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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:
27 Jan - 30 Mar 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:
adopted 2006, corrected 2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Dec 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Swiss Federal Office of Public Health, Consumer protection directorate, Notification authority for chemicals, Bern, Switzerland (16 Dec 2015)
Analytical monitoring:
yes
Remarks:
HPLC/UV
Details on sampling:
- Concentrations: All test concentrations at 0 h (without algae) and at 72 h (with algae).
- Sampling method: Duplicate samples were taken from test media and the control. For sampling at the end of the test, the treatment replicates were pooled.
- Sample storage conditions before analysis: Frozen at -20 ± 5 °C. It was shown that the test item is stable under these storage conditions.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 100 mg test item/L in test water (50.2 mg in 500 mL) was prepared using ultrasonic treatment three times (for 15, 15, and 10 min) in combination with two times intense stirring (for 15 and 30 min) at room temperature. The test media were prepared by dilution of this highest test concentration with test water just before test start.
- Controls: Test water without test item.
- Evidence of undissolved material: The highest test concentration was a clear solution.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular green microalgae
- Strain: 61.81 SAG
- Age of inoculum (at test initiation): 4 d, the inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.
- Method of cultivation: Standardized conditions according to guidelines.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.15 mmol/L = 15 mg/L CaCO3 (AAP medium, calculated)
Test temperature:
22 °C
pH:
7.2 (0 h), 7.4 - 7.6 (72 h)
Nominal and measured concentrations:
Control, 0.32, 1.0, 3.2, 10, 32, and 100 mg/L (nominal)
< LOQ, n.a., n.a. 3.08, 9.84, 31.5, and 97.5 mg/L (0 h)
< LOQ, n.a., n.a., 3.02, 9.72, 31.1, and 96.5 mg/L (72 h)
n.a.: not analyzed
Details on test conditions:
TEST SYSTEM
- Test vessel: 75 mL Erlenmeyer flasks with 30 mL test solution
- Type: Covered with glass lid
- Initial cells density: 5000 cells/mL (nominal) (corresponding to 0.47 * 10^4 relative fluorescence units)
- Control end cells density: 70.1 * 10^4 relative fluorescence units (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes (AAP medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: AAP medium was prepared by dissolving analytical grade salts in sterile, purified water according to guidelines.
- Culture medium different from test medium: AAP medium was used for algal cultivation and testing.
- Intervals of water quality measurement: The pH was measured at 0 and 72 h. Temperature was monitored and recorded continuously. The appearance of the test media was visually controlled and recorded daily.

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination
- Light intensity and quality: 66 µE*s^-1*m^-2 (Multitron-Pro, Infors HT)
- Other: The test flasks were randomly positioned in a temperature controlled orbital shaker and repositioned daily.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Fluorimeter (excitation at 440 nm, emission at 680 nm; SpectraMax I3x, Molecular Devices Ltd)
- Determination of biomass: A small volume (200 µL) of the algal suspension was withdrawn daily from each test flask for the determination of algal biomass (fluorescence measurements) and was not replaced.
- Visual inspection: The shape and size of the algal cells were inspected after 72 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 3.2
- Range finding study: Yes
- Test concentrations: Control, 0.1, 1.0, 10, and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes. After 72 h inhibition of average growth rate was 4.7% at 10 mg/L (nominal) and 130% at 100 mg/L (nominal).
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval = 19 - 26 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
40 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval = 37 - 46 mg/L
Details on results:
- Exponential growth in the control: Yes, the biomass in the control increased by a factor of 148 over 72 h.
- Observation of abnormalities: None
- Any observations that might cause a difference between measured and nominal values: No remarkable observations, all test media were clear solutions throughout the test period.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes, the sensitivity was within the range recommended by the guideline (0.9 - 1.5 mg/L).
- ErC50 (72 h): 0.9 mg/L
- Other: Tested twice yearly. Last test dating from October 2016.
Reported statistics and error estimates:
Statistical analysis was performed using ToxRat Professional. The 72-h EC10, EC20, and EC50 values for the inhibition of average growth rate and yield and the corresponding 95% confidence intervals were calculated by Probit Analysis using linear maximum likelihood regression. For the determination of the LOEC and NOEC values for average growth rate and yield, test concentrations were compared to control values by the Williams t-test or Welch t-test, where appropriate.

ANALYTICAL RESULTS

The samples from the nominal test concentrations of 0.32 and 1.0 mg/L were not analyzed, since these concentrations were below the NOEC

determined in this test and thus not relevant for the interpretation of the biological results. The measured concentrations of the test item in the test media (3.2 - 100 mg/L) were between 96 and 98% of the nominal values at the start of the test and between 94 and 97% at the end of the test (Table 1). Thus, the test media were correctly dosed and the test item was stable in the test media throughout the test period of 72 h. Therefore, the biological results were related to the nominal concentrations of the test item.

