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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:
10-12-2019 to 07-01-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met..
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23 (2000)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 0.32, 1.0, 3.2, 10.0 and 32.0 mg/L prepared as WSF (water soluble fraction ; the result of a WAF (water accommodated fractions) subject to a separation stage).
Singular samples for possible analysis were taken from all test concentrations and the control; Frequency at t=0 h and t=72 h; further maintenance of actual concentrations was demonstrated by running a test vessel at 3.2 mg/L (WSF) substance concentration but without algae (abiotic control) and samples for analysis were taken at the start of exposure and at the end of the test period.
- Sampling method: 1 mL volume samples were extracted; the replicates were not pooled at each concentration before sampling.
- Sample storage conditions before analysis: Stored in a freezer (≤ -15°C) until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The preparation procedure for test solutions was based on a non-GLP saturation test and range-finder (cited in the full study report). Preparation of test solutions started with loading rates individually prepared at 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. The test item was added to stirring test medium on a volume basis by means of an automated displacement pipette. The required volume of test item for each treatment group was determined based on the test item’s density of 0.978 g/cm3. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. The obtained mixtures were allowed to settle for a period of approximately two hours. Thereafter, the aqueous Water Soluble Fractions (WSFs) were collected by means of siphoning through glass wool and used as test concentrations. All test solutions were clear and colourless at the end of the preparation procedure. The test solutions preparation were performed under dimmed lighting and aluminium wrapped glassware with minimal headspace. After preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.8 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL. Thereafter, the replicates were closed airtight with minimal headspace to prevent potential vaporization of test item. Five test item concentrations in a geometric series with a separation factor of 3.2, were prepared as follows: 0 (control), 0.32, 1.0, 3.2, 10, 32 and 100 mg/L loading rates as WSF. For the definitive test: equivalent geometric mean measured concentrations were: 0 (control), 0.36, 0.76, 1.6, 3.9, 8.5 and 24 mg/L which were based on analysis during the definitive test period.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: For positive control - reference item: potassium dichromate were prepared in a separately conducted reference test (documented in the full study report). A negative/blank control without test item or reference item was also included.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Prior to start of the exposure, the test solutions were checked for undissolved test item. Presence of undissolved test item during preparation and during the test was not observed. Undissolved test item was excluded on the basis of application of loading rates as WSFs.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): 3 days (in pre-culture under the same conditions as the test).
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: 3 days pre-culture.
- Culturing media and conditions (same as test or not): No.
Stock Culture Medium M1 ; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis) with the following composition: NaNO3 500 mg/L; K2HPO4.3H2O 52 mg/L; MgSO4.7H2O 75 mg/L; Na2CO3.10H2O 54 mg/L; C6H8O7.H2O 6 mg/L; NH4NO3 330 mg/L; CaCl2.2H2O 35 mg/L; C6H5FeO7.xH2O 6 mg/L; H3BO3 2.9 mg/L; MnCl2.4H2O 1.81 mg/L; ZnCl2 0.11 mg/L; CuSO4.5H2O 0.08 mg/L; (NH4)6Mo7O24.4H2O 0.018 mg/L
Pre-Culture and definitive test adjusted-Medium M2 : NH4Cl 15 mg/L; MgCl2.6H2O 12 mg/L; CaCl2.2H2O 18 mg/L; MgSO4.7H2O 15 mg/L; KH2PO 1.6 mg/L; FeCl3.6H2O 64 µg/L; Na2EDTA.2H2O 100 µg/L; H3BO3 185 µg/L; MnCl2.4H2O 415 µg/L; ZnCl2 3 µg/L; CoCl2.6HO 1.5 µg/L; CuCl2.2H2O 0.01 µg/L; Na2MoO4.2H2O 7 µg/L; NaHCO3 300 mg/L; Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/l); pH 7.1 ± 0.3
- Any deformed or abnormal cells observed: None reported.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
In accordance with the OECD TG 201 guideline.
Hardness:
Ca+Mg: 0.24 mmol/l (24 mg CaCO3/l)
Test temperature:
During the exposure period the temperature measured in the incubator was maintained between 21 and 23 °C. Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).
pH:
0 hours: pH 7.3 ± 0.1 ; 72 hours: pH 7.5 - 8.1 (definitive test concentrations) and pH 8.2 (controls). pH did not vary more than 1.5 units.
Nominal and measured concentrations:
Range-finder test: 0 (Control), 1.0, 10.0 and 100.0 mg/L prepared as WSF
The preparation procedure for test solutions was based on a non-GLP saturation test and range-finder (cited in the full study report).
Final test: 0 (control), 0.32, 1.0, 3.2, 10.0 and 32.0 mg/L prepared as WSF (water soluble fraction ; the result of a WAF (water accommodated fractions) subject to a separation stage).
See table 3 for WSF and measured concentrations at initial exposure and during the course of the test.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass.
- Type (delete if not applicable): Closed - Static
- Material, size, headspace, fill volume: Glass, 40 mL, containing ca. 40 mL test solution (minimal headspace to prevent test item losses); closed airtight (capped)
- Aeration: Vessel shaken continuously (ca. 175 rpm)
- Initial cells density: 1 x 10^4 cells/mL.
- Control end cells density: Mean (of replicates after 72 hours) 174.5 x10^4 cells/mL (factor x175 increase)
- No. of vessels per concentration (replicates): 3 replicates of each test concentration
- No. of vessels per control (replicates): 6 replicates of the control ; 1 or 2 replicates of each test concentration without algae (abiotic control)
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used: Yes. adjusted-Medium M2.
- Detailed composition if non-standard medium was used: adjusted M2 according to OECD 201 using RO-water: NH4Cl 15 mg/L; MgCl2.6H2O 12 mg/L; CaCl2.2H2O 18 mg/L; MgSO4.7H2O 15 mg/L; KH2PO 1.6 mg/L; FeCl3.6H2O 64 µg/L; Na2EDTA.2H2O 100 µg/L; H3BO3 185 µg/L; MnCl2.4H2O 415 µg/L; ZnCl2 3 µg/L; CoCl2.6HO 1.5 µg/L; CuCl2.2H2O 0.01 µg/L; Na2MoO4.2H2O 7 µg/L; NaHCO3 300 mg/L; Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/l); pH 7.1 ± 0.3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis)
- Culture medium different from test medium: Yes. Medium M1 used for stock culture medium. adjusted-Medium M2 used for pre-culture medium and test medium. See above for more information.
- Intervals of water quality measurement: Not reported. Media pH was recorded at t=0 and t=72 hours

