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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:
07 September 2017 to 17 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- For the range-finding and definitive test, samples (approximately 150 mL) of all test solutions (exposure and control) were collected in 250 mL glass jars (pre-cleaned with Teflon lined caps) provided by ISI (no preservative). The vials and jars were filled, leaving some headspace.
- 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. All samples were stored at 4 ± 2°C in the dark prior to pick up and delivery to ISI’s laboratory for analysis
Vehicle:
no
Details on test solutions:
- Individual test solutions of the test material for both range-finding and definitive tests were prepared from stock solutions (1000 mg/L nominal concentration) prepared in algal nutrient media without the use of any solubilising agent. All stock solutions were prepared in 1 or 2 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. For the definitive test, the first ~ 100 mL of solution removed from of each glass aspirator was discarded.
- The 1000 mg/L stock solution used for the range-finding test was prepared by adding 1.0002 g of the test material to 1 L of algal nutrient media in a 1- L glass with 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 500 mL volumetric flask making this up to volume in algal nutrient media. The 1000 mg/L stock solution used for the definitive test was prepared by adding 2.0012 g of the test material to 2 L of algal nutrient media in a 2- L glass aspirator bottle and treated as noted above. 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).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: CPCC 37
- Source: 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)
- Method of cultivation: 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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 ± 1 °C
pH:
7.5 ± 0.1
Nominal and measured concentrations:
Nominal: 15.625, 31.25, 62.5, 125, 250, 500 and 1000 mg/L
Measured: 0.6, 0.5, 0.8, 1.9, 3.5, 6.5 and 14.1 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Clear glass 250-mL Erlenmeyer flasks covered with Jaece® non-toxic foam plugs
- Material, size, headspace, fill volume: 50 mL
- Agitation: Continuous shaking of test vessels on a rotary shaker set at 100 rpm
- Initial cells density: 5 x 10^3 to 1 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 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. The nutrient medium was filter sterilised prior to use in cultures and in testing.
- Elemental composition of the test media: N 4.20 mg/L, Mg 2.65 mg/L, Ca 1.20 mg/L, S 1.91 mg/L, P 0.19 mg/L, K 0.47 mg/L, Na 11.00 mg/L, C 2.14 mg/L, B 32.44 µg/L, Mn 115.38 µg/L, Zn 1.57 µg/L, Co 0.35 µg/L, Cu 0.004 µg/L, Mo 2.88 µg/L and Fe 33.1 µg/L.
- Culture medium different from test medium: no
- Intervals of water quality measurement: 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.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: Continuous (24 hours)
- Light intensity and quality: Cool-white fluorescent, measured at the surface of the liquid in the flasks: 4670-5880 Lux for testing

EFFECT PARAMETERS MEASURED:
- 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.

TEST CONCENTRATIONS
- The test consisted of seven nominal concentrations of the test material including 15.625, 31.25, 62.5, 125, 250, 500 and 1000 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 x 10^3 to 1 x 10^4 algal cells/mL), were established for each concentration. Each control replicate flask contained 49 mL of the same batch of algal nutrient medium and 1 mL of the same algal inoculum, without the addition of the test material.
- Range finding study: The test consisted of four nominal concentrations of the test item including 1, 10, 100 and 1000 mg/L plus a negative control (i.e., nutrient media with no added test item prepared the day of test initiation). 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 x 10^3 to 1 x 10^4 algal cells/mL), were established for each concentration. Each control replicate flask contained 49 mL of the same batch of algal nutrient medium and 1 mL of the same algal inoculum, without the addition of the test material. The environmental conditions and testing procedures were the same for all flasks.
Reference substance (positive control):
yes
Remarks:
Sodium chloride
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
657 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: cell yield
Remarks on result:
other: 95 % C.L: 590-723 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
62.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and cell yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
125 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and cell yield
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
547 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
RANGE-FINDING TEST
- Confirmation of the Exposure Concentrations: Nominal, measured and Time-Weighted Mean (TWM) concentrations of the test material determined on samples collected at the beginning and end of the test are provided in Table 1. Measured concentrations were below nominal concentrations due to the low solubility of the test material. Samples collected at the end of the test resulted in lower recoveries than at the test start. Nominal concentrations of 1000, 100 and 10 mg/L achieved TWM concentrations of 5.6, 0.9 and < MDL mg/L, respectively.
- Results of Range-Finding Test: Results of the range-finding test based on nominal concentrations are summarised in Table 2. The test met all of the test validity criteria. Test results expressed as the percent inhibition of growth in terms of the effects on average specific growth rate and cell yield after 72 hours of exposure. The NOEC and LOEC were estimated to be between 100 and 1000 mg/L, respectively for both average specific growth rate and cell yield. In terms of average specific growth rate, the 72- hour EC10 and EC20 were estimated to be in the range of 100 and > 1000 mg/L, while the EC50 was estimated to be above 1000 mg/L. For cell yield, the EC 10 was estimated to be between 10 and 100 mg/L, while the EC20 and 50 were both estimated to be between 100 and 1000 mg/L, respectively.

