2-(1,3-dioxoisoindolin-2-yl)ethane-1-sulfonamide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-08-28 to 2018-10-15

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:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC-DAD

Details on sampling:

- Concentrations: 12.5 – 25.0 – 50.0 – 100 – 200 mg/L (nominal)
- Sampling method: At the start, samples were taken from one additional replicate of each concentration and the control and at the end samples were taken from pooled replicates. The samples were transferred from the replicate directly into a 20 mL headspace vial, covered with aluminium tops with PTFE seals.
- Sample storage conditions before analysis: If necessary, all original samples were stored at room temperature before preparation. 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: 200 mg test item/L were freshly prepared with dilution water. The stock solution was treated for 5 minutes at room temperature with ultrasound and stirred for 1 hour (~1100 rpm, room temperature). The stock solution was checked via laser beam (Tyndall effect) for undissolved test item. A slight Tyndall effect was observed, therefore, the stirring period was prolonged for another 15 minutes. After this, no Tyndall effect was present. The stock solution and four dilution levels out of the stock solution in a geometric series with a factor of 2, prepared by dilution of the stock solution 200 mg/L with dilution water, were tested as follows: 12.5 – 25.0 – 50.0 – 100 – 200 mg/L.
- Controls: Six replicates (without test item) were tested under the same test conditions as the test item replicates.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: Pseudokirchneriella subcapitata HINDÁK, 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 amounted to 2590 - 5180 lux per day.

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

21 - 24°C, controlled at ± 2°C

pH:

7.89 to 9.45

Nominal and measured concentrations:

Nominal: 12.5 – 25.0 – 50.0 – 100 – 200 mg/L
Measured: 5.40, 7.76, 10.8, 15.2, 108 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): closed, sealed with cotton wool plugs
- Material, size, headspace, fill volume: Sterile glass Erlenmeyer flasks with 250 mL volume and 100 mL fill volume
- Initial cells density: Nominal:approximately 5 x E+03 - E+04 cells/mL; Actual: 6442 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:
TEST MEDIUM / WATER PARAMETERS
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: 24h day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was observed in the first definitive test at the concentration of 200 mg/L.
- Other: Microscopic evaluation of the cells at the start and the end of the incubation period 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: A first definitive test was carried out as a limit test with a nominal test concentration of 200 mg/L. The test resulted in mean inhibition values of 25% (growth rate) and 76% (yield) compared to the control. Therefore, the test was repeated as a dose-response study.
- Range finding study
- Test concentrations: 0, 1.0, 10.0, and 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

15.2 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.:

79.9 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.:

> 108 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.:

15.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

38.6 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.:

89.9 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
- Unusual cell shape: no
- Colour differences: no
- Adherence to test vessels: not reported
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: growth: 1.09 (0.842 - 1.31); valid range: 0.759 ± 0.555
- EC50: yield: 0.548 (0.439 - 0.660); valid range: 0.401 ± 0.297

Reported statistics and error estimates:

EC10-, EC20- and EC50-values with confidence intervals of growth analyses rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. The NOEC and LOEC were determined by calculation of statistical analyses significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The α-value (acceptable probability of incorrectly concluding that there is a difference) is α =0.05.

Table 1: MeasuredConcentration of 2‑Phthalimidoethanesulfonamide(TA-3) in the Definitive Test with Algae

Sampling date	Fresh Media, 0 hours		Old Media, 72 hours		Geometric mean measured concentration [mg/L]*^)
Nominal test item concentration [mg/L]	2‑Phthalimidoethanesulfonamide(TA-3)
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
200	186	93	63.0	31	108
100	92.2	92	< LOQ		15.2
50.0	46.4	93	< LOQ		10.8
25.0	24.1	96	< LOQ		7.76
12.5	11.7	93	< LOQ		5.40
Control	< LOQ		< LOQ

Meas. conc.= measured concentration of the test item, mean value of 2 injections, dilution factors taken into account

%                  = percentage of the nominal measured concentration of the test item

LOQ             = limit of quantification (5.00 mg/L of the test item)

*)                  = If the measured concentration was < LOQ, 1/2 LOQ (2.50 mg test item/L) was used for the calculation of the geometric mean measured concentration.

Table 2: Results of the Range Finding Test (0 - 72 hours)

Nominal test item concentration [mg/L]	Growth Rate Inhibition [%]	Yield Inhibition [%]
100	0	0
10.0	0	-2
1.00	0	1

Table 3: Measured Exposure Concentrations during the non-GLP Preliminary Range Finding Test Determination of the test item via HPLC-DAD

Sampling	0 hours Start of the exposure (with algae)		72 hours End of the exposure (with algae)
Nominal test item concentration [mg/L]	2‑Phthalimidoethanesulfonamide(TA-3)
	Measured concentration [mg/L]	% of the nominal concentration	Measured concentration [mg/L]	% of the nominal concentration
100	78.6	79	3.12	3
10.0	7.76	78	< LCL
1.00	0.616	62	< LCL
Control	< LCL		< LCL

Validity criteria fulfilled:

yes

Conclusions:

In this study, the effect values of 2 Phthalimidoethanesulfonamide (TA-3) on the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h were determined as follows (based on geometric mean measured test item concentrations): The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 h were > 108 mg/L and 89.9 (17.0 – 108) mg/L, respectively. The NOEC for both growth rate and yield was 15.2 mg/L, respectively.

Description of key information

The toxicity of 2‑Phthalimidoethanesulfonamide (TA-3) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201. The EC₅₀-values for inhibition of growth rate (E_rC₅₀) and yield (E_yC₅₀) after 72 hours were > 108 mg/L and 89.9 (17.0 – 108) mg/L, respectively. The NOEC for both growth rate and yield was 15.2 mg/L, respectively. All values are based on geometric mean measured test item concentrations.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

15.2 mg/L