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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 22.82% solids in water

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
One sample plus a back-up sample from each test concentration, including the blank control, on day 0 of the test before the test solutions were poured into the replicate test chambers. In addition to the neat test solution samples, one sample plus a back-up sample diluted 4x into methanol (5 mL test solution + 15 mL methanol) from each test concentration, including the blank control, on day 0 of the test before the test solutions were poured into the replicate test chambers. One sample plus a back-up sample from the pooled replicates at each test concentration, including the blank control, and the single abiotic control test chamber at test end (day 4). In addition to the neat test solution samples, one sample plus a back-up sample diluted 4x into methanol (5 mL test solution + 15 mL methanol) from each test concentration, including the blank control, and the single abiotic control test chamber at test end (day 4). All samples and back-up samples were stored refrigerated when not in use.

Test solutions

Vehicle:
no
Details on test solutions:
Test Solution Preparation
At test initiation (day 0) and 4 days prior to day 0 (day -4), test substance solutions were prepared by dilution from a stock solution of the test substance in filter-sterilized AAP nutrient medium. A primary stock solution with a nominal test substance concentration of 526 mg/L (equivalent to nominal 120 mg total solids/L) was prepared in a 1-L beaker by adding approximately 211 mg test substance to 400 mL of filter-sterilized AAP nutrient medium, stirring for 5 minutes (only at day -4), sonicating for 60 minutes, and stirring for 60 minutes. Test solutions were prepared in 250-mL beakers by adding the appropriate volume of the primary stock solution to filter-sterilized AAP nutrient medium to make nominal test substance concentrations of 6.49, 19.5, 58.4, and 175 mg/L (equivalent to nominal 1.48, 4.44, 13.3, and 40.0 mg total solids/L), and stirring each for 10 minutes. Aliquots of the primary stock solution were used for the nominal 526 mg/L test concentration solution and the abiotic (stability) control solution. Aliquots of the filter-sterilized AAP nutrient medium were used for the blank control solution. Each designated primary stock and test solution mixing vessel was used on both day 0 and day -4. The blank control and nominal 6.49, 19.5, and 58.4 mg/L test substance solutions were clear and colourless with no visible precipitate at test start. The nominal 175 mg/L test substance solution was clear and colourless with no visible precipitate but with surface film present at test start. The primary stock solution was clear and colourless with precipitate and surface film present at test start.

AAP Nutrient Medium Preparation
To prepare one litre of AAP nutrient medium, 1 mL of each of the 6 macronutrient stock solutions and 1 mL of the micronutrient stock solution were added to approximately 800 mL of Milli-Q (deionised) water with mixing after each addition. The final volume of the medium was brought to 1 litre with additional Milli-Q water.
The nutrient medium was adjusted to a pH of 7.49 with 1.0 N hydrochloric acid and filter-sterilized through 0.22-µm cellulose acetate filters into sterile containers. The containers with the resulting filter-sterilized AAP nutrient medium were stored in the refrigerator in the dark at approximately 4°C and acclimated to ambient temperature prior to use.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokirchneriella subcapitata, a freshwater, unicellular, non-motile, green alga, used in this study was cultured and maintained at the testing laboratory. The original culture source was the Department of Botany - Culture Collection of Algae - The University of Texas, Austin, Texas.

The culture method for P. subcapitata was based on published literature. Prior to the study, cultures were maintained under photoperiod, shaking speed, and temperature conditions similar to those used in the study. Illumination was maintained at 5005 ± 805 lux. The organisms were cultured in sterilized 250-mL Erlenmeyer flasks containing approximately 50 mL of filter-sterilized synthetic algal-assay procedure (AAP) nutrient medium and were aseptically transferred to fresh medium every 3 to 7 days. The flasks were fitted with sterilized foam stoppers to permit gas exchange. The P. subcapitata culture used to inoculate test vessels was aseptically transferred to fresh medium 4 days prior to use.

