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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:
28 September 2017 to 09 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- For the range-finding and definitive tests, 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 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 refrigerated until transported to ISI and kept refrigerated by ISI until analysis was conducted.
- All samples from the range-finding and definitive tests were analysed within 48 hours of sample collection.
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; corrected for purity) prepared in algal nutrient media without the use of any solubilising agent.
- All stock solutions were prepared in 500 mL glass volumetric flasks, stirred for approximately 0.5 hour using a stir bar. The stock solutions were then used to prepare the test concentrations.
- The 1000 mg/L stock solution used for the range-finding test was prepared by adding 0.62502 g of the test material to 500 mL of algal nutrient media in a 500 mL glass volumetric and stirred for 0.5 h. The individual test solutions were prepared in 400 mL of algal nutrient media (using 2 x 200 mL volumetric flask), and adding the required volume of the test material stock solution. The 1000 mg/L stock solution used for the definitive test was prepared by adding 0.62507 g of the test material to 500 mL of algal nutrient media in a 500 mL glass volumetric and stirred for 0.5 h, until the test material was visibly dissolved. The stock solution was refrigerated in complete darkness for approximately 19 hours, after which it was allowed to warm gradually to the ambient laboratory temperature. The individual test solutions were prepared as above for the range-finding test. All test solutions were stirred for 15 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
- Source: University of Waterloo Culture Collection (CPCC 37)
- Age of inoculum (at test initiation): 3 – 7 days old and in exponential growth

CULTURE CONDITIONS
- 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: 0.625, 1.25, 2.5, 5, 10, 20, 40 and 80 mg/L
Measured (0 hrs): 0.64, 1.50, 2.59, 5.39, 10.18, 20.49, 38.92 and 77.55 mg/L
Measured (72 hrs): 0.71, 1.13, 2.36, 5.72, 9.68, 19.78, 33.24 and 75.82 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 OECD. 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: 4650 - 6110 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 eight nominal concentrations of the test material including 0.625, 1.25, 2.5, 5, 10, 20, 40, and 80 mg/L plus a negative control.
- Range finding study: The test consisted of four nominal concentrations of the test material including 0.1, 1, 10 and 100 mg/L plus a negative control (i.e., nutrient media with no added test material 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.
Reference substance (positive control):
yes
Remarks:
Sodium chloride
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
31.02 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % C.L: 28.52-33.74 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
19.93 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: cell yield
Remarks on result:
other: 95 % C.L: 17.37-22.87 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10.18 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and cell yield
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
20.49 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and cell yield
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. Samples collected at the beginning of the test did not contain algae, whereas those collected at test termination did. Measured concentrations at test start were similar to nominal (both corrected for purity). However, the measured concentration at the end of the test was lower for the 100 mg/L (nominal), indicating that the test material was not recoverable over the period of the test. Nominal concentrations of 100 and 10 mg/L achieved TWM concentrations of 74 and 10 mg/L, respectively (i.e., approximately 74 and 100% of the nominal concentration).
- Results of Range-Finding Test: Results of the range-finding test 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 1 and 10 mg/L (TWM), respectively for both 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 between 10 and 74 mg/L, while for cell yield the same endpoints were estimated to be between <1 and 74 mg/L (TWM).

DEFINITIVE TEST
- Confirmation of the Exposure Concentrations: Nominal and measured concentrations of the test material determined on samples collected at the beginning and end of the test are provided in Table 3. As concentrations at the test end were similar to test start it was not appropriate to calculate Time-Weighted Mean concentrations for the definitive test. The initial concentration of 77.55 mg/L was achieved from preparation of a nominal 80 mg/L solution of the test material used for the highest concentration of the definitive test. The spike recovery was 104.4 – 105.3%, which was acceptable. Sample preparation procedures (e.g., stirring/settling time and mixing velocity) were similar for the range-finding and definitive tests; with the exception of an additional ~19 hours of refrigerated storage time for the stock solution during the definitive test.
- Definitive Test Results: 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 at and above 10.18 mg/L. The NOEC and LOEC were estimated to be 10.18 and 20.49 mg/L, respectively for both 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 14.00, 18.39 and 31.02 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 10.05, 12.71 and 19.93 mg/L, respectively.


TEST VALIDITY CRITERIA
All of the validity criteria for the range-finding and definitive tests were met as listed below:
- 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%.
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 fourteen reference toxicant tests.
- In conjunction with the definitive test, the 72-hour EC25 for the P. subcapitata reference toxicant test was 992 mg/L.
Reported statistics and error estimates:
- For the range-finding test, all endpoints for cell yield and average specific growth rate were generated using calculated Time-Weighted Mean (TWM) concentrations derived from measured concentrations at test start and end. For the definitive test, results were generated using measured concentrations of the test material at test initiation, corrected for purity.
- For all concentrations and the controls, average specific growth rate (0 to 3 days) and cell yield (72-hour) 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/ Williams Multiple Comparison Test (CETIS, 2013) (α=0.05). The ECx range endpoints, for cell yield (EyCx) and average specific growth rate (ErCx), were estimated from the inhibition of the average response at each test concentration compared to the control.
- 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

Nominal Concentration (mg/L)

Measured Concentration (mg/L)

Time-Weighted Mean Concentration (mg/L)

0 Hours

72 Hours

100

101.83d

51.42e

74

10

10.08

10.23

10

1

1.00

1.01

1

0.1

N/A

N/A

-

0 (Negative Control)

<MDL

<MDL

<MDL

d Average of duplicate analyses. The Relative Percent Difference (RPD) was 2.6%.

e Average of duplicate analyses. RPD was 0.23%.

