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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
Study period:
Range-finding test: May 11, 2017; Definitive test: June 22, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD 2011, 2002
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples were collected immediately prior to test commencement (0 hour before the addition of algae) and at test termination (72 hour, algae present, from pooled replicates) for analysis. Samples (40 to 150 mL) from all range-finding and definitive test solutions were collected in glass vials or jars fitted with teflon lined caps. The larger sample volumes were required to permit the concentration steps as noted above. The vials and jars were filled, leaving some headspace.
Vehicle:
no
Details on test solutions:
Preparation of the 200 mg/L nominal concentration stock solutions in algal nutrient media:

- All stock solutions were prepared in 1-L glass aspirator bottles, stirred for approximately 23 hours at a rate sufficient to maintain a vortex between approximately 10 – 35% of the solution depth using a stir bar. The solutions were then settled for approximately 1 hour. The first ~ 100 mL of solution removed from of each glass aspirator was discarded.

- The 200 mg/L stock solution used for the range-finding and definitive tests was prepared by adding 0.2005 g of the test substance to 1 L of algal nutrient media in a 1- L glass aspirator bottle and treated as noted above. The individual range-finding test solutions were prepared by adding an appropriate amount of the stock solution into a 250 mL volumetric flask making this up to volume in algal nutrient media.

The individual definitive test solutions were prepared by adding an appropriate amount of the stock solution into a 500 mL volumetric flask making this up to volume in algal nutrient media. All test solutions were stirred for 30 minutes using a stir bar and stir plate prior to being dispensed into the individual test vessels (i.e., 250 mL Erlenmeyer flasks covered with Jaece® non-toxic foam plugs).

Range-Finding Test
The test consisted of five nominal concentrations of the test substance including 0.01, 0.1, 1.0, 10, and 100 mg/L plus a negative control. For this test, two replicate 250 mL Erlenmeyer flasks, each containing 49 mL of the test solution (prepared in nutrient medium) and 1 mL of algal inoculum (5+E3 to 1+E4 algal cells/mL), were established for each concentration.

Definitive Test
The test consisted of seven nominal concentrations of the test substance including 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/L plus a negative control. For this test, four replicate 250 mL Erlenmeyer flasks, each containing 49 mL of the test solution (prepared in nutrient medium) and 1 mL of algal inoculum (5+E3 to 1+E4 algal cells/mL), were established for each concentration.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test organism:

Pseudokirchneriella subcapitata used in testing were obtained from an in-house culture. The starter culture was purchased from the University of Waterloo Culture Collection (CPCC 37).
Cultures of P. subcapitata were maintained according to test labolatory. Cultures were aseptically transferred twice weekly (typically from 3 – 7 day old donors) and maintained in temperature and light controlled environments isolated from all testing. The axenic nature of the stock culture was verified by plating on Trypticase Soy Agar (TSA) and Plate Count Agar (PCA). Algal growth curves conducted semi-annually ensured that algae were in an exponential growth phase, suitable for testing.

Nutrient medium:

Milli-Q water obtained from the University of Guelph was used in preparation of the nutrient medium for culture and testing of P. subcapitata. This water was free of particles, ions, organic molecules and microorganisms greater than 0.45 μm in diameter. Preparation of the medium was conducted according to Environment Canada 3 (2007). The nutrient medium was filtersterilized prior to use in cultures and in testing (details of the nutrient medium were provided in Section 3.2 of the Study Plan, Appendix A of this study report).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 ± 1 °C, as recorded daily with a maximum/minimum thermometer.
pH:
pH of the nutrient medium: 7.5 ± 0.1;
pH of the test solutions: pH was measured (but not adjusted) in all concentrations at the beginning and end of the test (pooled replicates).
Nominal and measured concentrations:
Range finding test: 0.01, 0.1, 1.0, 10, and 100 mg/L (nominal concentrations)
Definitive test: 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/L (nominal concentrations)
Details on test conditions:
For all replicates, cell counts were determined at approximately 24, 48, and 72 hours. Cell counts were conducted using a haemocytometer and a phase-contrast microscope (at 100 – 200 times magnification). Any changes in cell development or appearance, such as cell clumping, cell morphology, cell colour, cell shape, cell size, etc. (or lack thereof) were reported on the bench sheets.
Any additional observations relating to the test solutions, such as sedimentation of the test solution, precipitation of cells, solution appearance / colouration or other abnormalities, were also recorded on the bench sheets. At test commencement, pH was measured in the control and in each test solution. At the end of the test (72 hours), the pH was determined from the pooled replicate samples from the control and each test concentration.

Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.67 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.12 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.319 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.702 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.34 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.41 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.58 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.319 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.702 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Validity criteria:

Both the range-finding and definitive tests met the validity criteria (i.e., biomass, as determined by cell number, in control test vessels increased by a factor of at least 16 times after 72 hours; the mean Coefficient of Variation (CV; %) for section-by-section specific growth rates (days 0-1, 1-2, and 2-3) in the control cultures did not exceed 35% and the CV (%) of average specific growth rates during the whole test period (0-3 days) in replicate control cultures did not exceed 7%).

