Propanenitrile, 3-[[4-[[5,6(or 6,7)-dichloro-2-benzothiazolyl]azo]phenyl]ethylamino]-

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:

21 Dez - 24 Dez 2021

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:

adopted March 2006, corrected July 28, 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Duplicate samples from the freshly prepared test media of all test concentrations and from the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.
All samples were diluted by a factor of two with acetonitrile
- Sample storage conditions before analysis: All samples remained undiluted until sample preparation. All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
The test item was not well soluble in test water. In this study, only the inhibitory effects of dissolved test item were tested and thus no concentrations above the solubility limit of the test item in test water were tested. Therefore, a supersaturated stock solution of nominal 100 mg test item/L was prepared by suspending 99.9 mg test item in 999 mL test water for preparing the test concentrations. The stock suspension was stirred for 24 hours at room temperature in the dark to dissolve as much test item as possible. Then, non-dissolved fractions of the test item were separated from the test medium by membrane filtration (0.45 µm cellulose acetate filter). Prior to filtration, the filter was pre-saturated with the stock solution. The solution with dissolved test item was used as the test medium of the highest test concentration and to prepare the desired 1:3, 1:9, 1:27 and 1:81 dilutions.
The test media were prepared just before introduction of the algae (= start of the test).

Appearance of the Test Item in Test Medium: There were no remarkable observations.
Test concentration: Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:3, 1:9, 1:27 and 1:81 of this filtrate were tested, corresponding to the following nominal loading levels of 100, 33.3, 11.1, 3.7 and 1.2 mg test item/L (spacing factor 3). Additionally, a control was tested in parallel.

Test organisms (species):

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

Details on test organisms:

Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)

The algae were originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzen-wissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

TEST ORGANISM
- Name/Strain: Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
- Source (laboratory, culture collection): The algae were originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzen-wissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Age of inoculum (at test initiation): cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test start under the same conditions as in the test.
- Method of cultivation: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: no

Test type:

static

Water media type:

other: reconstituted water, OECD Medium

Limit test:

no

Total exposure duration:

72 h

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L)
as CaCO3

Test temperature:

Water temperature was: 21.5 to 22.3 °C

pH:

The pH was measured in all test item concentrations and the control at the start and the end of the test.
pH value in the control at test start: 8.0,
pH value in the control at test end: 8.2
pH values in the test item treatments at test start: 7.9,
pH values in the test item treatments at test end: 7.8 to 8.2

Nominal and measured concentrations:

Nominal concetration: Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:3, 1:9, 1:27 and 1:81 of this filtrate were tested, corresponding to the following nominal loading levels of 100, 33.3, 11.1, 3.7 and 1.2 mg test item/L (spacing factor 3). Additionally, a control was tested in parallel.

Details on test conditions:

Test Vessels: Erlenmeyer flasks of 50 mL volume with nominal 50 mL of test medium loosely covered with watch glasses.
Introduction of Algae: The test was started (0 hours) by inoculation of a biomass of nominal 5000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test start under the same conditions as in the test.
Replicates: The test was performed with three replicates per test concentration and six replicates in the control.
Test Procedure: The test units were continuously stirred by magnetic stirrers. The flasks were incubated in a water bath, were placed in a random order and were repositioned each day to minimize differences in test conditions.
Blanks: Additionally, one replicate of each test concentration and of the control was prepared without algae to provide a "blank" for the spectrophotometric measurements. The additional replicates were incubated under the same conditions as described above. The blank values were subtracted from the absorption measured in the samples containing algae in order to eliminate absorption caused by the test item.

OTHER TEST CONDITIONS
Light Regime: Continuous illumination
Light Intensity: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.
Mean light intensity: 5053 lux (range: 4750 to 5330 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Determination of the Cell Density: The cell density on each observation time was determined by spectrophotometric measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae).
Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.

Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

6.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

11.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

32.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

17.3 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.:

12.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

11.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Growth Inhibition: After 72 hours the inhibition of yield ranged from -8.4 to 99.6 % and the inhibition of growth rate ranged from -2.0 to 95.1 %.

Signs of Phytotoxicity evaluated by Microscopic Observations: The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested .

Results with reference substance (positive control):

Results of the Most Recent Test with Pseudokirchneriella subcapitata performed with the Reference Item Potassium Dichromate in Juli 2021

72-hour EC50 (Yield): 0.792 mg/L
72-hour NOEC (Yield): 0.20 mg/L
72-hour LOEC (Yield): 0.63 mg/L
72-hour EC50 (Growth rate): 1.62 mg/L
72-hour NOEC (Growth rate): 0.2 mg/L
72-hour LOEC (Growth rate): 0.63 mg/L

Reported statistics and error estimates:

Based on the calculated cell densities, the 72-hour ErL50 and the 72-hour EyL50 (see Definitions), the corresponding EL20 and EL10 values and where possible their 95 %-confidence limits were calculated by Probit analysis.
For the determination of the 72-hour LOEL and the 72-hour NOEL, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Bonferroni-Welch t-test (yield and growth rate).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH.

