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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:
2019-06-06 to 2019-07-19
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)
Version / remarks:
adopted March 23, 2006, corrected July 28, 2011
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No.: LSSI2A8010;
Purity: 99-101 % (nominal)/ 101 % (analytical)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1000, 320, 100, 32, 10 mg test item/L and control
- Sampling method: The samples were taken from the biological phase of the study. Collecting, storage and handing over of the samples were the study director’s responsibility. The information concerning the samples was provided by the study director.
Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test by pouring together the contents of the test beakers of each treatment.
All samples remained undiluted until analysis.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. Afterwards the samples were again stored deep frozen (≤ -20 °C) and will be kept stored up to the date of the final report.
- Analyses: The concentrations of the test item succinimide were analysed in the duplicate test media samples from all test concentrations, and in the duplicate control samples from both sampling times (0 and 72 hours).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1000 mg test item/L was prepared by dissolving 1007.3 mg test item into 1007.3 mL test water by intense stirring for 30 minutes. The pH of the stock solution was assessed from 7.1 to 8.1 with 1 M NaOH. Adequate volumes of this stock solution were diluted with test water to prepare the test media of the desired test concentrations.
The test media were prepared just before introduction of the algae (= start of the test).
- Controls: In the control, test water was used without addition of the test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle applied
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n.a.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): At the beginning of the test all test item was dissolved. At the end of the test in the replicates of the two highest concentrations some precipitate was observed lying at the bottom of the test vessel and at the surface of the test medium. This could either be the test item itself or a degradation product. The measured concentration for these concentrations were 86 and 85 % of the nominal value for the duplicate samples of 320 mg/L at test end and 93 and 94 % of the nominal value for the duplicate samples of 1000 mg/L.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
Species: Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
Origin: The algae were originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzen-wissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

The test was started (0 hours) by inoculation of a biomass of nominal 5000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 4 days prior to the test start under the same conditions as in the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol/L (= 24 mg/L) as CaCO3
Test temperature:
22.9 to 23.4 °C
pH:
pH value in the control at test start: 8.1,
pH value in the control at test end: 9.4;
pH values in the test item treatments at test start: 8.0 to 8.1,
pH values in the test item treatments at test end: 7.6 to 9.5
Nominal and measured concentrations:
Nominal: 1000, 320, 100, 32 and 10 mg test item/L and control
Geometric mean of measured concentrations: 962, 293, 83.7, 25.8 and 6.18 mg test item/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 50 mL volume with nominal 50 mL of test medium covered with air permeable glass dishes (watch glasses).
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): n.a.
- Renewal rate of test solution (frequency/flow rate): n.a.
- Initial cells density: 5000 algal cells per mL test medium
- Control end cells density: 1 097 450 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): n.a.

COURSE OF THE TEST
- Test Procedure: The test units were continuously shaken. The flasks were covered with glass dishes and incubated in a water bath. The flasks were placed in a random order and were repositioned each day to minimize differences in test conditions.
- Blanks: Additionally, one replicate of each test concentration and of the control was prepared without algae to provide a "blank" for the spectrophotometric measurements. The additional replicates were incubated under the same conditions as described above. The blank values were subtracted from the absorption measured in the samples containing algae to eliminate absorption caused by the test item.

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: n.a.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water
- MACRO-NUTRIENTS:
NaHCO3 = 50.0 mg/L
CaCl2 x 2 H2O = 18.0 mg/L
NH4Cl = 15.0 mg/L
MgSO4 x 7 H2O = 15.0 mg/L
MgCl2 x 6 H2O = 12.0 mg/L
KH2PO4 = 1.6 mg/L

- TRACE ELEMENTS:
Na2EDTA x 2 H2O = 100.0 µg/L
FeCl3 x 6 H2O= 64.0 µg/L
MnCl2 x 4 H2O = 415.0 µg/L
H3BO3 = 185.0 µg/L
Na2MoO4 x 2 H2O = 7.0 µg/L
ZnCl2 = 3.0 µg/L
CoCl2 x 6 H2O = 1.5 µg/L
CuCl2 x 2 H2O = 0.01 µg/L
The culture medium was prepared 17 days before test start to allow pH to stabilise.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: Yes with 1 M NaOH during medium preparation
- Photoperiod: Continuous
- Light intensity and quality: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.
Mean light intensity: 5355 lx (range: 5180 to 5670 lx)
- Salinity (for marine algae): n.a.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of the Cell Density: The cell density on each observation time was determined by spectrophotometric measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae). Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.

- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: Yes
- Test concentrations: 0.1, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no
Remarks:
For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 962 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 962 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
293 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
962 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): At the end of the exposure time the test water was slightly brown, and the test item was lying at the bottom and floating at the surface (nominal 320 and 1000 mg test item/L)
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the end of the exposure time the test water was slightly brown, and the test item was lying at the bottom and floating at the surface at the replicates with nominal 320 and 1000 mg test item/L.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
Results of the most recent test with Pseudokirchneriella subcapitata performed with the reference item potassium dichromate in January 2019 (Study No.: 129865210)
- Results with reference substance valid? Yes
- ErC50: 0.941 mg test item/L (95 % Conf. Interval 0.910 - 0.973)
- Other:
72-hour EC50 for Yield: 0.445 mg test item/L (95 % Conf. Interval 0.437 - 0.453)
72-hour EC50 for Biomass: 0.494 mg test item/L (95 % Conf. Interval 0.485 - 0.504)

72-hour NOEC for Yield < 0.094 mg test item/L
72-hour NOEC for Growth rate 0.094 mg test item/L
72-hour NOEC for Biomass < 0.094 mg test item/L

72-hour LOEC for Yield ≤ 0.094 mg test item/L
72-hour LOEC for Growth rate 0.21 mg test item/L
72-hour LOEC for Biomass ≤ 0.094 mg test item/L
Reported statistics and error estimates:
The 72-hour ErC50 and the 72-hour EyC50 (see Definitions), the corresponding EC20 and EC10 values could not be calculated as no significant effects were observed on the growth rate and regarding yield only the highest concentration displayed a significant effect.
For the determination of the 72 hour LOEC and the 72 hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Bonferroni-Welch t-test (yield) and Bonferroni-U-test (growth rate).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH.

Microscopic Observations: The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 1000 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.

 

Cell Density

Increase in Control Cultures: 219.5-fold increase within 72 hours and thus, the validity criterion of a factor of at least 16 within 72 hours was met.

Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures: 21.1 % and thus, the validity criterion (must not exceed 35 %) was met.

Coefficient of Variation of Average Growth between Control Replicates: 0.5 % and thus, the validity criterion (must not exceed 7 %) was met.

Table 1: Algal Cell Densities during the Test Period of 72 hours; m: mean value; s: standard deviation

Geometric mean of measured concentrations [mg test item/L]

Flask

No.

Number of algal cells [10 000 cells /mL] after

24 hours

48 hours

72 hours

Control

1

4.138

25.815

107.046

2

4.376

27.483

112.286

3

4.376

26.292

105.854

4

4.138

27.483

112.286

5

4.376

27.483

108.237

6

4.376

28.197

112.763

 

 

 

 

 

m

4.297

27.125

109.745

s

0.123

0.888

3.057

 

 

 

 

 

6.18

1

3.900

28.674

118.956

2

4.138

28.674

119.194

3

4.138

27.959

114.668

 

 

 

 

 

m

4.059

28.436

117.606

s

0.138

0.413

2.547

 

 

 

 

 

25.8

1

3.900

28.674

118.718

2

4.138

28.674

118.480

3

4.138

28.197

116.336

 

 

 

 

 

m

4.059

28.515

117.845

s

0.138

0.275

1.312

 

 

 

 

 

83.7

1

4.614

27.959

114.668

2

4.614

30.341

123.482

3

4.138

27.721

113.954

 

 

 

 

 

m

4.456

28.674

117.368

s

0.275

1.449

5.307

 

 

 

 

 

293

1

4.138

28.436

101.090

2

4.138

30.103

114.430

3

3.900

27.245

101.328

 

 

 

 

 

m

4.059

28.594

105.616

s

0.138

1.436

7.634

 

 

 

 

 

962

1

4.376

32.247

100.614

2

4.138

29.865

94.182

3

4.614

32.485

98.232

 

 

 

 

 

m

4.376

31.532

97.676

s

0.238

1.449

3.252

 

Table 2: Yield y and Percentage Inhibition of y during the Test Period

Geometric mean measured concentrations (mg test item/L)

 

Yield y (10 000 cells/mL) and inhibition of y in %

0 – 24 h

0 – 48 h

0 – 72 h

y

%

y

%

y

%

Control

3.797

/

26.625

/

109.245

/

6.18

3.559

6.3

27.936

-4.9

117.106

-7.2

25.8

3.559

6.3

28.015

-5.2

117.345

-7.4

83.7

3.956

-4.2

28.174

-5.8

116.868

-7.0

293

3.559

6.3

28.094

-5.5

105.116

3.8

962

3.876

-2.1

31.032

-16.6

97.176

11.0*

negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control

* mean value significantly different from the control(tested with Bonferroni-Welch,a = 0.05, one-sided smaller)