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 60 to 120 µE/m2/s (in 400 to 700 nm range), typically : The light intensity in the incubator was not measured at the start and at the end of the test. Consequently, it could not be verified whether the light intensity fell within the optimal range. This was not considered to be a significant deviation as control cultures indicated the conditions were adequate to support algal growth (factor x175 increase over the exposure period.).
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Spectrophotometer at 680 nm with immersion probe (pathlength = 10 mm). Algal medium used as blank.
- Other: Initial cell density: Cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2. In definitive test justified from the results of the range finding study.
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes.
- Test concentrations: Three replicates per concentration were exposed to dilutions representing 0 (Control), 1.0, 10.0 and 100.0 mg/L prepared as WSF in the range-finding test. In the definitive test 5 concentrations were used at 0.32, 1.0, 3.2, 10.0 and 32.0 mg/L prepared as WSF (water soluble fraction ; the result of a WAF (water accommodated fractions) subject to a separation stage).
- Results used to determine the conditions for the definitive study: Yes.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
7.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 6.6 - 8.5 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 2.7 - 3.2 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 2.1 - 3.8 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.98 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 0.91 - 1.0 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: NOEC based on biological relevance ; 0.76 mg/L when based on statistical significance
Remarks:
Growth rate inhibition was considered not to be biologically relevant at 1.6 mg/L, where the observed inhibition was below 10%.
Details on results:
- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells at the concentration closest to the EC50 when compared to the control.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels: Not reported.
- Aggregation of algal cells: No.
- Other: Noted considerable differences in algal cell densities measured between replicates of the WSFs prepared at 3.2, 10 and 32 mg/L. Based on the analytical results, it can be concluded that the higher cell density recorded in replicate 2 of the WSF prepared at 32 mg/L was most likely caused by a lower exposure concentration in the deviant replicate. Concentrations between replicates of the WSFs prepared at 3.2 and 10 mg/L were relatively close, and therefore, the actual cause for the lower cell density in these replicates could not be identified. Consequently, all replicates were included in the statistical analysis and no data was disregarded.
- Any stimulation of growth found in any treatment: Yes. At low concentrations 0.36 and 0.76 mg/L GMM growth inhibition was negative between 24-48h and/or for 0.76 mg/L GMM at 48-72h. This was low dose stimulation and was limited to < 14 % and was taken into account in the data analysis and effect level calculations.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Final test: During the exposure period the measured concentrations decreased in the range finder and definitive test from initial values. At the start of the test, the concentration measured in the solution without algae was slightly higher than the concentration in the solution with algae. The concentration in the solution without algae decreased to 25% relative to the initial value, while the concentration in the solution with algae was at 87% of initial at the end of the test. The presence of algae may have affected the test item concentration throughout the test. However, it should be considered that variation in concentrations between vessels may have been caused by volatility of the test item. Due to only a single sample per treatment being analysed (with and without algae) no reliable conclusions were drawn on the possible effects of algae on actual exposure.
- Effect concentrations exceeding solubility of substance in test medium: No.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes.
- EC50: The EC50 for growth rate inhibition (72h-ERC50) was within the expected range. The reference test was conducted in a separate study (cited in the full study report) under GLP.
- Other: The sensitivity of the test system was in agreement with the historical data.
Reported statistics and error estimates:
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (The Shapiro-Wilk´s Test on Normal Distribution and Levene´s Test on Variance Homogeneity (with Residuals) were performed. For the effect concentrations including NOEC: Step-down Jonckheere- Terpstra test, α=0.05, one-sided, smaller). Additionally, calculation of ERCx-values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition versus the logarithms of the corresponding average exposure concentrations of the test item. Calculation of EYCx-values was based on a 3-parameter normal cumulative distribution function (CDF) using non-linear regression analysis with the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test item.

