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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:
From 13 December 2019 to 16 January 2020
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Density (certified): 0,921 kg/dm3
- Water solubility: Insoluble
Analytical monitoring:
yes
Details on sampling:
All loading rates and the control were analytically verified via GCMS at the start (0 hours) and at the end of the exposure (72 hours) with algae.
At the start, samples were taken from one additional replicate of each test item loading and the control and at the end samples were taken from pooled replicates. For the sampling, at start of the first sampling interval, 50 g of the test solutions and from end of the first sampling interval on 50 g of the test solutions were directly weighted into the sampling vials.

- Sampling method: 50 mL of the test item concentrations and the control were added with approx. 10% sodium chloride and liquid extracted with 2.5 mL cyclohexane by manual agitation for 2 min (enrichment factor 20). Before analysis, the organic phase was separated and dried with a small quantity of sodium sulfate. If necessary, to reach the calibration range, samples at test start were further diluted factor 10 with cyclohexane prior to analysis. 0.1 mL of internal standard solution (Undecanoic acid, derivatised, at 0.5 mg/L) were added to 1 mL of each sample prior to analysis.
- Sample storage conditions before analysis: All samples were immediately extracted and the extracts were stored at room temperature. Until the start of the analysis the prepared samples were stored on an autosampler.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Five water accommodated fractions (WAF) were prepared separately with nominal loading rates of the test item in the range of 100 to 10 000 mg/L. For each loading rate, an appropriate amount of the test item was pipetted onto the surface of the demineralized water in a glass flask (rinsed with cyclohexane and demineralized water). For this purpose, the density was taken into account. A slow stirring procedure was applied for 120 ± 1 hour at room temperature. The WAF was prepared in a tightly closed glass flask with a side-arm near the bottom. After a separation phase of 1 hour, the aqueous phase or WAFs were removed via the side-arm of the flask (from the approximate bottom of the glass flask).
- Eluate: dilution water
- Differential loading: 100 – 316 – 1 000 – 3 160 – 10 000 mg/L
- Controls: Six replicates (without test item) were exposed under the same conditions as the test concentrations
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The WAF was checked via laser beam (Tyndall effect) for undissolved test item and microscopically for dispersed droplets (phase-contrast microscope, magnification: 400 times). The Tyndall effect of the three highest loadings were negative. The Tyndall effect of the two lowest loadings were positive, but no dispersed droplets or undissolved test item were observed by microscopically check.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK CCAP 278/4 (axenic)
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
- Age of inoculum (at test initiation): A four-day old preculture, prepared in dilution water, was used as inoculum
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567 – 5130 lux for 24 hours per day. Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
47 mg CaCO3/L
Test temperature:
Min: 21.5°C; Max: 22.5°C; Mean value: 22.0°C
pH:
From 7.43 to 8.32
Dissolved oxygen:
No data
Salinity:
Not applicable
Conductivity:
232 µS/cm
Nominal and measured concentrations:
- Nominal test item loadings: 100, 316, 1000, 3160 and 10000 mg/L
- Measured concentrations: See Table 6.1.5/3 in "Any other information on results incl. tables".
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks (vol. 250 mL) with cotton wool plugs
- Material, size, headspace, fill volume: test volume 100 mL
- Initial cells density: 5416 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Threefold concentrated AAP medium according to the guidelines
- pH: the pH of the test medium has to be 7.5 +/- 0.1.
- Total organic carbon: < LOQ (limit of quantification = 2.00 mg C/L)
- Acidity: 0.1 mmol/L
- Alkalinity: 0.5 mmol/L
- Total hardness: 47 mg CaCO3/L
- Conductivity: 232 µS/cm

