2-isopropyl-N,2,3-trimethylbutyramide

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:

May-December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test"

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Method C.3 of Commission Regulation (EC) No 761/2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: WS-23
CAS number: 51115-67-4
Physical state/Appearance: White solid
Batch: 80100005
Purity: 99.80%
Expiry Date: 13 October 2018
Storage Conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

Range-Finding Test
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test solutions. Two replicate flasks were used for the control and each test concentration. A 100 mg/L stock solution was prepared by dissolving 50 mg of the test item in 500 mL of AAP media (with the aid of ultrasonication for approximately 5 minutes). Serial dilutions of the stock solution were prepared to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with 2.4 mL P. subcapitata suspension (with an initial biomass/cell count of 9.26 x 105 cells/mL) to give the required test solutions of 0.10, 1.0, 10 and 100 mg/L. The stock solutions and each of the prepared test concentrations were inverted several times to ensure adequate mixing and homogeneity. The control group was prepared under identical conditions but not exposed to the test item.

Limit test and Definitive test
The test was conducted in 250 mL glass conical flasks. Six flasks each containing 100 mL of test solution were prepared for the control and 100 mg/L treatment groups. A 100 mg/L stock solution was prepared by dissolving 100 mg of test item in 1 liter of AAP media (with the aid of ultrasonication for approximately 5 minutes). The stock solution was inoculated with 21.3 mL P. subcapitata suspension (with an initial biomass/cell count of 2.34 x 105 cells/mL) to give the required test solution of 100 mg/L. The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by daily replenishment of the growth media. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 2°C. Four days prior to the start of the test a sufficient volume of the master culture was added to approximately 100 mL of AAP growth media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 10^4 – 10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ± 1 ºC

pH:

The pH value of the control test solution was observed to increase from pH 7.9 at 0 hours to pH 8.9 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guideline.

Nominal and measured concentrations:

Range finding test.
Analytical determination of the 100 mg/L test solutions at 0 and 72 hours showed measured test concentrations of 102% and 103% of nominal respectively were obtained indicating that the test item was stable under test conditions.

Definitive Test
Analytical determination of the test solutions at 0 and 72 hours showed measured test concentrations of 97% and 99% of nominal respectively were obtained and so the results are based on nominal test concentrations only.

Details on test conditions:

Range finding test.
At the start of the range-finding test a 2 mL sample from each test and control solution was removed and the algal cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs to reduce evaporation and incubated at 24 ± 1 ºC (INFORS Multitron® Version 2 incubator). Test vessels were kept under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours. After 72 hours the algal cell density of each test vessel was determined using a Coulter® Multisizer Particle Counter.

Definitive Test
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test solution were prepared for the control and 100 mg/L treatment groups. The control group was prepared under identical conditions but not exposed to the test item. Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 2.34 x 10^5 cells/mL. Inoculation of 1 litre of test medium with 21.3 mL of this primary algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells/mL and had no significant dilution effect on the final test concentration. The flasks were then plugged with polyurethane foam bungs to reduce evaporation and incubated at 24 ± 1 ºC (INFORS Multitron® Version 2 incubator). Test vessels were kept under continuous illumination (intensity approximately 7000 lux ± 15%) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Test Organism Observations
A 1 mL sample was taken from all test vessels at 24, 51 and 72 hours and algal cell density determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density. To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other:

Remarks:

Yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other:

Remarks:

Yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other:

Remarks:

Yield

Details on results:

Range Finding Test
There was no effect on algal growth at the test concentrations of 0.10, 1.0, 10 and 100 mg/L. As no toxic effects were seen at concentrations up to 100 mg/L a limit test using a single concentration of 100 mg/L was selected for the definitive test. This experimental design conforms to the OECD Test Guideline. Analytical determination of the 100 mg/L test solutions at 0 and 72 hours (see Annex 4) showed measured test concentrations of 102% and 103% of nominal respectively were obtained indicating that the test item was stable under test conditions.

Initial Experiments
Based on the result of the range-finding test two initial experiments were conducted at a concentration of 100 mg/L. In both instances significant inhibition of yield (19% and 17% respectively) was observed to have occurred preventing the calculation of a No Observed Effect Concentration (NOEC) value based on yield.

Definitive test
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 1. Daily specific growth rates for the control and 100 mg/L cultures are given in Table 2. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 3. From the data given, it is clear that the growth rate (r) and yield (y) of P. subcapitata (CCAP 278/4) were not affected by the presence of the test item at a nominal test concentration of 100 mg/L over the 72-hour exposure period. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Inhibition of growth rate
ErC10 (0 - 72 h) >100 mg/L
ErC20 (0 - 72 h) >100 mg/L
ErC50 (0 - 72 h) >100 mg/L
where ErCx is the test concentration that reduced growth rate by x%.

Inhibition of Yield
EyC10 (0 - 72 h) >100 mg/L
EyC20 (0 - 72 h) >100 mg/L
EyC50 (0 - 72 h) >100 mg/L
where EyCx is the test concentration that reduced yield by x%.


Validity Criteria
The test is considered valid as all validity criteria were met. The algal cell concentration of the control cultures increased by a factor of 123 within the 72-hour test period, corresponding to a specific growth rate of 1.60 day-1. This increase was in line with the OECD Guideline that states the biomass must increase by a factor of at least 16 after 72 hours.
Nominal cell density of control at 0 hours 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 6.15 x 10^5 cells per mL
The mean coefficient of variation of section by section specific growth rate (days 0 -1, 1-2 and 2-3) for the control cultures was 5% and hence satisfied the validity criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation of average specific growth rate for the control cultures over the test period (0 – 72 h) was 3% and hence satisfied the validity criterion given in the OECD Guideline which states that this must not exceed 7%.

