2-(4-(diethylaminopropylcarbamoyl)phenylazo)-3-oxo-N-(2,3-dihydro-2-oxobenzimidazol-5-yl)butyramide

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2020-08-28 to 2020-09-3

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-(diethylaminopropylcarbamoyl)phenylazo)-3-oxo-N-(2,3-dihydro-2-oxobenzimidazol-5-yl)butyramide

EC Number:

404-910-2

EC Name:

2-(4-(diethylaminopropylcarbamoyl)phenylazo)-3-oxo-N-(2,3-dihydro-2-oxobenzimidazol-5-yl)butyramide

Cas Number:

164578-14-7

Molecular formula:

C25H31N7O4

IUPAC Name:

N-[3-(diethylamino)propyl]-4-[(1E)-2-{2-oxo-1-[(2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-yl)carbamoyl]propyl}diazen-1-yl]benzamide

Test material form:

solid: bulk

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

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

Details on test organisms:

Pseudokirchneriella subcapitata HINDÁK CCAP 278/4 (axenic)
Origin: Culture Collection of Algae and Protozoa (CCAP)
SAMS Research Services Ltd
Dunstaffnage Marine Laboratory
Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Total hardness [mg CaCO3/L =33

Test temperature:

min.22.0°C, max.23.5 °C, mean value 22.8 °C

pH:

see table in the rich text field "Any other information on results incl.tables"

Dissolved oxygen:

no data

Salinity:

not relevant

Conductivity:

no data

Nominal and measured concentrations:

see table in the rich text field "Any other information on results incl.tables"

Details on test conditions:

The study was conducted under static conditions with an initial cell density of 5375 cells/mL. A
stock solution with a nominal concentration of 3.00 mg test item/L was prepared once 72 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out with an appropriate amount of demineralized water. The stock solution was stirred for 72 ± 1 hours (1100 rpm, room temperature) with a magnetic stirrer. Undissolved test item was observed after stirring via Tyndall effect. The stock solution was centrifuged at 17000 g for 20 minutes at
20 ± 1 °C to separate the undissolved test item. Then the components of the dilution were added
to the stock solution . Out of the stock solution five concentrations were tested in a
geometrical series with a dilution factor of 3: 0.0370 - 0.111 - 0.333 - 1.00 - 3.00 mg/L,
corresponding to the geometric mean measured test item concentrations of: 0.0219 – 0.0684 –
0.181 – 0.463 – 1.53 mg test item/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.
The concentrations of the test item were analytically verified via LC-MS/MS in
the fresh media at the start (0 hours) and at the end (72 hours) of the exposure in all concentration levels and the control.
The measured concentrations of the test item in the fresh media were in the
range of 57 to 61% of the nominal values at the start of the exposure (0 hours). The measured
concentrations of the active substance test item in the old media were in the range of 38 to 62%
of the nominal values. Therefore, all effect values are given are based on geometric mean
measured test item concentrations.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.068 mg/L

Basis for effect:

yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.068 mg/L

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.022 mg/L

Basis for effect:

yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.022 mg/L

Basis for effect:

growth rate

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.108 mg/L

95% CI:

0.093 - 0.124

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

Undissolved test item was observed after stirring via Tyndall effect. The stock solution was centrifuged at 17000 g for 20 minutes at 20 ± 1 °C to separate the undissolved test item.

Basis for effect:

yield

Remarks:

EyC50 range (0.0934 - 0.124) that means EC50=0.108 mg/L

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.591 mg/L

95% CI:

0.502 - 0.881

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

Undissolved test item was observed after stirring via Tyndall effect. The stock solution was centrifuged at 17000 g for 20 minutes at 20 ± 1 °C to separate the undissolved test item.

Basis for effect:

growth rate

Remarks:

ErC50 -range (0.502 - 0.881 mg/L) that means EC50=0.591 mg/L

Details on results:

All calculations were based on the fluorescence values of each individual replicate.
The fluorescence values were related to cell density values according to a calibration curve.
The growth rate, the growth rate inhibition, the yield and the
inhibition of yield after 72 hours were calculated from the cell density values according to the formulas.