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- pH: The pH-values at the start of exposure were measured in one additional replicate of each test item loading and the control. At the end of exposure, the pH-values were measured from pooled samples of each test item loading and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure.
- Photoperiod: 24 hours/day light
- Light intensity and quality: mean value 5337 lux; range of the measured values: 4687 to 6015 lux (CV 7.35%)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The cell density was measured daily via Chlorophyll afluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was
observed in the preliminary range finding test at the loading rate of 10 000 mg/L. Microscopic evaluation of the cells at the start and end of the incubation was carried out. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation and adherence of algae to test containers or aggregation.
EL10-, EL20- and EL50-values with confidence intervals of yield inhibition after 72 hours were calculated by sigmoidal doseresponse regression. The NOEL / LOEL was determined by calculation of statistical significant differences of growth rate and yield using ANOVA. A Shapiro-Wilk’s Normality test and a Brown-Forsythe’s Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: A non-GLP preliminary range finding test under static conditions over a period of 72 hours was conducted at the test facility with two water accommodated fractions (WAFs) of the test item with nominal loadings of 1 000 – 10 000 mg/L. Two replicates per concentration and four for the control were tested. All concentration levels were clear throughout the exposure period. Based on the results of this preliminary test, 8% and 5% inhibition of growth rate were observed at the nominal loading rates of 1 000 and 10 000 mg/L, respectively.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: EL10 based on yield inhibition cannot be calculated
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Details on results:
Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities. All effects values are given based on the nominal test item loadings.
Results with reference substance (positive control):
- Results with reference substance valid? yes
72h-ErC50 = 0.940 mg/L (95% CI: 0.732 - 1.33 mg/L)
72h-EyC50 = 0.660 mg/L (95% CI: 0.393 - 0.700 mg/L)

Table 6.1.5/1: Cell densities

Nominal test item loading (mg/L)

Replicate No.

Cell density (cells/mL)

0 hours

24 hours

48 hours

72 hours

10000

1

2

3

5416

5416

5416

17866

20220

21057

110740

120349

130241

791223

774769

844698

Mean

5416

19714

120443

803563

3160

1

2

3

5416

5416

5416

17128

18905

20385

116679

119181

131010

755151

773333

783434

Mean

5416

18806

122290

770639

1000

1

2

3

5416

5416

5416

16775

21084

19346

108218

127447

117984

692208

891577

765155

Mean

5416

19068

117883

782980

316

1

2

3

5416

5416

5416

18852

18720

20120

119298

109163

100196

846816

751768

605436

Mean

5416

19231

109552

734673

100

1

2

3

5416

5416

5416

22089

22004

22456

123183

132471

136492

837177

805000

854580

Mean

5416

22183

130715

832252

Control

1

2

3

4

5

6

5416

5416

5416

5416

5416

5416

20770

22035

22581

19516

21926

18384

133211

126191

122365

120681

109367

120807

868186

902092

859740

806168

826614

710779

Mean

5416

20869

122104

828930

Table 6.1.5/2: Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Nominal test item loading (mg/L)

Replicate No.

Growth rate (d-1)

Inhibition of growth rate (%)

Yield (cells/mL)

Inhibition of yield (%)

10000

1

2

3

1.66

1.65

1.68

1

1

0

785807

769353

839282

5

7

-2

Mean

(-) 1.67

1

(-) 798147

3

3160

1

2

3

1.65

1.65

1.66

2

1

1

749735

767917

778018

9

7

6

Mean

(-) 1.65

1

(-) 765223

7

1000

1

2

3

1.62

1.70

1.65

4

-2

2

686792

886161

759739

17

-8

8

Mean

(-) 1.66

1

(-) 777564

6

316

1

2

3

1.68

1.64

1.57

0

2

6

841400

746352

600020

-2

9

27

Mean

(-) 1.63

3

(-) 729257

11

100

1

2

3

1.68

1.67

1.69

0

1

-1

831761

799584

849164

-1

3

-3

Mean

(-) 1.68

0

(-) 826836

0

Control

1

2

3

4

5

6

1.69

1.71

1.69

1.67

1.68

1.63

 

862770

896676

854324

800752

821198

705363

 

Mean

1.68

 

823514

 

Table 6.1.5/3: Measured concentrations of the test item during the definitive test

Sampling date

Fresh medium

0 hours

Old medium

72 hours

Nominal loading rate of the test item (mg/L)

Calculated concentration of the test item (µg/L)*

Calculated concentration of the test item (µg/L)*

Group I

Group II

Group III

Sum**

Group I

Group II

Group III

Sum**

10000

3160

1000

316

100

Control

0.619

0.609

0.688

0.546

0.556

<LOQ

0.303

<LOQ

0.287

<LOQ

<LOQ

<LOQ

0.132

<LOQ

0.116

<LOQ

<LOQ

<LOQ

1.05

0.609

0.976

0.546

0.556

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

QC

RR: 101%

-

-

-

RR: 109%

-

-

-

* Group I (Peak 1 to 3); group II (peak 4 to 6); group III (peak 7 to 9). Calculated concentration taking enrichment, dilution factor and the percentage of each group into account.