Results with reference substance (positive control):

A positive control study (Envigo Study Number FP48BQ; conducted between 28 November 2016 and 01 December 2016) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for this study were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h): 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h): 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out for the control and 100 mg/L test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance. There were no statistically significant differences (P->0.05), between the control and 100 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate and yield were both 100 mg/L.

Table 1. Algal Cell Counts and Percentage Inhibition of Growth from the Range‑finding Test

Nominal Concentration (mg/L)		Cell Densities* (cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	6.03E+03	7.13E+05	-	-
	R2	5.58E+03	5.46E+05
	Mean	5.81E+03	6.29E+05
0.10	R1	5.22E+03	6.45E+05	-6	-10
	R2	4.67E+03	7.32E+05
	Mean	4.94E+03	6.88E+05
1.0	R1	5.87E+03	6.89E+05	-5	-21
	R2	5.14E+03	8.37E+05
	Mean	5.50E+03	7.63E+05
10	R1	6.08E+03	8.56E+05	-6	-41
	R2	6.00E+03	9.13E+05
	Mean	6.04E+03	8.85E+05
100	R1	5.92E+03	5.98E+05	2	7
	R2	5.79E+03	5.79E+05
	Mean	5.86E+03	5.89E+05

*    Cell densities represent the mean number of cells/mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks. R₁and R₂= Replicates 1 and 2

Table 2. Algal Cell Counts and pH Values in the Definitive Test

Nominal Concentration (mg/L)		pH	Cell Count*(cells/mL)			pH
		0 h	24 h	51 h	72 h	72 h
Control	R1	7.9	2.85E+04	1.77E+05	6.90E+05	8.9
	R2		2.23E+04	1.20E+05	4.62E+05
	R3		2.13E+04	1.48E+05	6.34E+05
	R4		2.33E+04	1.53E+05	6.18E+05
	R5		2.95E+04	1.74E+05	6.73E+05
	R6		2.46E+04	1.44E+05	6.10E+05
	Mean		2.49E+04	1.53E+05	6.15E+05
	SD		3.36E+03	2.10E+04	8.10E+04
100	R1	7.9	2.70E+04	1.85E+05	6.47E+05	8.9
	R2		2.56E+04	1.59E+05	6.66E+05
	R3		2.92E+04	1.83E+05	6.63E+05
	R4		2.77E+04	1.56E+05	6.56E+05
	R5		2.85E+04	1.65E+05	6.68E+05
	R6		2.82E+04	1.86E+05	6.52E+05
	Mean		2.77E+04	1.72E+05	6.59E+05
	SD		1.27E+03	1.39E+04	8.33E+03

*    Cell counts represent the mean number of cells/mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks. R₁- R₆= Replicates 1 to 6

Table.3            Daily Specific Growth Rates for the Control Cultures in the Definitive Test.

Nominal Concentration (mg/L)		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3	Day 0 - 3
Control	R1	0.072	0.068	0.065	0.068
	R2	0.062	0.062	0.064	0.063
	R3	0.060	0.072	0.069	0.067
	R4	0.064	0.070	0.067	0.067
	R5	0.074	0.066	0.064	0.068
	R6	0.066	0.065	0.069	0.067
	Mean	0.066	0.067	0.066	0.067
100	R1	0.070	0.071	0.060	0.068
	R2	0.068	0.068	0.068	0.068
	R3	0.073	0.068	0.061	0.068
	R4	0.071	0.064	0.069	0.068
	R5	0.073	0.065	0.067	0.068
	R6	0.072	0.070	0.060	0.068
	Mean	0.071	0.068	0.064	0.068

R₁- R₆= Replicates 1 to 6

Table4            Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.068		6.85E+05
	R2	0.063		4.57E+05
	R3	0.067		6.29E+05
	R4	0.067	-	6.13E+05	-
	R5	0.068		6.68E+05
	R6	0.067		6.05E+05
	Mean	0.067		6.10E+05
	SD	0.002		8.12E+04
100	R1	0.068	-1	6.42E+05
	R2	0.068	-1	6.61E+05
	R3	0.068	-1	6.58E+05
	R4	0.068	-1	6.51E+05
	R5	0.068	-1	6.63E+05
	R6	0.068	-1	6.47E+05
	Mean	0.068	-1	6.54E+05	-7
	SD	0.000		8.34E+03

*       In accordance with the OECD test guideline only the mean value for yield is calculated R₁-R₆= Replicates 1 to 6 SD= Standard Deviation

Validity criteria fulfilled:

yes

Conclusions:

EC50 for growth rate and yield was found to be >100 mg/L after 72 hours. The NOEC for both growth rate and yield was found to be 100 mg/L.

Executive summary:

The toxicity to algae was assessed following the methods outlined in the OECD 201 guidelines. Test concentrations were measured analytical by GC-HPLC and measured results were found to be 97 -99% of nominal concentrations. Therefore, nominal concentrations were used to report the results. EC50 for growth rate and yield was found to be >100 mg/L after 72 hours. The NOEC for both growth rate and yield was found to be 100 mg/L.

Description of key information

The toxicity to algae was assessed following the methods outlined in the OECD 201 guideline. Test concentrations were measured analytical by GC-HPLC and measured results were found to be 97 -99% of nominal concentrations. Therefore, nominal concentrations were used to report the results. EC50 for growth rate and yield was found to be >100 mg/L after 72 hours. The NOEC for both growth rate and yield was found to be 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L