MDL (Method Detection Limit) = 0.5 mg/L

N/A = not analysed (nominal concentration was < MDL)

 

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 Test Material

Test Endpoint

Nominal Concentration (mg/L)

0.1

1

10

100

% Inhibition in Average Specific Growth Rate (0 to 72 hours)

-2.98a

2.22

6.99

76.42

% Inhibition in Cell Yield (72 hour)

-17.63

11.34

31.57

98.81

a A negative value for % inhibition indicates stimulation as compared to the control.

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/)

0 hours

72 hours

80

77.55d

75.82e

40

38.92

33.24

20

20.49

19.78

10

10.18

9.68

5

5.39

5.72

2.5

2.59

2.36

1.25

1.50

1.13

0.625

0.64

0.71

0 (Negative control)

<MDL

<MDL

d Average of duplicate analyses, Relative Percent Difference (RPD) = 1.4 %.

e Average of duplicate analyses, RPD = 4.1 %.

MDL = 0.5 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)

Measured Concentrations at 0 hours (mg/L)

% Inhibition on Average Specific Growth Rate (0 to 72 hours)

% Inhibition on

Cell Yield

(72 hour)

80

77.55

58.47

95.07

40

38.92

40.27

86.78

20

20.49

16.42

55.50

10

10.18

2.49

10.76

5

5.39

-1.56*

-7.74

2.5

2.59

2.92

12.67

1.25

1.50

0.62

3.36

0.625

0.64

0.36

1.57

0 (Negative control)

<MDL

0.00

0.00

*A negative value for % inhibition indicates stimulation as compared to the control.

Table 5: Effects of the Test Material on Growth Inhibition (Average Specific Growth Rate and Cell Yield) of P. subcapitata Based on a 72-Hour Static Exposure

Endpoint

ECxa (95% Confidence Limits) (Nominal; mg/L)

NOEC (mg/L)

LOEC (mg/L)

EC10

EC20

EC50

Average Specific Growth Rate

(0 to 72 hours)

14.00

(11.04 - 16.38)

18.39

(16.00 - 20.68)

31.02

(28.52 - 33.74)

10.18

20.49

Cell Yield (72 hour)

10.05

(4.98 - 12.60)

12.71

(9.59 - 15.40)

19.93

(17.37 - 22.87)

10.18

20.49

a Effective concentration causing a specified x % inhibition.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the 72 h EC50 for the specific growth rate and cell yield were 31.02 and 19.93 mg/L, respectively.
Executive summary:

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

The test material was investigated by testing on the freshwater green alga, Pseudokirchneriella subcapitata under static test conditions for 72 hours. A range-finding test was performed to determine suitable concentrations for the definitive test.

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

Results of the range-finding test were based on measured concentrations, or more specifically, Time-Weighted Mean (TWM) concentrations. All test solutions and results were corrected for purity of the test material (i.e., 80% test material; 20% water). From the range-finding test, in terms of average specific growth rate and cell yield, the 72-hour EC10s, EC20s and EC50s were estimated to be in the range of <1 - 74 mg/L. The NOEC and LOEC from the range-finding test were estimated at 1 and 10 mg/L, respectively for both average specific growth rate and cell yield.

Results of the definitive test were generated using measured concentrations of the test material at test initiation, also corrected for purity. 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 14.00, 18.39 and 31.02 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 10.05, 12.71 and 19.93 mg/L, respectively. The NOEC and LOEC were estimated to be 10.18 and 20.49 mg/L, respectively for both average specific growth rate and cell yield.

Under the conditions of this study, the 72 h EC50 for the specific growth rate and cell yield were 31.02 and 19.93 mg/L, respectively.

Description of key information

Under the conditions of the study, the 72 h EC50 for the specific growth rate and cell yield were 31.02 and 19.93 mg/L, respectively.

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
31.02 mg/L
EC10, LC10 or NOEC for freshwater algae:
10.18 mg/L

Additional information

The potential of the test material to cause toxicity to aquatic 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).

The test material was investigated by testing on the freshwater green alga, Pseudokirchneriella subcapitata under static test conditions for 72 hours. A range-finding test was performed to determine suitable concentrations for the definitive test.

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

Results of the range-finding test were based on measured concentrations, or more specifically, Time-Weighted Mean (TWM) concentrations. All test solutions and results were corrected for purity of the test material (i.e., 80% test material; 20% water). From the range-finding test, in terms of average specific growth rate and cell yield, the 72-hour EC10s, EC20s and EC50s were estimated to be in the range of <1 - 74 mg/L. The NOEC and LOEC from the range-finding test were estimated at 1 and 10 mg/L, respectively for both average specific growth rate and cell yield.

Results of the definitive test were generated using measured concentrations of the test material at test initiation, also corrected for purity. 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 14.00, 18.39 and 31.02 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 10.05, 12.71 and 19.93 mg/L, respectively. The NOEC and LOEC were estimated to be 10.18 and 20.49 mg/L, respectively for both average specific growth rate and cell yield.

Under the conditions of this study, the 72 h EC50 for the specific growth rate and cell yield were 31.02 and 19.93 mg/L, respectively.