ToxRat Professional v 3.2.1 was the software package utilised.

Statistically significant inhibition of growth rate was found at GMM test concentrations of 1.6 mg/L and higher. A NOEC of 0.76 mg/L is suggested by the program. Note that the 8.2% inhibition as found for 1.6 mg/L can be considered as not biologically relevant based on the fact that the effect is below 10%.

Inhibition of yield increased with increasing GMM concentration of test item from 1.6 mg/L (30% inhibition) upwards resulting in 93% inhibition at 8.5 mg/L and 199% inhibition at 24 mg/L. Statistically significant inhibition of yield was found at test concentrations of 1.6 mg/L and higher. A NOEC of 0.76 mg/L is suggested by the program.

Table 1. Mean cell densities (x10^4 cells/mL) during the range-finding test

Time (h)

Test item, WSF loading rate concentration (mg/L)

 

Control

1.0

10.0

100

0

1.0

1.0

1.0

1.0

72

214

187

66

1.1

 

 

 

 

 

 

Table 2. Percentage inhibition of growth rate and inhibition of yield during the range-finding test

 

Growth rate (0 – 72h)

Yield (0 – 72h)

Test item, WSF loading rate concentration (mg/L) 

mean

Std. Dev.

n

% inhibition

mean

Std. Dev.

n

% inhibition

Control

1.788

0.0212

3

-

213.2

13.82

3

-

1.0

1.743

0.0061

3

2.6

185.5

3.41

3

13

10.0

1.394

0.0251

3

22

64.7

4.84

3

70

100.0

0.041

0.0616

3

98

0.1

0.22

3

100

 

 

 

 

 

 

 

 

 

 

Table 3. Measured concentrations versus nominal concentrations: final test

Test item, WSF loading rate concentration (mg/L)

Measured concentrations (mg/L) at time point

Geometric Mean Measured average (mg/L)

t=0h

t=72h

0 (Control)

n.d.

n.d.