** Sum of group I to III, only measured values > LOQ taken into account.

LOQ = Limit of quantification, group I: 0.1 µg test item/L, corresponding to 0.024 µg a.i./L; group II: 0.3 µg test item/L, corresponding to 0.14 µg a.i./L; Group III: 0.6 µg test item/L, corresponding to 0.13 µg a.i./L.

QC = Quality control (0.1 µg test item/L), recovery calculated for group I to nominal concentration.

Validity criteria fulfilled:
yes
Conclusions:
The test substance was not found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values based on nominal test item loadings: The EL50 and EL10-values with 95% confidence intervals for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were > 10 000 mg/L.
Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD Guideline 201 from 2020-01-02 to 2020-01-16, with the definitive exposure phase from 2020-01-07 to 2020-01-10 at the test facility. The study was conducted under static conditions with an initial cell density of 5416 cells/mL. Five water accommodated fractions (WAF) were prepared separately with nominal loading rates of the test item in the range of 100 to 10 000 mg/L set up in a geometric series: 100 – 316 – 1 000 – 3 160 – 10 000 mg/L. For each loading rate, an appropriate amount of the test item was pipetted onto the surface of the demineralized water in a glass flask (rinsed with cyclohexane and demineralized water). For this purpose, the density was taken into account. A slow stirring procedure was applied for 120 ± 1 hour at room temperature. The WAF was prepared in a tightly closed glass flask with a side-arm near the bottom. After a separation phase of 1 hour, the aqueous phase or WAFs were removed via the side-arm of the flask (from the approximate bottom of the glass flask). The WAF was checked via laser beam (Tyndall effect) for undissolved test item and microscopically for dispersed droplets (phase-contrast microscope, magnification: 400 times). The Tyndall effect of the three highest loadings were negative. The Tyndall effect of the two lowest loadings was positive, but no dispersed droplets or undissolved test item were shown by microscopically check. Three replicates were tested for each test item loading rate and six replicates for the control. The environmental conditions were within the acceptable limits. The test media were clear throughout the test period. As the active ingredient of the test item shows 9 significant peaks, a group evaluation of three major groups took place: group I with peaks 1 to 3, group II with peaks 4 to 6 and group III with peaks 7 to 9. Since the test item is a UVCB, all effect values given are based on the nominal loading rates of the test item.

According to the results of this study, the test substance was not found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values based on nominal test item loadings: The EL50 and EL10-values with 95% confidence intervals for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were > 10 000 mg/L.

Description of key information

OECD Guideline 201, GLP, key study, validity 1:

72h-ErL50 (Pseudokirchneriella subcapitat) > 10000 mg/L based on nominal loading rates

72h-ErL10 (Pseudokirchneriella subcapitat) > 10000 mg/L based on nominal loading rates

Key value for chemical safety assessment

Additional information

One key study is available to assess the toxicity of the registered substance to aquatic algae.

In this toxicity test with Pseudokirchneriella subcapitata (NOACK, 2020), the effects of five water accommodated fractions (WAF) with nominal loading rates of 100 - 316 - 1000 - 3160 - 10000 mg/L of the registered substance were determined at the test facility according to OECD Guideline 201 with GLP compliance. This study was conducted under static and closed conditions with an initial cell density of 5416 cells/mL. No undissolved test item was present in any of the tested WAFs. The test item concentrations were analytically verified via GC-MS/MS at the start and the end of the exposure in all WAFs and in the control. According to the results of this study, the test substance was not found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours. Therefore, the ErL50 and ErL10-values for inhibition of growth rate after 72 hours were > 10 000 mg/L, based on nominal loading rates.

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