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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:
2018-08-03 to 2018-09-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Remarks:
HPLC-DAD
Details on sampling:
- Concentrations: Nominal: 250 mg/L and 0 mg/L
- Sampling method: The concentrations of 2H-lsoindole-2-ethanesulfonic acid, 1,3-dihydro-1,3-dioxo-, potassium salt (1:1) (TA-1) were determined in the fresh media (0 h) and old media (72 h) in the limit concentration and in the control via HPLC-DAD. The sample and the control were diluted (factor 2) with acetonitrile directly after sampling. Further dilution was made prior to analysis with dilution medium for the limit concentration.
- Sample storage conditions before analysis: All samples were stored at room temperature until the start of analysis, if necessary. Prepared samples were stored in an autosampler at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution = Limit concentration: 250 mg test item/L, freshly prepared with dilution water. The stock solution was agitated until the test item was completely dissolved.
- Controls: Six replicates (without test item) were exposed under the same conditions as the test item replicates. Reference item: Potassium dichromate was tested as a reference item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No, all solutions were visually clear.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: Pseudokirchneriella subcapitata HINDAK CCAP 278/4
- 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 amounts to 2567 - 5130 lux for 24 h per day.

ACCLIMATION
- Acclimation period: N/A
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: not reported
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
0.24 mmol Ca+Mg/L
Test temperature:
Nominal range: 21 - 24 °C, controlled at ± 2 °C
pH:
8.11 to 9.43
Nominal and measured concentrations:
Nominal: 250 mg/L and 0 mg/L
Geometric measured mean: 186 mg/L and < LOQ
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed
- Material, size, headspace, fill volume: Total volume: 250 mL and fill volume: 100 mL
- Aeration: No, test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density: 5E03-E04 cells/mL, (actual: 6442 cells/mL)
- No. of vessels per concentration (replicates): 6 replicates
- No. of vessels per control (replicates): 6 replicates

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Concentration [mg/L]
NH4Cl 15
MgClg x 6 H2O 12
CaCl2 x 2H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl x4 H2O 0.415
ZnCl2 3E-03
Na2MoO4 x 2 H2O 7E-03
COCl2 x 6 H2O 1.5E-03
CuCl2 x 2 H2O 1E-05
NaHCO3 50
pH 8.1 ± 0.2
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 24 h/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 pE*m-2*s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter
- Chlorophyll measurement:Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm
- Other: Microscopic evaluation of the cells at the start and the end of exposure was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: N/A
- Justification for using less concentrations than requested by guideline: Limit Test
- Range finding study: yes including analysis of the nominal concentrations of 1.00, 10.0, 100 mg/L
- Test concentrations range finding study: 1.00, 10.0, 100 mg/L
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 186 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 186 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 186 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 186 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 186 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 186 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: growth: 1.09 CI (0.842-1.31);
yield: 0.548 CI (0.439 - -0.660)
Reported statistics and error estimates:
Protesting
Shapiro-Wilk's Test on Normal Distribution:
Normality check was passed (Shapiro-Wilk's; p > 0.01).
Levene's Test on Variance Homogeneity:
Variance homogeneity check was passed (p > 0.01).
STUDENT-t test for Homogeneous Variances with growth rate at 0 - 72 h: Two-sample comparison of treatments with "Control". Significance was Alpha = 0.050. one-sided smaller; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable
difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for Ho: p1 = p2; the differences are significant in case p(t) <= Alpha (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).

Table 1: Nominal and measured concentrations for 72 h

Sampling date

0h

72h

Geometric mean measured test item concentration [mg/L]

Nominal test tiem conce3ntration [mg/L]

2H-lsoindole-2-ethanesulfonic acid, 1,3-dihydro-1,3-dioxo-, potassium salt (1:1) (TA1)

Meas. Conc. [mg/L]

%

Meas. Conc. [mg/L]

%

250

275

110

126

50

186

Control

< LOQ

< LOQ

-

Meas. conc. = measured concentration of the test item, dilution factors taken into account

% = percentage of the nominal concentration of the test item

LOQ = limit of quantification of the analytical method (25.0 mg test item/L)

Validity criteria fulfilled:
yes
Conclusions:
In this study, 2H-isoindole-2-ethanesulfonic acid, 1,3-dihydro-1,3-dioxo-, potassium salt (1:1) (TA-1) did not inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h. The EC50-values tor inhibition of growth rate (ErC50) and yield (EyC50) after 72 h were > 186 mg/L (all values based on geometric mean measured test item concentration). The NOEC-values for inhibition of growth rate and yield after 72 h were ≥ 186 mg/L, respectively.

Description of key information

The toxicity of 2H-lsoindole-2-ethanesulfonic acid, 1, 3-dihydro-1, 3-dioxo-, potassium salt ( 1: 1) (TA-1) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to guideline OECD 201 (2011).

2H-lsoindole-2-ethanesulfonic acid, 1,3 -dihydro-1,3 -dioxo-, potassium salt (1:1) (TA-1) did not inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours. The EC50 -values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were >186 mg/L (all values based on geometric mean measured test item concentration). The NOEC values for inhibition of growth rate and yield after 72 hours were 186 mg/L, respectively.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
186 mg/L
EC10 or NOEC for marine water algae:
186 mg/L

Additional information