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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:
22 Nov - 25 Nov 2016
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)
Version / remarks:
2006, corrected 2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
No 2016/266
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim, Germany
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Sampling method: at test start and test end with algae; samples and the control were stabilized (factor 2) with acetonitrile containing 0.2% formic acid
- Sample storage conditions before analysis: stored at room temperature
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: preparation of stock solution (400 mg test item/L with dilution water); stock solution was stirred 15 minutes with 1100 rpm at room temperature; stock solution was diluted to achieved the required test item concentrations
- Controls: yes, test medium control
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: SAG 61.81
- Source: Experimental Phycology and Culture Collection of Algae at the University of Goettingen, Germany
- Age of inoculum (at test initiation): A four days old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux for 24 h per day. Stock cultures are cultivated on 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:
0.24 mmol Ca+Mg/L
Test temperature:
22.5 °C (22.0 - 23.0 °C)
pH:
7.78 - 7.92 (0 h test treatment); 8.09 - 9.18 (72 h test treatment)
7.96 (0 h control); 9.20 (control test end 72 h)
Nominal and measured concentrations:
nominal: control; 10.24; 25.6; 64.0; 160 and 400 mg/L
measured: refer to "Any other information on results incl. tables"
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: open (sealed with cotton wool plugs)
- Material, size, headspace, fill volume: Sterile 250 mL-Erlenmeyer flasks; sealded with cotton wool plugs; test volume: 100 mL; headspace 150 mL
- Aeration: no
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm; positioned randomly and repositioned daily
- Initial cells density: Nominal: approx 5 x 10 E+03 - 10 E+04 cells/mL; Current: 6305 cells/mL
- Control end cells density: 1932289 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: OECD medium according to the guideline OECD 201
- Culture medium different from test medium: yes; stock cultures are cultivated on nutrient medium Z according to Lüttge et al. (1994)
- Intervals of water quality measurement: The pH-values were measured at the start of exposure in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-values were measured from pooled samples of each test item concentration and the control.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 24 hours/day light
- Light intensity and quality: Fluorescent tubes, Osram L 36 W/865, cool daylight; light homogeneity: within ± 15% over incubation area; mean value 5725 lux (4881 - 6291 lux)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm
- Other: Microscopic evaluation of algae cells at test start and test end.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5

Range finding study
- Test concentrations: control; 1.0; 100 and 400 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Growth rate inhibition (%): 1.0 mg/L: 1%; 100 mg/L: 4% and 400 mg/L: 100%; results in details are summarized within the table 1 in section "Any other information on material and methods incl. tables".
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
81.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval
Remarks:
64.6 -135 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
68.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidence interval
Remarks:
44.9 - 130 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10.24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
25.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10.24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
25.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
< 10.24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
75.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % confidence intervals
Remarks:
64.7 - 139 mg/L
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities.
- Other: Analytical and biological results are summarized within the tables 1-2 in the section "Any other information on results incl. tables".
Results with reference substance (positive control):
- EC50 (growth rate): 0.436 mg/L (95% confidence interval: 0.414 - 0.458 mg/L)
Reported statistics and error estimates:
ECx-values with confidence intervals of growth rate inhibition and for yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Shapiro-Wilk’s test on normal distribution was calculated with a significance level 0.01. Levene’s test on variance homogeneity was calculated with a significance level 0.01. Monotonicity of Concentration/Response was calculated by trend analysis by contrasts (significance level 0.05). SD Jonckheere-Terpsta test procedure rate was done for growth with a significance level 0.05. Calculations were carried out using software: Excel, Microsoft Corporation; SigmaPlot, Spss Inc.; GraphPad Prism, GraphPad Software, Inc. and ToxRat Version 3.2.1, ToxRat Solutions GmbH

Table 1: Cell Densities

Nominal item concentration [mg/L]

Replicate No.