Table 1. Results for test samples (rounded).

Sampling day/sample age

[d/h]

Nominal concentration of test item

[mg/L]

Measured concentration of test item

[mg/L]

Sample preparation factor

Determined concentration of test item

[mg/L]

% of nominal concentration

0/0

(fresh media)

Control

n.d.

1.1

< LOQ

n.a.

3.2

2.80

1.1

3.08

96

10

8.95

1.1

9.84

98

32

28.6

1.1

31.5

98

100

88.6

1.1

97.5

97

3/72

(aged media)

Control

n.d.

1.1

< LOQ

n.a.

3.2

2.74

1.1

3.02

94

10

8.84

1.1

9.72

97

32

28.3

1.1

31.1

97

100

87.7

1.1

96.5

97

n.d. = no test item detected

n.a. = not applicable

LOQ = 0.08 mg/L

BIOLOGICAL RESULTS

After 72 h, the test item showed significant inhibitory effects on the yield and growth rate of the algae at the nominal test concentrations of 10 and 100 mg/L (Table 2). The NOEC (72 h) and LOEC (72 h) for yield and growth rate is 3.2 mg/L and 10 mg/L, respectively.

Table 2. Section-by section growth rates (day^-1) and inhibition of growth rates (Ir).

Nominal

test item concentration

 

[mg/L]

 Section-by-Section Growth Rates (day^-1)

and Inhibition of the growth rates (Ir)

0 – 24 h

24 – 48 h

48 – 72 h

µ

Ir [%]

µ

Ir [%]

µ

Ir [%]

Control

1.551

0.0

1.803

0.0

1.643

0.0

0.32

1.536

0.9

1.849

-2.5

1.605

2.3

1.0

1.456

6.1

2.005

-11.2

1.548

5.8

3.2

1.709

10.2

1.660

7.9

1.531

6.8

10

1.570

-1.2

1.665

7.7

1.547

5.8

32

1.471

5.1

1.065

40.9

0.979

40.4

100

0.608

60.8

-0.247

113.7

-0.389

123.7

Note: Percentage inhibition values in excess of 100% are obtained when the biomass at the end of the interval is lower than at the start of the interval.

Table 3. Average Growth Rates (µ)

Nominal test item concentration

[mg/L]

 

Average growth rate µ (day -1) and Inhibition of µ (Ir)

0 – 24 h

0 – 48 h

0 – 72 h

µ

Ir [%]

µ

Ir [%]

µ

Ir [%]

Control

1.551

0.0

1.677

0.0

1.666

0.0

0.32

1.536

0.9

1.693

-0.9

1.663

0.1

1.0

1.456

6.1

1.731

-3.2

1.670

-0.2

3.2

1.709

-10.2

1.684

-0.4

1.633

1.9

10

1.570

-1.2

1.617*

3.6

1.594#

4.3

32

1.471

5.1

1.268*

24.4

1.172#

29.7

100

0.608*

60.8

0.181*

89.2

-0.009#

100.5

#: Mean value statistically significantly lower than in the control (according to Welch t-test one-sided smaller, α = 0.05).

*: Mean value statistically significantly lower than in the control (according to a Williams t-test one-sided smaller, α = 0.05).

Note: Percentage inhibition values in excess of 100% are obtained when the biomass at the end of the interval is lower than at the start of the interval.

 

Validity criteria fulfilled:
yes
Executive summary:

This study resulted in an ErC10 (72 h) of 24 mg/L and an ErC50 (72 h) of 40 mg/L (nominal) based on the inhibition of the growth rate of P. subcapitata (OECD 201, GLP).

Description of key information

ErC10 (72 h) = 24 mg/L (nominal, OECD 201, P. subcapitata)

ErC50 (72 h) = 40 mg/L (nominal, OECD 201, P. subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:
40 mg/L
EC10 or NOEC for freshwater algae:
24 mg/L

Additional information

There is one study available, in which the toxicity of the substance to aquatic algae was investigated according to OECD guideline 201 and GLP.

In a static test, Pseudokirchneriella subcapitata at an initial concentration of 5000 cells/mL (nominal) were exposed to nominal test item concentrations of 0.32, 1.0, 3.2, 10, 32, and 100 mg/L for 72 h under controlled conditions. The test item concentrations in the test media were analytically verified at the beginning (0 h) and at the end of the test (72 h) by HPLC-UV.

The measured concentrations of the test item in the test media were between 96 and 98% of the nominal values at the start of the test (0 h) and between 94 and 97% at the end of the test (72 h). Thus, the test item was correctly dosed and stable throughout the duration of the test. The biological results were therefore based on nominal test item concentrations. 

After 72 h, the test item significantly inhibited cell growth in the 10 to 100 mg/L treatments. The obtained ErC10 (72 h) is 24 mg/L and the ErC50 (72 h) is 40 mg/L.