Study design

Test type:
static
Water media type:
freshwater
Total exposure duration:
96 h

Test conditions

Test temperature:
air temperature in the environmental chamber: 23 ± 2°C
solution temperature: 23°C
pH:
7.51 to 8.56
Nominal and measured concentrations:
Nominal concentrations, mg/L: 0, 6.49, 19.5, 58.4, 175, 526, and abiotic control (526)
Nominal total solids concentrations, mg total solids/L: 0, 1.48, 4.44, 13.3, 40.0, 120, and abiotic control (120)
Mean measured concentrations, mg total solids/L: not detected, 1.42, 3.77, 5.72, 34.3, 105, and 102 (abiotic control)
One major active component was used to determine concentrations of the test substance because it eluted as a well-resolved peak with a retention time of approximately 5.15 minutes and is considered representative of the total solids content. The limit of detection was 0.007 mg total solids/L after application of a 4x dilution factor.
Details on test conditions:
The study was conducted with a blank control and 5 concentrations of test substance at a mean lighting intensity of 4304 lux (range of 3870 to 4540 lux), air temperature of 23 ± 2°C, a liquid temperature of 23°C, and a shaking speed of 100 rpm.

Definitive Test:
Nominal test substance concentrations of 6.49, 19.5, 58.4, 175, and 526 mg/L (equivalent to nominal concentrations of 1.48, 4.44, 13.3, 40.0, and 120 mg total solids/L were chosen for the definitive test based on the results of the first range-finding study. An abiotic (stability) control was included at a nominal concentration of 526 mg/L (equivalent to 120 mg total solids/L) to demonstrate the stability of the test substance in AAP nutrient medium under the test conditions without the presence of algae. Test solutions were not renewed.
The blank control was tested as 6 replicates and each test concentration was tested as 3 replicates. The abiotic control was tested as a single flask (no replicates), and was included at the high test concentration to demonstrate the stability of the test substance in AAP nutrient medium under the test conditions without the presence of algae. Each flask, excluding the abiotic control, was randomly assigned a number to position the test flasks on the shaker table and to eliminate bias while counting. The individual flasks were labelled with concentration, replicate, and random number. The abiotic control was labelled as “abiotic control.” At definitive test initiation, flasks were inoculated with approximately 10,000 P. subcapitata cells/mL by aseptically transferring the appropriate volume of algal inoculum from a pre-counted, logarithmically growing stock culture to each flask. Cell count (biomass) measurements were conducted at approximately 24, 48, 72, and 96 hours after the definitive test initiation. Measurements of pH were conducted at test start in an aliquot taken directly from the appropriate mixing vessel and at 96 hours in an aliquot taken after pooling all replicates, where appropriate, of each control or test concentration. The pH of the abiotic control was taken from the single flask. Test solutions were appropriately discarded at test end.