Cell density [cells/mL]

 

 

 

0 hours

24 hours

48 hours

72 hours

400

1

6305

4738

<LOQ cell density

<LOQ cell density

 

2

6305

5575

<LOQ cell density

<LOQ cell density

 

3

6305

4315

<LOQ cell density

<LOQ cell density

 

Mean

6305

4876

n.a.

n.a.

160

1

6305

9141

2995

<LOQ cell density

 

2

6305

8712

<LOQ cell density

<LOQ cell density

 

3

6305

9299

3021

<LOQ cell density

 

Mean

6305

9051

n.a.

n.a.

64.0

1

6305

16879

124088

1151699

 

2

6305

21381

180790

1623007

 

3

6305

17677

134034

1296595

 

Mean

6305

18646

146304

1357100

25.6

1

6305

20547

137744

1181044

 

2

6305

21643

187787

1655172

 

3

6305

22762

181111

1448511

 

Mean

6305

21651

168881

1428242

10.24

1

6305

21928

161626

1688408

 

2

6305

19464

154088

1276496

 

3

6305

21666

207649

1840775

 

Mean

6305

21019

174454

1601893

Control

1

6305

28020

235518

1943358

 

2

6305

25229

204255

1837956

 

3

6305

27755

265157

2322035

 

4

6305

21542

240141

1560792

 

5

6305

24815

174667

2027900

 

6

6305

24107

221694

1901693

 

Mean

6305

25245

223572

1932289

LOQ cell density = Limit of quantification of the cell density (2736 cells/mL)

n.a. = not applicable

Table 2: Measured Concentrations and Percentage of the Nominal Concentration of the test item in Fresh Medium (0 hours) and Old Medium (72 hours) with Algae

Sampling date

0 hours fresh medium

72 hours old medium

Start of analysis

Nominal concentration of the test item [mg/L]

Test Item

Meas. conc. [mg/L]

[%]

Meas. conc. [mg/L]

[%]

400

365

91

364

91

160

184

115

161

100

64.0

64.3

100

53.5

84

25.6

22.4

87

21.0

82

10.24

10.7

105

9.29

91

Control

< LOQ

< LOQ

 

 

Meas. Cone. = Measured concentration of the test item (dilution factor taken into account), mean value of two injections

% = Percentage of the nominal concentration of the test item

LOQ = Limit of quantification (2.00 mg test item/L)

Validity criteria fulfilled:
yes
Conclusions:
The algae study with Pseudokirchneriella subcapitata was performed according to OECD 201 and resulted in an ErC50 (72 h) of 81.3 mg/L, an ErC10 (72 h) of 68.7 mg/L and an NOEC of 10.24 mg/L. All effect values were based on the nominal concentrations.

Description of key information

ErC50 (72 h): 81.3 mg/L (OECD 201, Pseudokirchneriella subcapitata, nominal)

ErC10 (72 h): 68.7 mg/L (OECD 201, Pseudokirchneriella subcapitata, nominal)

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
81.3 mg/L
EC10, LC10 or NOEC for freshwater algae:
68.7 mg/L

Additional information

One experimental study is available investigating the toxicity of the substance to aquatic freshwater algae. The study was conducted according to OECD 201 (GLP) using the freshwater algae Pseudokirchneriella subcapitata. The definitive test was carried out with five concentrations in a geometrical series with a dilution factor of 2.5: 10.24, 25.6, 64.0, 160 and 400 mg/L including a control. The measured concentrations of the test item at test start (0 h) were between 87 and 115% of the nominal values. The measured concentrations in the old media (72 h) were in the range of 82 to 100% of the nominal values. Thus, it is justified that the effect concentrations are based on the nominal concentrations. Inhibition of growth was recorded resulting in an ErC10 (72 h) of 68.7 mg/L (95 % confidence limits: 44.9 – 130 mg/L) and an ErC50 (72 h) 81.3 mg/L (95 % confidence limits: 64.6 – 135 mg/L). Inhibition of yield was recorded resulting in an EyC50 (72 h) of 75.8 mg/L (95 % confidence limits: 64.7 – 139 mg/L) and an EyC10 (72 h) of <10.24 mg/L.