3-[ethyl[4-[(4-nitrophenyl)azo]phenyl]amino]propiononitrile

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:

30 October 2017 to 2 November 2017

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidelines for studies on the new chemical substance

Version / remarks:

Guidelines for studies on the new chemical substance as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973, amended 2009 under the reference of YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002 and partially amended 2006 as the joint ordinance of The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE)

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-7-3

Version / remarks:

JMAFF 2-7-3. The guidelines related to the study reports for the registration application of pesticide (Ref. No. 12-Nousan-8147 on 24 November 2000) & Ref. No.13-Seisan-3986 on 10 October 2001.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: because the test material was very poorly soluble in water, a test solution was prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23. A saturated test material solution (nominal loading rate of 100 mg/L) was prepared by dispersing/dissolving the amount of test material into the test medium (ISO Medium) two days before the start of the experiment. This solution was shaken for approximately 24 hours at approximately 30°C (Day -2) and then was equilibrated for approximately 24 hours at approximately 20°C (Day -1). The non-dissolved test materials were removed by filtration through a fine (0.22 μm) filter to give the 100% saturated solution. As only limit test was carried out, further dilution of this saturated solution was not performed. Test solution was distributed into test vessels prior to introduction of algae.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG
- Source (laboratory, culture collection): SAG: Collection of Algal Cultures, Inst. Plat Physiology and University of Göttingen, Germany.
- Method of cultivation: under standardised conditions (see OECD 201).

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22.7 - 22.9°C

pH:

7.7 - 8.9

Nominal and measured concentrations:

100% saturated solution (selected on the basis that a significant toxic reponse was not observed during the range-finding test)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Material, size, headspace, fill volume: 100 mL test solution
- Initial cells density: 10xE4 cells/mL/test medium
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes. Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: 8334.6 lux (equivalent to ~112.6 µE/m²/s)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: counting chamber at 24, 48 and 72 hours
- Other: Microscopic observations at 24, 48 and 72 h in each concentration and the control to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0, 0.1, 1, 10 and 100% saturated solution
- Replicates: 2 per test concentration and 3 in the control
- Results used to determine the conditions for the definitive study: yes

CALCULATIONS
Area under the growth curve (biomass), average specific growth rate and yield were calculated for each test flask. Then the mean area under the growth curve, the growth rate and mean yield were determined as arithmetic mean value over all test flasks per treatment.

CALCULATION OF AVERAGE SPECIFIC GROWTH RATE
The average specific growth rate (μ) for individual cultures are calculated from the following relationship:

µ = (ln(Nn) - ln(N0)) / (tn - t0)

where:
ln(Nn) = natural logarithm of measured number of cells/mL at time tn
ln(N0) = natural logarithm of measured number of cells/mL at time t0
t0 = time (h) of the beginning of the test
tn = time (h) of nth measurements after the beginning of the test

The percentage inhibition of growth rate (% Iµ):

% Iµ = ((µc - µt) / µc)) · 100%

where:
% Iµ = percent inhibition in average specific growth rate
µc = mean growth rate of the control
µt = mean growth rate of test concentration t

CALCULATION OF AREAS UNDER THE GROWTH CURVE

A = ((N1 - N0) / 2) · t1 + ((N1 + N2 - 2N0) / 2) · (t2 - t1) + ((Nn-1 + Nn - 2N0) / 2) · (tn - tn-1)

Where:
N0 = nominal number of cells/mL at time t0 (start of the test)
N1 = mean measured number of cells/mL at t1 (24 hours)
N2 = mean measured number of cells/mL at t2 (48 hours)
Nn = mean measured number of cells/mL at tn
t1 = time of first measurement after start of the test
t2 = time of second measurement after start of the test
tn = time of nth measurement after start of the test

The percentage inhibition of area (% Iᴀ):

% Iᴀ = ((Ac - At) / Ac) · 100%

Where:
% Iᴀ = percent inhibition in area under the growth curve
Ac = mean area of the control
At = mean area of test concentration t

CALCULATION OF YIELD

Percentage inhibition in yield (% Iy):

Iy = ((yc - yi) / yc) · 100%

Where:
yc = mean value for yield in the control group
yi = mean value for yield for the test concentration

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): yes. No morphological deviations observed
- Average specific growth rates: The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h average specific growth rate was not statistically significantly different from the untreated control value, accordingly the NOEC was determined as 100% saturated solution (from a nominal loading rate of 100 mg/L).
- Areas under growth curves: The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h areas were not statistically significantly different from the untreated control value, accordingly the NOEC was determined as 100% saturated solution (from a nominal loading rate of 100 mg/L).
- Yield - The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h yield was not statistically significantly different from the untreated control value; accordingly the NOEC was determined as 100% saturated solution (from a nominal loading rate of 100 mg/L).

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period. The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test. Statistical comparisons of biomass, average specific growth rates and yield in control and treated groups were carried out using 2 Sample t-Test (α = 0.05). For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested in significant difference to the control values by 2 Sample t-Test (α= 0.05).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the test material had no toxic effect at saturation; the EC50 results are higher than the solubility of the test material in the test medium.

Executive summary:

The effect of the test material on algal growth, using the unicellular green alga Pseudokirchneriella subcapitata, was assessed over 72 hours in a study which was conducted under GLP conditions and in line with standardised guidelines OECD 201, EU Method C.3, US EPA OCSPP 850.5400 and JMAFF 2-7 -3.

As a significant toxic response was not observed during the preliminary range-finding test, only one test concentration of 100% saturated solution (from a loading rate of 100 mg/L) and one control group was tested. The limit test was performed without analytical measurements because of the very poor water solubility of the test material and because there were no toxic effects at the solubility level of the test material in the test medium during the range-finding test. The test included six replicates at test concentration and six replicates for the untreated control. Statistical comparisons of biomass, average specific growth rates and yield were performed. The NOEC, ErC50, EbC50 and EyC50 of the test material were 100% saturated solution.

Under the conditions of the study, the test material had no toxic effect at saturation; the EC50 results are higher than the solubility of the test material in the test medium.

Description of key information

Under the conditions of the study, the test material had no toxic effect at saturation; the EC50 results are higher than the solubility of the test material in the test medium.

Additional information

The effect of the test material on algal growth, using the unicellular green alga Pseudokirchneriella subcapitata, was assessed over 72 hours in a study which was conducted under GLP conditions and in line with standardised guidelines OECD 201, EU Method C.3, US EPA OCSPP 850.5400 and JMAFF 2-7 -3.

As a significant toxic response was not observed during the preliminary range-finding test, only one test concentration of 100% saturated solution (from a loading rate of 100 mg/L) and one control group was tested. The limit test was performed without analytical measurements because of the very poor water solubility of the test material and because there were no toxic effects at the solubility level of the test material in the test medium during the range-finding test. The test included six replicates at test concentration and six replicates for the untreated control. Statistical comparisons of biomass, average specific growth rates and yield were performed. The NOEC, ErC50, EbC50 and EyC50 of the test material were 100% saturated solution.

Under the conditions of the study, the test material had no toxic effect at saturation; the EC50 results are higher than the solubility of the test material in the test medium.