0

0.32

0.38

0.34

0.36

1.0

0.86

0.67

0.76

3.2

1.7

1.6#

1.6

3.2 #WA

2.0

0.50

1.0*

10

4.3

3.5#

3.9

32

11

6.8#

8.5

100

28

21

24

 

 

 

 

n.d. = not detected

n.a. = not applicable

#WA = without algae

* = Abiotic control, incubated without algae

# = Mean value of concentrations analysed in replicate 1 and 2.

 

Table 4. Percentage reduction of growth rate (total test period) and percentage inhibition of yield during the final test

Test item, WAF concentration (mg/L) 

Geometric Mean Measured average (mg/L)

Growth rate (0 – 72h)

Yield (0 – 72h)

mean

Std. Dev.

n

% inhibition

mean

Std. Dev.

n

% inhibition

0 (Control)

0

1.720

0.0144

6

-

173.5

7.45

6

-

0.32

0.36

1.706

0.0062

3

0.82

166.2

3.13

3

4.2

1.0

0.76

1.710

0.0046

3

0.58

168.3

2.36

3

3.0

3.2

1.6

1.580

0.1657

3

8.2#

122.1

50.88

3

30*

10

3.9

1.408

0.1958

3

18*

74.6

35.67

3

57*

32

8.5

0.764

0.2632

3

56*

11.3

10.15

3

93*

100

24

0.000

0.0000

3

100*

0.0

0.00

3

100*

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

* = effect was statistically significant

# = effect not biologically relevant (< 10%)

 

Table 5. Mean cell densities (x10^4 cells/mL) during the final test

 

Replicate

Geometric Mean Measured average (mg/L)

0 (Control)

0.36

0.76

1.6

3.9

8.5

24

t=0h

1

1.000

1.000

1.000

1.000

1.000

1.000

1.000

2

1.000

1.000

1.000

1.000

1.000

1.000

1.000

3

1.000

1.000

1.000

1.000

1.000

1.000

1.000

4

1.000

 

 

 

 

 

 

5

1.000

 

 

 

 

 

 

6

1.000

 

 

 

 

 

 

n

6

3

3

3

3

3

3

Mean

1.0

1.0

1.0

1.0

1.0

1.0

1.0

Std. Dev.

0.0

0.0

0.0

0.0

0.0

0.0

0.0

 

CV

0.0

0.0

0.0

0.0

0.0

0.0

0.0

t=24h

1

9.995

4.092

3.800

4.933

3.717

2.714

2.094

2

7.809

4.577

3.412

4.785

3.329

3.532

1.877

3

4.628

5.640

5.062

4.734

5.044

2.557

2.404

4

5.132

 

 

 

 

 

 

5

3.953

 

 

 

 

 

 

6

3.250

 

 

 

 

 

 

n

6

3

3

3

3

3

3

Mean

5.8

4.8

4.1

4.8

4.0

2.9

2.1

Std. Dev.

2.6

0.8

0.9

0.1

0.9

0.5

0.3

CV

44.6

16.6

21.1

2.1

22.3

17.8

12.5

t=48h

1

29.459

31.600

29.395

26.454

19.954

11.068

4.244

2

31.189

29.053

28.317

18.747

10.842

8.733

3.722

3

31.161

29.792

31.628

28.484

19.862

6.440

4.221

4

31.623

 

 

 

 

 

 

5

30.574

 

 

 

 

 

 

6

30.648

 

 

 

 

 

 

n

6

3

3

3

3

3

3

Mean

30.8

30.1

29.8

24.6

16.9

8.7

4.1

Std. Dev.

0.8

1.3

1.7

5.1

5.2

2.3

0.3

CV

2.4

4.3

5.7

20.9

31.0

26.5

7.3

t=72h

1

162.235

170.737

167.274

149.988

100.866

7.762

1.000

2

180.071

164.949

168.606

64.467*

34.804*

23.930*

1.000

3

175.601

165.795

171.870

154.972

91.166

5.224

1.000

4

172.711

 

 

 

 

 

 

5

172.623

 

 

 

 

 

 

6

183.904

 

 

 

 

 

 

n

6

3

3

3

3

3

 

Mean

164.1

174.5

167.2

169.3

123.1

75.6

12.3

Std. Dev.

15.8

7.5

3.1

2.4

50.9

35.7

10.1

CV

9.6

4.3

1.9

1.4

41.3

47.2

82.5

* Algal cell density in this replicate was considered as remarkably deviating from the cell densities in the other replicates at this treatment.