Validity Criteria

Criterion	value required	value obtained	fullfilled
Cell Density Increase in control Cultures	at least by factor of 16 within 72 h	58.1-fold within 72 h	yes
Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures	< 35%	13.0 %	yes
Coefficient of Variation of Average Growth between Control Replicates	< 7%	2.0 %	yes

Validity criteria fulfilled:

yes

Conclusions:

The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyL50 was calculated to be 14.2 mg test item/L and the 72-hour ErL50 value was calculated to be 32.4 mg test item/L. The 72-hour NOEyL was determined to be 3.7 mg test item/L and the associated 72-hour LOEyL was 11.1 mg test item/L. The 72-hour NOErL was determined to be 3.7 mg test item/L and the associated 72-hour LOErL was 11.1 mg test item/L.
The initial concentrations and the exposure concentrations during the test were determined in the analytical part.
Since no quantifiable amount of the test item was observed in any of the test samples, the results are reported based on the nominal loading levels.

Executive summary:

Test Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV).

Test Design: This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test media were inoculated with nominal 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometric measurement.

Endpoints: Yield and growth rate of the algae

Test Concentrations:Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:3, 1:9, 1:27 and 1:81 of this filtrate were tested, corresponding to the following nominal loading levels of 100, 33.3, 11.1, 3.7 and 1.2 mg test item/L (spacing factor 3). Additionally, a control was tested in parallel.

Test Conditions: Water temperature: 21.5 to 22.3 °C; pH value in the control at test start: 8.0, pH value in the control at test end: 8.2; pH values in the test item treatments at test start: 7.9, pH values in the test item treatments at test end: 7.8 to 8.2; continuous illumination; mean light intensity: 5053 lux (4750 to 5330 lux).

Biological results

Parameter	Yield	Growth Rate
	mg test item/L	mg test item/L
72-hour EC50	14.2	32.4
72-hour EC20	6.45	17.3
72-hour EC10	4.28	12.4
72-hour NOEL	3.7	3.7
72-hour LOEC	11.1	11.1

Analytical results:The quantification of the test item  in the test samples was performed using liquid chromatography with UV detection.The concentrations of the test item were determined in the duplicate test media samples from all test concentrations (test item stock solution and its dilutions) and the duplicate control samples from all sampling times. No signal above the Limit of Quantification was observed in any of the test samples.

Description of key information

The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyL50 was calculated to be 14.2 mg test item/L and the 72-hour ErL50 value was calculated to be 32.4 mg test item/L. The 72-hour NOEyL was determined to be 3.7 mg test item/L and the associated 72-hour LOEyL was 11.1 mg test item/L. The 72-hour NOErL was determined to be 3.7 mg test item/L and the associated 72-hour LOErL was 11.1 mg test item/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

32.4 mg/L

EC10 or NOEC for freshwater algae:

3.7 mg/L

Additional information

The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test media were inoculated with nominal 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometric measurement. Yield and growth rate of the algae were evaluated.

Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:3, 1:9, 1:27 and 1:81 of this filtrate were tested, corresponding to the following nominal loading levels of 100, 33.3, 11.1, 3.7 and 1.2 mg test item/L (spacing factor 3). Additionally, a control was tested in parallel. The water temperature was 21.5 to 22.3 °C; pH value in the control at test start: 8.0, pH value in the control at test end: 8.2; pH values in the test item treatments at test start: 7.9, pH values in the test item treatments at test end: 7.8 to 8.2; continuous illumination; mean light intensity: 5053 lux (4750 to 5330 lux).

The following growth rate and yield were found after 72 hours.

Parameter	Yield	Growth Rate
	mg test item/L	mg test item/L
72-hour EC50	14.2	32.4
72-hour EC20	6.45	17.3
72-hour EC10	4.28	12.4
72-hour NOEL	3.7	3.7
72-hour LOEC	11.1	11.1

The quantification of the test item  in the test samples was performed using liquid chromatography with UV detection.The concentrations of the test item were determined in the duplicate test media samples from all test concentrations (test item stock solution and its dilutions) and the duplicate control samples from all sampling times. No signal above the Limit of Quantification was observed in any of the test samples. Since no quantifiable amount of the test item was observed in any of the test samples, the results are reported based on the nominal loading levels.

The 72-hour EyL50 was calculated to be 14.2 mg test item/L and the 72-hour ErL50 value was calculated to be 32.4 mg test item/L. The 72-hour NOEyL was determined to be 3.7 mg test item/L and the associated 72-hour LOEyL was 11.1 mg test item/L. The 72-hour NOErL was determined to be 3.7 mg test item/L and the associated 72-hour LOErL was 11.1 mg test item/L.