Pentanediamide, N1,N5-dibutyl-2-[(2-ethyl-1-oxohexyl)amino]-, (2S)-

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:

11 April 2011 - 14 April 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analytical measurements were performed at the control and at the applied test concentration level at the beginning and at the end of the test.

Vehicle:

no

Details on test solutions:

Because the test item is poorly soluble in water, a supersaturated test item stock solution (nominally 100 mg/L) was prepared by dispersing/dissolving the test item amount without the use of any organic solvent into the test medium (OECD medium) two days before the start of the study (on day -2). This solution was shaken for approximately 48 hours and thereafter the non-dissolved test material was removed by filtration through a fine (0.45 μm) filter to give the 100 % v/v saturated solution.

Test organisms (species):

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

Details on test organisms:

EXPERIMENTAL ORGANISMS

Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of LAB Research Ltd.
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
Initial cell number: The initial cell number in the test cultures was 10^4 cells/mL.
Pre-culturing: The pre-culture was intended to give an amount of alga suspension suitable for the inoculation of test cultures. The pre-culture was incubated under the conditions of the study in an aerated Algal Growth Medium and used when still exponentially growing (after an incubation period of 3 days). The cell count of above culture was determined by microscopic method and this cell suspension was diluted with Algal Growth Medium to 10^7 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not reported

Test temperature:

Culture temperature was checked at the beginning of the study and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 22.9 – 23.3 °C measured in the flask and between 22.6 and 23.7 °C measured within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the study, in the control and test concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.75 – 9.10 during the experiment.

Dissolved oxygen:

Not reported

Salinity:

Not reported

Nominal and measured concentrations:

Because no toxic effect was observed in the preliminary range-finding test, only one test concentration at the solubility level of the test item in the test medium (100 % v/v saturated solution) and one control group were tested in a limit test.

Measured geometric mean test item concentration was: 10.0 mg/L.

The biological results are based on the measured geometric mean concentration.

Details on test conditions:

DILUTION WATER

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

LIGHT INTENSITY

The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 8097 lux (equivalent to 109 μE/m^2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: biomass, growth rate and yield

Details on results:

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 No Observed Effect Concentration (NOEC) was determined as 10.0 mg/L.

AREAS UNDER THE 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 No Observed Effect Concentration (NOEC) was determined as 10.0 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 No Observed Effect Concentration (NOEC) was determined as 10.0 mg/L.

Results with reference substance (positive control):

The date of the last study with the reference item Potassium dichromate is: 07 - 10 December 2010.
The 72h ErC 50 : 1.40 mg/L, (95 % confidence limits: 1.29 – 1.53 mg/L)
The 72h EbC 50 : 0.92 mg/L, (95 % confidence limits: 0.85 – 0.99 mg/L)
The 72h EyC 50 : 0.92 mg/L, (95 % confidence limits: 0.85 – 1.00 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 using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).

The ErC50, EbC50 and EyC50 values of the test item were determined from the raw data.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (α = 0.05) by TOXSTAT software.

For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by 2 Sample t-Test (α = 0.05) by TOXSTAT software.

Table 1: Growth Rates (μ) and Percentage Inhibition of μ during the Test Period

Test Group	Growth rate µ and % inhibition of µ
	0 – 24 h		0 – 48 h		0 – 72 h
	µ	%	µ	%	µ	%
Control	0.0553	0.0	0.0588	0.0	0.0599	0.0
Saturated solution (10.0 mg/L measured)	0.0553	3.6	0.0586	0.4	0.0593	1.1

 

Table 2: Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Test Group	Areas under the Growth Curves (A) and % inhibition of A
	0 – 24 h		0 – 48 h		0 – 72 h
	A	%	A	%	A	%
Control	34.0	0.0	258.0	0.0	1336.0	0.0
Saturated solution (10.0 mg/L measured)	32.0	5.9	252.0	2.3	1286.0	3.7

 

Table 3: Yield (Y) and Percentage Inhibition of Y during the Test Period

Test Group	Yield (Y) and % inhibition of Y (0-72 h)
	Y	%
Control	74.0	0.0
Saturated solution (10.0 mg/L measured)	70.5	4.7

Validity criteria fulfilled:

yes

Remarks:

see overall remarks

Conclusions:

Under the conditions of this algal growth inhibition test the observed endpoints for the effect of the test material were the following:

The 72h EbC50 value (biomass): > 10.0 mg/L
The 72h ErC50 value (growth rate): > 10.0 mg/L
The 72h EyC50 value (yield): > 10.0 mg/L

The No-Observed Effect Concentration (NOEC): 10.0 mg/L
The Lowest Observed Effect Concentration (LOEC): > 10.0 mg/L

Based on the results of this study, the test item had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the test item in the test medium.

Executive summary:

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

Because no significant inhibition of algal growth was observed during the preliminary range-finding test, a limit test was carried out using only one concentration at the solubility level of the test substance in the test medium (100 % v/v saturated solution) and one control group.

Concentration of test item was analytically determined during the experiment. Measured concentration was 10.3 mg/L at the start and 9.8 mg/L at the end of the test. Geometric mean of start and end concentration was 10.0 mg/L. Biological results are based on this concentration.

The test design included six replicates at test concentration and six replicates for the untreated control.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (α = 0.05) by TOXSTAT software.

The ErC50, EbC50 and EyC50 values of the test item were determined directly from the raw data.

Under the conditions of this algal growth inhibition test the observed endpoints for the effect of the test material were the following:

The 72h EbC50 value (biomass): > 10.0 mg/L

The 72h ErC50 value (growth rate): > 10.0 mg/L

The 72h EyC50 value (yield): > 10.0 mg/L

The No-Observed Effect Concentration (NOEC): 10.0 mg/L

The Lowest Observed Effect Concentration (LOEC): > 10.0 mg/L

Based on the results of this study, the test item had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the test item in the test medium.

Description of key information

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

Under the conditions of this algal growth inhibition test the observed endpoints for the effect of the test material were the following:

The 72h EbC50 value (biomass): > 10.0 mg/L

The 72h ErC50 value (growth rate): > 10.0 mg/L

The 72h EyC50 value (yield): > 10.0 mg/L

The No-Observed Effect Concentration (NOEC): 10.0 mg/L

The Lowest Observed Effect Concentration (LOEC): > 10.0 mg/L

Based on the results of this study, the test item had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the test item in the test medium.

Additional information

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

Because no significant inhibition of algal growth was observed during the preliminary range-finding test, a limit test was carried out using only one concentration at the solubility level of the test substance in the test medium (100 % v/v saturated solution) and one control group.

Concentration of test item was analytically determined during the experiment. Measured concentration was 10.3 mg/L at the start and 9.8 mg/L at the end of the test. Geometric mean of start and end concentration was 10.0 mg/L. Biological results are based on this concentration.

The test design included six replicates at test concentration and six replicates for the untreated control.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (α = 0.05) by TOXSTAT software.

The ErC50, EbC50 and EyC50 values of the test item were determined directly from the raw data.

Under the conditions of this algal growth inhibition test the observed endpoints for the effect of the test material were the following:

The 72h EbC50 value (biomass): > 10.0 mg/L

The 72h ErC50 value (growth rate): > 10.0 mg/L

The 72h EyC50 value (yield): > 10.0 mg/L

The No-Observed Effect Concentration (NOEC): 10.0 mg/L

The Lowest Observed Effect Concentration (LOEC): > 10.0 mg/L

Based on the results of this study, the test item had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the test item in the test medium.