Results of analytical analysis:

Range finding study:

Nominal conc. (mg/L)

Measured conc. at 0 h (mg/L)

Measured conc. at 72 h (mg/L)

100

102.3

32.2

10

11.1

below detection limit

1

1.1

below detection limit

0.1

0.1

Not analysed

0.01

below detection limit

Not analysed

Negative contrl.

below detection limit

below detection limit

Definitive study:

Nominal conc. (mg/L)

Measured conc. at 0 h (mg/L)

Measured conc. at 72 h (mg/L)

2

1.58

0.418

1

0.702

below detection limit

0.5

0.319

below detection limit

0.25

0.196

below detection limit

0.125

0.094

below detection limit

0.0625

0.055

not analysed

0.03125

0.029

not analysed

Negative control

below detection limit

below detection limit

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the results of the definitive test based on the measured initial concentrations were following, the NOEC and LOEC were estimated to be 0.319 and 0.702 mg/L, respectively for average specific growth rate and cell yield. In terms of average specific growth rate, the 72 h EC10, EC20 and EC50 were estimated to be 0.50, 0.67 and 1.12 mg/L, respectively. The 72 h EC10, EC20 and EC50 for cell yield were estimated to be 0.34, 0.41 and 0.58 mg/L, respectively.
Executive summary:

A study was conducted to determine the toxicity of the test substance to Pseudokirchneriella subcapitata according to OECD Guideline 201 (OECD 2002 and 2011), in compliance with GLP. Two valid experiments were performed. The range finding study with two replicates was conducted using 5 concentrations ranging from 0.01 to 100 mg/L (nominal). The definitive study with four replicates was conducted using 7 concentrations ranging from 0.03125 to 2 mg/L (nominal). The incubation time was 72 h. 200 mg/L nominal concentration stock solutions for two tests were prepared in algal nutrient media (stirred for approximately 23 h). Subsequently test solutions were created. Each contained an appropriate amount of the test substance (prepared in nutrient medium) and algal inoculum (5+E3 to 1+E4 algal cells/mL). The cell concentration of each replicate was determined by measuring the cell numbers every 24 h with an electronic particle counter. Growth rate μ and the yield were determined from the cell number at the respective observation time. The NOEC and LOEC from the range-finding test were estimated to be between 0.1 and 1.1 mg/L, respectively for both average specific growth rate and cell yield. In terms of average specific growth rate and cell yield, the 72 h EC10s and EC20s were estimated to be in the range of 0.1 – 1.1 mg/L. The EC50s for average specific growth rate and cell yield were estimated to be between 1.1 and 11.1 mg/L and 0.1 to 1.1 mg/L, respectively. Test results for the definitive test were based on measured concentrations of the test substance at t=0 h, since at test termination measured concentrations in most test solutions were below the method detection limit (MDL). Under the study conditions, the results of the definitive test based on the measured initial concentrations were following, the NOEC and LOEC were estimated to be 0.319 and 0.702 mg/L, respectively for average specific growth rate and cell yield. In terms of average specific growth rate, the 72 h EC10, EC20 and EC50 were estimated to be 0.50, 0.67 and 1.12 mg/L, respectively. The 72 h EC10, EC20 and EC50 for cell yield were estimated to be 0.34, 0.41 and 0.58 mg/L, respectively (Holtze, 2017).

Description of key information

Key value for chemical safety assessment

EC50 for freshwater algae:
1.12 mg/L
EC10 or NOEC for freshwater algae:
0.5 mg/L

Additional information

A study was conducted to determine the toxicity of the test substance to Pseudokirchneriella subcapitata according to OECD Guideline 201 (OECD 2002 and 2011), in compliance with GLP. Two valid experiments were performed. The range finding study with two replicates was conducted using 5 concentrations ranging from 0.01 to 100 mg/L (nominal). The definitive study with four replicates was conducted using 7 concentrations ranging from 0.03125 to 2 mg/L (nominal). The incubation time was 72 h. 200 mg/L nominal concentration stock solutions for two tests were prepared in algal nutrient media (stirred for approximately 23 h). Subsequently test solutions were created. Each contained an appropriate amount of the test substance (prepared in nutrient medium) and algal inoculum (5+E3 to 1+E4 algal cells/mL). The cell concentration of each replicate was determined by measuring the cell numbers every 24 h with an electronic particle counter. Growth rate μ and the yield were determined from the cell number at the respective observation time. The NOEC and LOEC from the range-finding test were estimated to be between 0.1 and 1.1 mg/L, respectively for both average specific growth rate and cell yield. In terms of average specific growth rate and cell yield, the 72 h EC10s and EC20s were estimated to be in the range of 0.1 – 1.1 mg/L. The EC50s for average specific growth rate and cell yield were estimated to be between 1.1 and 11.1 mg/L and 0.1 to 1.1 mg/L, respectively. Test results for the definitive test were based on measured concentrations of the test substance at t=0 h, since at test termination measured concentrations in most test solutions were below the method detection limit (MDL). Under the study conditions, the results of the definitive test based on the measured initial concentrations were following, the NOEC and LOEC were estimated to be 0.319 and 0.702 mg/L, respectively for average specific growth rate and cell yield. In terms of average specific growth rate, the 72 h EC10, EC20 and EC50 were estimated to be 0.50, 0.67 and 1.12 mg/L, respectively. The 72 h EC10, EC20 and EC50 for cell yield were estimated to be 0.34, 0.41 and 0.58 mg/L, respectively (Holtze, 2017).