DEFINITIVE TEST RESULTS
- Confirmation of the Exposure Concentrations: Nominal, measured and TWM concentrations of the test material determined on samples collected at the beginning and end of the test are provided in Table 3. The TWM concentration of 14.1 mg/L was achieved from preparation of the nominal 1000 mg/L stock solution of the test material used for the highest concentration of the definitive test. The spike recovery was 78.8 to 83.9% which was acceptable (average > 80 %). The measured concentrations were lower at test end as compared to test start.
- Results of the Definitive Test: Results of the definitive test are summarised in Table 4. The test met all of the test validity criteria. Results of the definitive test showed that there were adverse effects on growth of algae in terms of average specific growth rate and cell yield at concentrations of the test material above 125 mg/L. The NOEC and LOEC were estimated to be 62.5 and 125 mg/L, respectively for average specific growth rate and cell yield. In terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be 547, 813 and > 1000 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 131, 263 and 657 mg/L, respectively.
Results with reference substance (positive control):
- Sodium chloride was the reference toxicant used in this study. The 72-hour EC25 was compared to historical data and was deemed acceptable based on results falling within the 95% confidence interval (warning limit) calculated for the last thirteen reference toxicant tests.
- In conjunction with the definitive test, the 72-hour EC25 for the P. subcapitata reference toxicant test was 857 mg/L.
Reported statistics and error estimates:
- Although analytical concentrations were verified, due to the nature of the test material (UVCB), measured concentrations were not necessarily representative of the whole substance. Therefore, statistical analysis and end points are expressed here in terms of nominal concentrations.
- For all concentrations and the controls, average specific growth rate (0 to 3 days) and cell yield (72-hour) and were calculated for each replicate and then averaged. Percent inhibition (I) and Coefficient of Variation (CV; %) were also calculated for average specific growth rate and cell yield. For the control flasks, the section-by-section specific growth rates (e.g., 0-1, 1-2, and 2-3 days) and mean % CV were determined. Concentration response curves were plotted for average specific growth rate and for cell yield.
- All data satisfied the assumptions for normality and homogeneity of variance. For the range-finding test, the NOEC and LOEC endpoints for average specific growth rate and cell yield were determined using ANOVA/ William’s Test (CETIS, 2013) (α=0.05). Growth inhibition and ranges were estimated from the inhibition of average specific growth rate (ErCx) and cell yield (EyLCx) compared to the average control response but no statistical analyses were conducted.
- For the definitive test, the NOEC and LOEC endpoints for average specific growth rate and cell yield were determined using ANOVA/Dunnett Multiple Comparison Test (CETIS, 2013) (α=0.05). Growth inhibition values were estimated from inhibition of average specific growth rate (ErCx) and cell yield (EyCx) compared to the control, and were calculated using Non-Linear Regression Analysis (CETIS, 2013).

Table 1: Summary of the Nominal and Measured (at T=0 hours) Concentrations of the Test Material in the Range-Finding Test Solutions

Nominal Concentration

(mg/L)

Measured Concentration (mg/L)

Time-Weighted Mean Concentration (mg/L)

0 Hours

72 Hours

1000

6.90c

4.42d

5.6

100

0.92

0.84

0.9

10

< MDL

< MDL

< MDL

1

0.17

N/A

0.2g

0 (Negative Control)

< MDL

< MDL

< MDL

c Result is the average of duplicate samples. The relative percent difference (RPD) was 7.1%

d Result is the average of duplicate samples. The RPD was 5.0%

Method Detection Limit (MDL) = 0.1 mg/L

N/A = Not analysed

g Based on a single measurement (not a TWM)

 

Table 2: Summary of Range-Finding Test Results Showing Percent Inhibition of Growth of P. subcapitata in Terms of Average Specific Growth Rate and Cell Yield After 72 Hours of Exposure to the Test Material

Test Endpoint

Nominal Concentration (mg/L)

1

10

100

1000

Average Specific Growth Rate

(0 to 72 hours)

-0.52a

0.90

3.23

15.90

Cell Yield (72 hour)

-2.51

5.03

16.35

57.14

 

Table 3: Summary of the Nominal and Measured Concentrations of the Test Material in the Definitive Test Solutions

Nominal Concentration

(mg/L)

Measured Concentration (mg/L)

Time-Weighted Mean Concentration (mg/L)

0 Hours

72 Hours

1000

25.71c

6.60d

14.1

500

11.09

3.43

6.5

250

5.27

2.15

3.5

125

2.61

1.28

1.9

62.5

0.87

0.78

0.8

31.25

0.35

0.7

0.5

15.625

0.58

0.64

0.6

0 (Negative Control)