 

Table 2. Section by section growth rate (per day) during the final test

 

Replicate

Geometric Mean Measured average (mg/L)

0 (Control)

0.36

0.76

1.6

3.9

8.5

24

t=0h - t=24h

1

2.302

1.409

1.335

1.596

1.313

0.998

0.739

2

2.055

1.521

1.227

1.565

1.203

1.262

0.630

3

1.532

1.730

1.622

1.555

1.618

0.939

0.877

4

1.635

 

 

 

 

 

 

5

1.374

 

 

 

 

 

 

6

1.179

 

 

 

 

 

 

n

6

3

3

3

3

3

3

Mean

1.680

1.553

1.395

1.572

1.378

1.066

0.749

Std. Dev.

0.4234

0.1628

0.2039

0.0214

0.2153

0.1719

0.1240

% Inh.

-

7.5

17

6.4

18

37

55

t=24h - t=48h

1

1.081

2.044

2.046

1.679

1.681

1.406

0.706

2

1.385

1.848

2.116

1.366

1.181

0.905

0.685

3

1.907

1.664

1.832

1.795

1.371

0.924

0.563

4

1.818

 

 

 

 

 

 

5

2.046

 

 

 

 

 

 

6

2.244

 

 

 

 

 

 

n

6

3

3

3

3

3

3

Mean

1.747

1.852

1.998

1.613

1.411

1.078

0.651

Std. Dev.

0.4340

0.1899

0.1478

0.2221

0.2523

0.2837

0.0773

% Inh.

-

-6.0

-14

7.6

19

38

63

t=48h - t=72h

1

1.706

1.687

1.739

1.735

1.62

-0.355

-1.446

2

1.753

1.737

1.784

1.235

1.166

1.008

-1.314

3

1.729

1.717

1.693

1.694

1.524

-0.209

-1.440

4

1.698

 

 

 

 

 

 

5

1.731

 

 

 

 

 

 

6

1.792

 

 

 

 

 

 

n

6

3

3

3

3

3

3

Mean

1.735

1.713

1.739

1.555

1.437

0.148

-1.400

Std. Dev.

0.0342

0.0249

0.0457

0.2776

0.2392

0.7484

0.0743

% Inh.

-

1.2

-0.21

10

17

91

181

Validity criteria fulfilled:
yes
Conclusions:
The test item 72h-ErC50 for growth rate reduction was 7.4 (C.I. 6.6 – 8.5) mg/L based on geometric mean measured concentrations. The corresponding ErC10 was 3.0 (C.I. 2.1 – 3.8) mg/L. The NOEC based on statistical significance was 0.76 mg/L, however based on biological relevance the NOEC was 1.6 mg/L. This was the concentration where the observed inhibition was below 10%.
Executive summary:

The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. Following a range finding study at 0 (Control), 1.0, 10.0 and 100.0 mg/L prepared as WSF, a definitive study was conducted under static conditions with an initial cell density of 1.0x10^4 cells/mL. Five test item solutions were employed in a geometric series with a separation factor of 3.2, were prepared as follows: 0 (control), 0.32, 1.0, 3.2, 10.0 and 32.0 mg/L prepared as WSF (water soluble fraction ; the result of a WAF (water accommodated fractions) subject to a separation stage). The preparation procedure for the definitive test solutions was based on a non-GLP saturation test and the range-finder test. Preparation of test solutions started with respective loading rates were individually prepared. The test item was added to stirring test medium on a volume basis by means of an automated displacement pipette. The required volume of test item for each treatment group was determined based on the test item’s density of 0.978 g/cm3.A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. The obtained mixtures were allowed to settle for a period of approximately two hours. Thereafter, the aqueous Water Soluble Fractions (WSFs) were collected by means of siphoning through glass wool and used as test concentrations. All test solutions were clear and colourless at the end of the preparation procedure. The test solutions were performed under dimmed lighting and aluminium wrapped glassware with minimal headspace. After preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.8 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL. Thereafter, the replicates were closed airtight with minimal headspace to prevent potential vaporization of test item. Three replicates were tested for each test item concentration and six replicates for the control under constant illumination and shaking at a temperature of 22 ± 1 °C. Test vessels were completely filled and closed to prevent any volatilization of the test item. Environmental conditions were within the acceptable limits. The concentrations of the test item were analytically verified via UPLC-MS at 0 hours (test start) and at 72 hours (test end) of the exposure. At the start of the test, the concentration measured in the solution without algae was slightly higher than the concentration in the solution with algae. The concentration in the solution without algae decreased to 25% relative to the initial value, while the concentration in the solution with algae was at 87% of initial at the end of the test. The equivalent geometric mean measured concentrations were: 0 (control), 0.36, 0.76, 1.6, 3.9, 8.5 and 24 mg/L which were based on analysis during the definitive test period. The study met the acceptability criteria prescribed by the protocol and was considered valid. The test item 72h-ErC50 for growth rate reduction was 7.4 (C.I. 6.6 – 8.5) mg/L based on geometric mean measured concentrations. The corresponding ErC10 was 3.0 (C.I. 2.1 – 3.8) mg/L. The NOEC based on statistical significance was 0.76 mg/L, however based on biological relevance the NOEC was 1.6 mg/L. This was the concentration where the observed inhibition was below 10%.

