working stock solution and diluting with filter-sterilized AAP nutrient medium to make nominal concentrations of 6.25, 12.5, 25, and 50 mg/L. Aliquots of the nominal 100 mg/L working stock solution were used for the 100 mg/L test concentration solution and the abiotic (stability) control solution.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Selenastrum capricornutum
-Culture Maintenance: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.
-Test Culture Preparation:For each definitive control and test solution, 3 aliquots (50 mL each) were placed in separate sterilized 250 mL Erlenmeyer flasks fitted with sterilized foam stoppers. Each flask, excluding the abiotic control, was randomly assigned a number to eliminate bias while counting. To achieve the desired nominal concentration of approximately 10000 Selenastrum capricornutum cells/mL at test initiation, an approximate 0.550 mL (= 550 μL) aliquot of algal inoculum from a logarithmically growing stock culture was aseptically transferred to each flask, except the abiotic control.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Recovery Test: 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 50 and 100 mg/L) were selected for the recovery test. A blank control from a randomly selected single blank control replicate was prepared for comparison by diluting an aliquot 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 replicate from each test solution selected for the recovery test was exposed to untreated filter-sterilized AAP nutrient medium for 6 days. For each of the nominal 50 and 100 mg/L test concentrations, approximately 48.5 mL of filter-sterilized AAP nutrient medium was placed in separate sterilized 250 mL Erlenmeyer flasks each fitted with a foam stopper.
Cell counts were made approximately 72 and 144 hours from recovery test initiation.
Hardness:
Not reported
Test temperature:
25.0 to 25.2 ºC
pH:
For the definitive test range 7.42-7.62 at 0-hour and 7.35 to 7.67 at the 72-hour interval
Dissolved oxygen:
Not reported
Salinity:
freshwater
Nominal and measured concentrations:
Nominal concentrations: 6.25, 12.5, 25, 50, and 100 mg/L
Measured concentrations (initial): 6.20, 12.3, 24.5, 49.3, and 99.5 mg/L
Measured concentrations (final): 4.68, 9.18, 19.2, 37.4, and 76.8 mg/L

Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass Erlenmeyer flasks; because of the volatile nature of the test substance, test vessels were sealed and were not opened during the exposure period.
- Initial cells density: 10000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: synthetic algal-assay-procedure (AAP) nutrient medium

OTHER TEST CONDITIONS
- Photoperiod: 24 hour light and 0 hour dark
- Light intensity and quality: range 6000-10000 lux
-Test flasks were arranged on a lighted shaker table in a chamber in a random design and were re-positioned each working day.
-Recovery Test: The ability of Selenastrum capricornutum to recover after exposure to the test substance was determined by visually counting the number of cells taken from an approximate 0.2 mL sample from each flask at approximately 72 hours from the recovery test initiation. The counts were conducted as described for the definitive test. Observations and counts of healthy and unhealthy cells (e.g., deformed, scenescent, stunted) were recorded in the study records. The 72- and 144-hour counts were made at approximately the same time that the recovery test was initiated. If cell growth was evident (logarithmic growth of healthy cells prior to 240 hours), the recovery test was terminated and the test substance concluded to be algistatic.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Selenastrum capricornutum growth measurement was determined by visually counting the number of cells taken from an approximate 0.2 mL sample from each flask at approximately 24, 48, and 72 hours after the definitive test initiation. The counts were conducted using a hemacytometer and a compound microscope

Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
67 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.I. = 60.5 to 74.1 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
38.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 95% C.I. = 32.6 to 44.6 mg
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Reported statistics and error estimates:
The mean healthy cell count, area under the growth curve, and growth rate at the 72-hour interval for each test concentration were expressed relative to the blank control. All calculations were based on the nominal concentrations. The means and standard errors of the healthy cell counts, areas under the growth curve, and growth rate for each test concentration and the blank control were calculated using standard procedures. The healthy cell counts, areas under the growth curve, and growth rates were used to calculate the appropriate EC50 values. This “effective concentration” was defined as the concentration producing a 50% inhibition of growth relative to the blank control. The appropriate EC50 values and associated 95% confidence limits were determined by weighted least-squares non-linear regression of the log of the test concentration against the measured parameter. The NOEC, defined as the highest concentration of test substance that had no significant effect on the measured parameter relative to the blank control, was determined from a trend test (Jonckheere-Terpstra) applied in a step-down manner. Such a procedure assumed a monotone dose response. In some events, the NOEC was determined by appropriate parametric or non-parametric multiple comparisons methods. All tests of significance were at α = 0.05.
Validity criteria fulfilled:
yes
Conclusions:
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
Healthy Cell Count
72-hour EC50 = 38.1 mg/L
72-hour NOEC= 12.5 mg/L
The most sensitive parameter based on nominal test concentrations was area under the growth curve with an EC50 of 31.3 mg/L and a NOEC of 12.5 mg/L. The ability to recover was assessed at nominal concentrations of 50 and 100 mg/L. The test substance was determined to be algistatic at nominal concentrations less than or equal to 100 mg/L.
Executive summary:

This study was conducted to determine the effect of the test substance on the growth and growth rate of the green alga, Selenastrum capricornutum. The algae were exposed to nominal concentrations of 6.25, 12.5, 25, 50, and 100 mg/L of nutrient medium. For the definitive (dose-response) test, the organisms were exposed for 72 hours (3 days) without test medium renewal. The effect was expressed as percent inhibition in growth based on healthy cell count (cell density), area under the growth curve, and growth rate relative to the blank (normal culture medium) control for the 72-hour (day 3) interval of the test. The 72-hour results, including the 95% confidence interval (C.I.), based on nominal concentrations are as follows: Healthy Cell Count EC50 of 38.1 mg/L (95% C.I. = 32.6 to 44.6 mg/L) and NOEC of 12.5 mg/L; Area Under the Growth Curve EC50 of 31.3 mg/L (95% C.I. = 23.6 to 39.1 mg/L) and NOEC of 12.5 mg/L; Growth Rate EC50 of 67.0 mg/L (95% C.I. = 60.5 to 74.1 mg/L) and NOEC of 12.5 mg/L. The ability of the organisms to recover was assessed for each definitive test concentration with 50% or greater growth inhibition (i.e., nominal 50 and 100 mg/L). The effects on growth and growth rate of Selenastrum capricornutum were found to be algistatic, i.e., logarithmic growth resumed within 144 hours (6 days), at concentrations less than or equal to 100 mg/L.

Description of key information

A static, 72-hour study in Selenastrum capricornutum with the test substance was performed. The 72-hour EC50 in Selenastrum capricornutum was 67 mg/L (measured initial) based on growth rate. The NOEC based on growth rate was 12.5 mg/L (measured initial).

Key value for chemical safety assessment

EC50 for freshwater algae:
67 mg/L
EC10 or NOEC for freshwater algae:
12.5 mg/L

Additional information