 

 

Table 3: Growth Rates µ and Percentage Inhibition of µ during the Test Period

Geometric mean measured concentrations (mg test item/L)

 

Growth rate µ (1/day) and inhibition of µ in %

0 – 24 h

0 – 48 h

0 – 72 h

µ

%

µ

%

µ

%

Control

2.151

-

1.997

-

1.797

-

6.18

2.094

2.7

2.020

-1.2

1.820

-1.3

25.8

2.094

2.7

2.022

-1.3

1.821

-1.3

83.7

2.186

-1.6

2.024

-1.4

1.819

-1.2

293

2.094

2.7

2.023

-1.3

1.784

0.7

962

2.168

-0.8

2.072

-3.8

1.758

2.2

negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control

Validity criteria fulfilled:
yes
Conclusions:
The 72h-NOErC (endpoint growth rate) of succinimide was determined to be above the highest concentration tested, i.e. 960 mg test item/L based on geometric mean of measured concentrations. The corresponding 72h-LOErC must therefore be above 960 mg test item/L.
Executive summary:

In a 72-hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata were exposed to succinimide at measured concentrations of 0, 962, 293, 83.7, 25.8 and 6.18 mg test item/L under static conditions in accordance with the OECD guideline 201.  The NOErC value based on growth rate is equal to or above 960 mg test item/L, and the LOErC must be above 960 mg test item/L, respectively. 

The NOEyC value based on yield is equal to 293 mg test item/L, and the LOEyC equals 960 mg test item/L, respectively. 

The 72-hour EyC50and the 72-hour ErC50value could not be calculated as no significant effect was seen for growth and for yield only the highest concentration displayed a significant effect. Therefore, the EC50values for both endpoints was estimated to be above the highest concentration tested (i.e. > 962 mg test item/L).

According to Chapter R.7b of the Guidance on Information Requirements and Chemical Safety Assessment from the ECHA, Version 4.0, January 2017: “The preferred observational endpoint in this study [Comm.: referring to i.a. OECD 201] is algal growth rate inhibition because it is not dependent on the test design, whereas biomass depends both on growth rate of the test species as well as test duration and other elements of test design.” Therefore, the NOEC for growth rate is reported as key value.

 

There were no compound-related phytotoxic effects regarding growth rate.

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for the Freshwater Alga and Cyanobacteria, Growth Inhibition Test.

 

Results Synopsis

 

Test Organism: P. subcapitata

Test Type (Flowthrough, Static, Static Renewal): Static

 

72-h NOErC ≥ 960 mg test item/L

72-h LOErC > 960 mg test item/L

 

72-h NOEyC = 293 mg test item/L

72-h LOEyC = 960 mg test item/L

 

Endpoint(s) Effected: Yield, Growth rate

Description of key information

In a 72 -h freshwater Alga and Cyanobacteria, Growth Inhibition Test conducted according to OECD guideline 201 the freshwater algae Pseudokirchneriella subcapitata was exposed to succinimide at measured concentrations of 962, 293, 83.7, 25.8 and 6.18 mg test item/L. There were no compound-related effects on the growth rate. A significant effect was revealed at the highest concentration regarding yield.

Growth rate is the prefered endpoint for tests on the algal growth inhibition according to the guidance on information requirements and safety assessment (Chapter R.7b, June 2017). Therefore the results on growth rate are presented as key value.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
960 mg/L

Additional information

In a 72-hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata were exposed to succinimide at measured concentrations of 0, 962, 293, 83.7, 25.8 and 6.18 mg test item/L under static conditions in accordance with the OECD guideline 201.  The NOErC value based on growth rate is equal to or above 960 mg test item/L, and the LOErC must be above 960 mg test item/L, respectively. 

The NOEyC value based on yield is equal to 293 mg test item/L, and the LOEyC equals 960 mg test item/L, respectively. 

The 72-hour EyC50and the 72-hour ErC50value could not be calculated as no significant effect was seen for growth and for yield only the highest concentration displayed a significant effect. Therefore, the EC50values for both endpoints was estimated to be above the highest concentration tested (i.e. > 962 mg test item/L).

According to Chapter R.7b: Endpoint specific guidance Draft Version 4.0 (Public) – January 2017: “The preferred observational endpoint in this study [Comm.: referring to i.a. OECD 201] is algal growth rate inhibition because it is not dependent on the test design, whereas biomass depends both on growth rate of the test species as well as test duration and other elements of test design.” Therefore, the NOEC for growth rate is reported as key value.