Description of key information

72h-EC50 (aquatic algae; growth rate) = 7.4 (C.I. 6.6 – 8.5) mg/L based on GMM concentrations, 72 hour, freshwater, OECD TG 201, 2020

72h-EC10 (aquatic algae; growth rate) = 3.0 (C.I. 2.1 – 3.8) mg/L based on GMM concentrations, 72 hour, freshwater, OECD TG 201, 2020

72h-NOEC (aquatic algae; growth rate) = 1.6 mg/L based on GMM concentrations and biological significance, 72 hour, freshwater, OECD TG 201, OECD TG 201, 2020

Key value for chemical safety assessment

EC50 for freshwater algae:
7.4 mg/L
EC10 or NOEC for freshwater algae:
1.6 mg/L

Additional information

Key study : OECD TG 201, 2020 : The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. Following a range finding study at 0 (Control), 1.0, 10.0 and 100.0 mg/L prepared as WSF, a definitive study was conducted under static conditions with an initial cell density of 1.0x10^4 cells/mL. Five test item solutions were employed in a geometric series with a separation factor of 3.2, were prepared as follows: 0 (control), 0.32, 1.0, 3.2, 10.0 and 32.0 mg/L prepared as WSF (water soluble fraction ; the result of a WAF (water accommodated fractions) subject to a separation stage). The preparation procedure for the definitive test solutions was based on a non-GLP saturation test and the range-finder test. Preparation of test solutions started with respective loading rates were individually prepared. The test item was added to stirring test medium on a volume basis by means of an automated displacement pipette. The required volume of test item for each treatment group was determined based on the test item’s density of 0.978 g/cm3.A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. The obtained mixtures were allowed to settle for a period of approximately two hours. Thereafter, the aqueous Water Soluble Fractions (WSFs) were collected by means of siphoning through glass wool and used as test concentrations. All test solutions were clear and colourless at the end of the preparation procedure. The test solutions were performed under dimmed lighting and aluminium wrapped glassware with minimal headspace. After preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.8 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL. Thereafter, the replicates were closed airtight with minimal headspace to prevent potential vaporization of test item. Three replicates were tested for each test item concentration and six replicates for the control under constant illumination and shaking at a temperature of 22 ± 1 °C. Test vessels were completely filled and closed to prevent any volatilization of the test item. Environmental conditions were within the acceptable limits. The concentrations of the test item were analytically verified via UPLC-MS at 0 hours (test start) and at 72 hours (test end) of the exposure. At the start of the test, the concentration measured in the solution without algae was slightly higher than the concentration in the solution with algae. The concentration in the solution without algae decreased to 25% relative to the initial value, while the concentration in the solution with algae was at 87% of initial at the end of the test. The equivalent geometric mean measured concentrations were: 0 (control), 0.36, 0.76, 1.6, 3.9, 8.5 and 24 mg/L which were based on analysis during the definitive test period. The study met the acceptability criteria prescribed by the protocol and was considered valid. The test item 72h-ErC50 for growth rate reduction was 7.4 (C.I. 6.6 – 8.5) mg/L based on geometric mean measured concentrations. The corresponding ErC10 was 3.0 (C.I. 2.1 – 3.8) mg/L. The NOEC based on statistical significance was 0.76 mg/L, however based on biological relevance the NOEC was 1.6 mg/L. This was the concentration where the observed inhibition was below 10%.