< MDL

< MDL

< MDL

c Average of duplicate analyses, Relative Percent Difference (RPD) = 2.2%

d Average of duplicate analyses, RPD = 6.2%

MDL (Method Detection Limit) = 0.1 mg/L

 

Table 4: Summary of Definitive Test Results Showing Percent Inhibition of Growth of P. subcapitata in Terms of Average Specific Growth Rate and Cell Yield After 72 Hours of Exposure to the Test Material

Nominal Concentration

(mg/L)

Average Specific Growth Rate

(0 to 72 hours)

Cell Yield

(72 hour)

1000

29.96

79.36

500

9.82

40.50

250

6.13

27.78

125

3.74

18.12

62.5

1.77

8.91

31.25

4.05

19.17

15.625

3.15

15.42

TEST VALIDITY CRITERIA

All of the validity criteria for the range-finding and definitive tests were met:

- Biomass, as determined by cell number, in control test vessels increased by a factor of at least 16 times after 72 hours.

- The mean CV (%) for section-by-section specific growth rates (days 0-1, 1-2, and 2-3) in the control cultures did not exceed 35%

- The CV (%) of average specific growth rates during the whole test period (0-3 days) in replicate control cultures did not exceed 7%

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, from the results of the definitive test, in terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be 547, 813 and > 1000 mg/L, respectively. The 72- hour EC10, EC20 and EC50 for cell yield were estimated to be 131, 263 and 657 mg/L, respectively. All ECx values exceeded the OECD limit of 100 mg/L. The NOEC and LOEC were estimated to be 62.5 and 125 mg/L, respectively for both average specific growth rate and cell yield.
Executive summary:

The toxicity of the test material to algae was determined in accordance with the standardised guideline OECD 201, under GLP conditions.

Both the range-finding and definitive tests met the validity criteria.

Test solution preparation included continuous stirring for approximately 24 hours, followed by a settling period of approximately 1 hour. Analytical concentrations were verified, however, due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, the results are expressed here in terms of nominal concentrations only.

For the range-finding test, in terms of average specific growth rate and cell yield, the 72-hour EC10s and EC20s were estimated to be in the range of 100 and > 1000 mg/L. The EC50s for average specific growth rate and cell yield were estimated to be > 1000 mg/L and between 100 to 1000 mg/L, respectively. The NOEC and LOEC from the range-finding test were estimated to be between 100 and 1000 mg/L, respectively for both average specific growth rate and cell yield.

Under the conditions of this study, from the results of the definitive test, in terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be 547, 813 and > 1000 mg/L, respectively. The 72- hour EC10, EC20 and EC50 for cell yield were estimated to be 131, 263 and 657 mg/L, respectively. All ECx values exceeded the OECD limit of 100 mg/L. The NOEC and LOEC were estimated to be 62.5 and 125 mg/L, respectively for both average specific growth rate and cell yield.

Description of key information

Under the conditions of this study, from the results of the definitive test, in terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be 547, 813 and > 1000 mg/L, respectively. The 72- hour EC10, EC20 and EC50 for cell yield were estimated to be 131, 263 and 657 mg/L, respectively. All ECx values exceeded the OECD limit of 100 mg/L. The NOEC and LOEC were estimated to be 62.5 and 125 mg/L, respectively for both average specific growth rate and cell yield.

Key value for chemical safety assessment

EC50 for freshwater algae:
1 000 mg/L
EC10 or NOEC for freshwater algae:
547 mg/L

Additional information

The toxicity of the test material to algae was determined in accordance with the standardised guideline OECD 201, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Both the range-finding and definitive tests met the validity criteria.

Test solution preparation included continuous stirring for approximately 24 hours, followed by a settling period of approximately 1 hour. Analytical concentrations were verified, however, due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, the results are expressed here in terms of nominal concentrations only.

For the range-finding test, in terms of average specific growth rate and cell yield, the 72-hour EC10s and EC20s were estimated to be in the range of 100 and > 1000 mg/L. The EC50s for average specific growth rate and cell yield were estimated to be > 1000 mg/L and between 100 to 1000 mg/L, respectively. The NOEC and LOEC from the range-finding test were estimated to be between 100 and 1000 mg/L, respectively for both average specific growth rate and cell yield.

Under the conditions of this study, from the results of the definitive test, in terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be 547, 813 and > 1000 mg/L, respectively. The 72- hour EC10, EC20 and EC50 for cell yield were estimated to be 131, 263 and 657 mg/L, respectively. All ECx values exceeded the OECD limit of 100 mg/L. The NOEC and LOEC were estimated to be 62.5 and 125 mg/L, respectively for both average specific growth rate and cell yield.