Recovery Test:
At the end of the 96-hour exposure period, the blank control and those test concentrations exhibiting an approximate 50% inhibition of healthy biomass relative to the blank control were selected for the recovery test. Each blank control and test concentration was tested as a single flask (no replicates) and was exposed to untreated filter-sterilized AAP nutrient medium for up to 10 days without test medium renewal. Cell count (biomass) measurements were conducted every 3-4 days after the recovery test initiation. Recovery test flasks were prepared by diluting an approximate 0.5-mL aliquot from a single, randomly selected replicate of the blank control or the combined approximate 0.5-mL aliquots from each of the replicate flasks of the applicable concentration to a total of approximately 50 mL with fresh nutrient medium, resulting in a concentration that theoretically would not inhibit algal growth and growth rate based on visual observations during termination of the definitive test. The individual flasks were labelled with concentration and “recovery”. If cell growth was evident (based on a 16x increase in cell density prior to 10 days), the recovery test was terminated and the test substance concluded to be algistatic. If cell growth was not evident, the recovery test was terminated and the test substance concluded to be algicidal.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 526 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass, yield, and growth rate, all based on cell count
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
526 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass, yield, and growth rate, all based on cell count
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 526 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass, yield, and growth rate, all based on cell count
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 105 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: total solids
Basis for effect:
other: biomass, yield, and growth rate, all based on cell count
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
105 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: total solids
Basis for effect:
other: biomass, yield, and growth rate, all based on cell count
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 105 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: total solids
Basis for effect:
other: biomass, yield, and growth, all based on cell count
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 526 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass, yield, and growth rate, all based on cell count
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
175 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass, yield, and growth, all based on cell count
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
526 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass, yield, and growth, all based on cell count
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 105 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: total solids
Basis for effect:
other: biomass, yield, and growth, all based on cell count
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
34.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: total solids
Basis for effect:
other: biomass, yield, and growth rate, all based on cell count
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
105 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: total solids
Basis for effect:
other: biomass, yield, and growth, all based on cell count
Details on results:
Biomass over 72- and 96-hours in the blank control increased by a factor of approximately 68 and 215, respectively; the coefficient of variation of the average specific growth rate over 72- and 96-hours in the blank control replicates was 6.0 and 2.9%, respectively; and the mean coefficient of variation over 72- and 96-hours for section-by-section specific growth rates (days 0-1, 1-2, 2-3, and 3-4) in the blank control replicates was 26.5 and 25.5%, respectively; thereby satisfying the appropriate OECD test acceptance criteria for a 72-hour test. Algae remaining in all test concentrations and the blank control were normal in appearance at test termination.
Recovery was assessed for the mean, measured 105 mg total solids/L concentration. Biomass in the blank control and recovery test concentration increased over three days by factors ranging from approximately 41 to 44.
Reported statistics and error estimates:
Analyses are reported based on nominal and mean, measured total solids concentrations and were conducted using SAS Version 8.2. All statistical tests were calculated at a significance level of p = 0.05. No statistical analysis was performed for the 72-hour data to determine the EC50 values since there was <50% growth inhibition relative to the blank control based on biomass, yield, and growth rate, all based on cell count, at any test concentration. No statistical analysis was performed to determine the 96-hour ErC50 value since there was <50% growth inhibition relative to the blank control based on growth rate, based on cell count, at any test concentration.
The 72- and 96-hour data for biomass, yield, and growth rate, all based on cell count, were determined to be normally distributed (Shapiro-Wilk test) with equal variances (Levene’s test). No outliers, as determined by the Tukey outlier rule, were found in the data for biomass, yield, and growth rate. All data were determined to be normal, homogeneous, and not consistent with monotonicity. Therefore, the Dunnett method was used to determine the NOEC and LOEC values.
The 96-hour EbC50 and EyC50 values for biomass and yield, respectively, each based on cell count and nominal test substance concentrations, were obtained by the OECD Model 2 inverse regression model.
The 96-hour EbC50 and EyC50 values for biomass and yield, respectively, each based on cell count and mean, measured total solids concentrations, were obtained by the OECD Model 2 inverse regression model.

Any other information on results incl. tables

Inhibition of growth of P. subcapitata exposed to mean, measured total solids concentrations as compared to the blank control for 72 and 96 hours:

 

Parameter

(Cell Count)

 

72-hour percent inhibition in mean, measured total solids concentrations of 1.42, 3.77, 5.72, 34.3, and 105 mg total solids/L

Biomass

17, -8, -37, -1, and 11%

Yield

18, -8, -38, -1, and 11%

Growth Rate

5, -2, -8, 0, and 3%

 

Parameter

(Cell Count)

 

72-hour percent inhibition in mean, measured total solids concentrations of 1.42, 3.77, 5.72, 34.3, and 105 mg total solids/L

Biomass

-16, -23, -33, -17, and 49%

Yield

-16, -23, -34, -17, and 49%

Growth Rate

5, -2, -8, 0, and 3%

Note: Negative values of inhibition indicate stimulation of growth

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
72-hour NOEC and LOEC values based on nominal test substance concentrations and biomass, yield, and growth rate, all based on cell count = 526 mg/L and >526 mg/L, respectively.
72-hour EbC50, EyC50, and ErC50 values based on nominal test substance concentrations and biomass, yield, and growth rate, all based on cell count, respectively = >526 mg/L.