EC10-,EC20- and EC50- values with confidence intervals of growth rate inhibition and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression with the GraphPad Prism Software.

The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield.
The following statistical tests were conducted:
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) for growth rate.
Williams Multiple Sequential t-test Procedure was performed with
a significance level of 0.05 for growth rate.
Multiple Sequentially-rejective Welch-t-test after Bonferroni-Holm
was done with a significance level of 0.05 for yield.

Calculations were carried out using software
• Excel, MICROSOFT CORPORATION
• SigmaPlot, SPSS INC.
• GraphPad Prism, GRAPHPAD SOFTWARE, INC.
• ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH

Results with reference substance (positive control):

EC50-values of the Reference Item based on nominal conc. (0-72 hours:

Growth Rate Inhibition
ErC50= 1.12 mg/L
95% confidence interval= 1.08 - 1.15 mg/L
Valid Range (average +/- 3x Standard deviation)= 0.771 +/- 0.563

Yield Inhibition
EyC50=0.594 mg/L
95% confidence interval=0.518 - 0.664 mg/L
Valid Range (average +/- 3x Standard deviation)= 0.420 +/- 0.344

Reported statistics and error estimates:

1. ECx-Calculation - Growth Rate (Geometric Mean Measured Test ItemConcentrations)
Equation: Sigmoidal dose-response (variable slope)
Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HillSlope))

Transform x= log(x)
log mg/L Grow th Rate Inhibition
Y1 Y2 Y3
-1.660 0.460 0.720 -0.230
-1.165 5.970 5.730 5.500
-0.742 26.440 23.290 23.620
-0.334 28.900 30.760 31.770
0.185 100.000 100.000 100.000

2. ECx-Calculation - Yield (Geometric Mean Measured Test Item Concentrations)
Equation: Sigmoidal dose-response (variable slope)
Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HillSlope))

Transform x= log(x)
log mg/l Yield Inhibition
Y1 Y2 Y3
-1.660 2.670 4.130 -1.210
-1.165 28.910 27.940 26.970
-0.742 77.990 73.620 74.100
-0.334 80.910 82.850 83.820
0.185 100.000 100.000 100.000

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The study meets the validity criteria of the guideline.

Conclusions:

In this study, Pigment Additiv C was found to inhibit the growth of the freshwater green alga
Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on the
geometric mean measured test item concentrations): The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.591 (0.502 – 0.881) mg/L and 0.108 (0.0934 – 0.124) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 0.0219 mg/L.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2020-08-28 to 2020-09-03 at the test facility, with the definitive exposure phase from 2020-08-31 to 2020-09-03. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 5375 cells/mL. A stock solution with a nominal concentration of 3.00 mg test item/L was prepared once 72 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out with an appropriate amount of demineralized water. The stock solution was stirred for 72 ± 1 hours (1100 rpm, room temperature) with a magnetic stirrer. Undissolved test item was observed after stirring via Tyndall effect. The stock solution was centrifuged at 17000 g for 20 minutes at
20 ± 1 °C to separate the undissolved test item. Then the components of the dilution were added to the stock solution (see Table 2). Out of the stock solution five concentrations were tested in a geometrical series with a dilution factor of 3: 0.0370 - 0.111 - 0.333 - 1.00 - 3.00 mg/L, corresponding to the geometric mean measured test item concentrations of: 0.0219 – 0.0684 – 0.181 – 0.463 – 1.53 mg test item/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The concentrations of the test item were analytically verified via LC-MS/MS in the fresh media at the start (0 hours) and at the end (72 hours) of the exposure in all concentration levels and the control.

The measured concentrations of the test item in the fresh media were in the range of 57 to 61% of the nominal values at the start of the exposure (0 hours). The measured concentrations of the active substance test item in the old media were in the range of 38 to 62% of the nominal values. Therefore, all effect values are given are based on geometric mean measured test item concentrations.