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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:
2017-06-01 to 2017-06-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light, in the tightly closed original container.
- Stability under test conditions: Assumed to be stable.
- Solubility and stability of the test substance in the solvent/vehicle: 1.76 g/L in water (20°C, pH 6.5)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A stock solution with a nominal loading of 320 mg/L test item was freshly prepared with dilution water and agitated until the solution was visually clear.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentration levels and the control were analytically verified via GC-MS after 0 (start of exposure), 24, 48 and 72 hours (end of the exposure).
- Sampling method: Separate replicates for each test item concentration and control for the test item analysis at the beginning of the exposure were prepared without algae. The samples for the test item analysis after 24, 48 and 72 hours were prepared with algae and incubated under test conditions. Additionally, the concentration levels 40.0 and 160 mg/L were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) after 0, 24, 48 hours and at the end of the exposure. Separate replicates for the TOC measurement were prepared without algae and without MES buffer and incubated under test conditions.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution with a nominal loading of 320 mg/L test item was freshly prepared with dilution water and agitated until the solution was visually clear. The test item was pipetted onto the surface of the dilution water. For this purpose, the density was taken into account. Additionally a stock solution of 320 mg/L was prepared with dilution water (without the compounds MES and NaHCOs). Out of this stock solution the dilution levels 40.0 and 160 mg/L were prepared for analysis of total organic carbon (TOC).
- Controls: Six replicates (without test item) were tested under the same conditions as the test item replicates
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green freshwater algae
- Strain: SAG 61.81
- Age of inoculum (at test initiation): 4 d
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux for 24 hours per day.

ACCLIMATION
- Acclimation period: 96 hours.
- Culturing media and conditions (same as test or not): The culturing conditions were not the same as the test conditions (the light intensity in the test was higher: 4440 to 8880 lux, corresponding to 60 to 120 pE*m'2*s_1).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
mean 22.8 °C; range 22.0 - 23.5 °C
pH:
after 72 h: 7.60 - 8.79 (concentrations), 8.89 (control)
Nominal and measured concentrations:
nominal: 0, 20.0, 40.0, 80.0, 160 and 320 mg/L
measured (72 h): < LOQ, 20.6, 32.2, 67.1, 137 and 278 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile headspace flasks, volume: 59 mL, with aluminium tops with PTFE seals.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Filled without headspace.
- Initial cells density: Nominal: approximately 5 x 10^3 - 10^4 cells/mL, Actual : 6839 cells/mL
- Control end cells density: 1547320 cells/mL
- No. of vessels per concentration (replicates): 3 replicates per concentration. Separate replicates for each measuring time were prepared.
- No. of vessels per control (replicates): 6 replicates, Separate replicates for each measuring time were prepared.

GROWTH MEDIUM
- Standard medium used: yes, threefold concentrated AAP medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to OECD 201.
- Culture medium different from test medium: Yes

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 24 hours/day light.
- Light intensity and quality: mean 6181 lux, range 5424 - 7004 lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Via Chlorophyll-a-fluorescence.
- Other: 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, adherence of algae to test containers or aggregation of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 20.0, 40.0, 80.0, 160 and 320 mg/L
Reference substance (positive control):
yes
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
124 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: (113-134 mg/L)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
73.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: (62.8 - 86.0 mg/L)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
40 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
75.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CI: (62.8 - 92.4 mg/L)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
43.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CI: (34.5 -53.4 mg/L)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
40 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- All measured concentrations of the test item were in the range of 80 and 120% of the nominal values. The test item concentrations 40.0 and 160 mg/L and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start of the exposure, after 24 and 48 hours and the end of the exposure. The TOC remained stable throughout the exposure period.
Results with reference substance (positive control):
- Results with reference substance valid? Yes, the reference item toxicity is in the valid range following test facility SOPs.
- ErC50: with headspace: 0.559 mg/L; without headspace: 0.811 mg/L
Reported statistics and error estimates:
EC10-, EC20- and EC50-value with confidence intervals of yield inhibition after 72 hours was calculated by sigmoidal dose-response regression.
The NOEC / LOEC was determined by calculation of statistical significance of growth rate and yield. The following statistical tests were conducted:
Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01.
Levene’s test on variance homogeneity was done with a significance level of 0.01.
Monotonicity was done by trend analysis by contrasts (significance level 0.05).
Step-down Jonckheere-Terpstra test procedure was done for with a significance level to 0.05.