72-hour NOEC and LOEC values based on mean, measured total solids concentrations and biomass, yield, and growth rate, all based on cell count = 105 mg total solids/L and >105 mg total solids/L, respectively.
72-hour EbC50, EyC50, and ErC50 values based on mean, measured total solids concentrations and biomass, yield, and growth rate, all based on cell count, respectively, were all >105 mg total solids/L.

96-hour NOEC and LOEC values based on nominal test substance concentrations and biomass, yield, and growth rate, all based on cell count = 175 mg/L and 526 mg/L, respectively.
96-hour EbC50, EyC50, and ErC50 values based on nominal test substance concentrations and biomass, yield, and growth rate, all based on cell count, respectively, were all >526 mg/L.

96-hour NOEC and LOEC values based on mean, measured total solids concentrations and biomass, yield, and growth rate, all based on cell count = 34.3 mg total solids/L and 105 mg total solids/L, respectively.
96-hour EbC50, EyC50, and ErC50 values based on mean, measured total solids concentrations and biomass, yield, and growth rate, all based on cell count, respectively, were all >105 mg total solids/L.

The test substance was determined to be algistatic at mean, measured concentrations less than or equal to 105 mg total solids/L.
Executive summary:

The toxicity of the test substance to the green algae, Pseudokirchneriella subcapitata, was determined in a 96-hour, static toxicity test. The study was conducted with a blank control and 5 concentrations of the test substance at a mean lighting intensity of 4304 lux (range of 3870 to 4540 lux), air temperature of 23 ± 2°C, a liquid temperature of 23°C, and a shaking speed of 100 rpm. Synthetic algal-assay-procedure (AAP) nutrient medium was used as the test diluent and blank control. Test solutions were not renewed. Six replicates were used for the blank control and 3 replicates were used per test concentration. A single test flask was used for the abiotic (stability) control at the high test concentration that was included to demonstrate the stability of the test substance in AAP nutrient medium under the test conditions without the presence of algae. Biomass, yield, and growth rate, all based on cell count, were determined at 24-hour intervals over the 96-hour test.

The reductions in biomass, yield, and growth rate, all based on cell count, for Pseudokirchneriella subcapitata at 96 hours indicated a concentration-dependent response for exposure to the test substance.

The 72-hour NOEC and LOEC values based on nominal test substance concentrations and biomass, yield, and growth rate, all based on cell count, were determined to be 526 mg/L and >526 mg/L, respectively. The 72-hour EbC50, EyC50, and ErC50 values based on nominal test substance concentrations and biomass, yield, and growth rate, all based on cell count, respectively, were all >526 mg/L.

The 72-hour NOEC and LOEC values based on mean, measured total solids concentrations and biomass, yield, and growth rate, all based on cell count, were determined to be 105 mg total solids/L and >105 mg total solids/L, respectively. The 72-hour EbC50, EyC50, and ErC50 values based on mean, measured total solids concentrations and biomass, yield, and growth rate, all based on cell count, respectively, were all >105 mg total solids/L.

The 96-hour NOEC and LOEC values based on nominal test substance concentrations and biomass, yield, and growth rate, all based on cell count, were determined to be 175 mg/L and 526 mg/L, respectively. The 96-hour EbC50, EyC50, and ErC50 values based on nominal test substance concentrations and biomass, yield, and growth rate, all based on cell count, respectively, were all >526 mg/L.

The 96-hour NOEC and LOEC values based on mean, measured total solids concentrations and biomass, yield, and growth rate, all based on cell count, were determined to be 34.3 mg total solids/L and 105 mg total solids/L, respectively. The 96-hour EbC50, EyC50, and ErC50 values based on mean, measured total solids concentrations and biomass, yield, and growth rate, all based on cell count, respectively, were all >105 mg total solids/L.

The test substance was determined to be algistatic at mean, measured concentrations less than or equal to 105 mg total solids/L.