Table 1: Measured concentrations and percentage of the nominal concentrations of Ethyl isovalerate.

Sampling date

0 h

24 h

48 h

72 h

Nominal concentration of the test item (mg/L)

Meas. conc. (mg/L)

%

Meas. conc. (mg/L)

%

Meas. conc. (mg/L)

%

Meas. conc. (mg/L)

%

320

266

83

286

89

298

93

278

87

160

142

89

138

86

143

89

137

86

80

70.9

89

68.4

85

70.1

88

67.1

84

40

34.3

86

33.1

83

33.6

84

32.2

81

20

21.6

108

21.2

106

21.6

108

20.6

103

Control

< LOQ

< LOQ

< LOQ

< LOQ

 

Meas. conc.= Measured concentration of the test item, dilution and enrichment factor taken into account

% = Percentage of the nominal concentration of the test item

LOQ = Limit of quantification (0.2 mg test item/L)

Table 2: Evaluation after 72 hours

Geometric mean measured test item concentration
[mg/L]
Replicate
No.
Growth rate
[d-1]
Inhibition of growth rate
[%]
Yield
[cells/mL]
Inhibition of yield
[%]
320 1 n.a. 100 n.a. 100
2 n.a. 100 n.a. 100
3 n.a. 100 n.a. 100
mean (+)        n.a 100 (+)           n.a. 100
160 1 0.436 76 18422 99
2 0.629 65 38255 98
3 0.313 83 10654 99
mean (+)   0.459 75 (+)         22444 99
80.0 1 1.70 6 1111207 28
2 1.36 25 397205 74
3 1.63 10 891699 42
mean (+)     1.56 14 (+)       800037 48
40.0 1 1.76 3 1317724 14
2 1.80 0 1495157 3
3 1.77 2 1395263 9
mean (-)      1.78 2 (-)     1402715 9
20.0 1 1.89 -5 1977069 -28
2 1.86 -3 1816139 -18
3 1.90 -5 2036876 -32
mean (-)      1.88 -4 (-)     1943361 -26
control 1 1.80 1509085
2 1.80 1484857
3 1.77 1357752
4 1.78 1425167
5 1.84 1676861
6 1.86 1789161
mean 1.81 1540481
Validity criteria fulfilled:
yes
Conclusions:
The toxicity of the test item to the green freshwater algae Pseudokirchneriella subcapitata was determined according to OECD Test Guideline 201 and GLP. The results (based on nominal concentrations) are as follows:
The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 124 (113 - 134) mg/L and 75.3 (62.8 - 92.4) mg/L, respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 73.6 (62.8 - 86.0) mg/L and 43.5 (34.5 - 53.4) mg/L, respectively. The NOEC and LOEC values for both inhibition of growth rate and yield after 72 hours were 40.0 mg/L and 80 mg/L, respectively.

Description of key information

ErC50 (72 h) = 124 mg/L (OECD 201, Pseudokirchneriella subcapitata)

ErC10 (72 h) = 73.6 mg/L (OECD 201, Pseudokirchneriella subcapitata)

(based on nominal concentrations).

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
124 mg/L
EC10, LC10 or NOEC for freshwater algae:
73.6 mg/L

Additional information

The toxicity of the test substance to the green freshwater algae Pseudokirchneriella subcapitata was determined in a reliable study according to OECD guideline 201 and GLP. The Algae were exposed to the test substance in concentrations of 0, 20, 40, 80, 160 and 320 mg/L, equivalent to < LOQ (limit of quantification), 20.6, 32.2, 67.1, 137 and 278 mg/L measured concentrations (after 72 hours), for 72 hours. The concentrations and the control were analytically verified via GC-MS after 0, 24, 48 and 72 hours. The effect values determined were the growth rate inhibition (ErCx) and the inhibition of yield (EyCx) as well as the NOEC and LOEC after 72 hours. The respective values were ErC50 = 124 mg/L, ErC10 = 73.6 mg/L, EyC50 = 75.3 mg/L, EyC10 = 43.5 mg/L, NOEC = 40 mg/L (based on nominal concentrations).