N-isopropylacrylamide

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:

The study was conducted between 22 August 2017 and 08 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)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical state/Appearance: White flakes
Batch: 2161004
Purity: 99.41 %
Expiry Date: 31 January 2018
Storage Conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Range-Finding Test
The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours.

Definitive Test
Based on the result of the range-finding test a "limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no effect on algal growth was observed.

Vehicle:

no

Details on test solutions:

Range-Finding Test
A nominal amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made 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 algal suspension (3.8 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis 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 the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ± 1 °C.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104 – 105 cells/mL.

Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

7.4-7.5

Nominal and measured concentrations:

Range-finding test (nominal concentration): 0.10, 1.0, 10 and 100 mg/L.
Definitive test (limit test): 100 mg/L

Details on test conditions:

Range-Finding Test
The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours.
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.
A nominal amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made 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 algal suspension (3.8 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 ºC 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 cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
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.

Definitive Test
Based on the result of the range-finding test a "limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no effect on algal growth was observed.

Experimental Preparation
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution. This stock solution was inoculated with algal suspension (4.3 mL) to give the required test concentrations of 100 mg/L.
The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.

Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and 100 mg/L treatment group.
The control group was maintained 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 1.17 x 106 cells per mL. Inoculation of 1 liter of test medium with 4.3 mL of this algal suspension gave an initial nominal cell density of 5.00 x 103 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 °C 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.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

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

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Range-finding Test
The results showed no effect on growth rate at the test concentrations of 0.10, 1.0, 10 and 100 mg/L.
Based on this information a single test concentration of six replicates, of 100 mg/L was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the maximum test concentration given in the OECD/EC Test Guidelines no effect on growth was observed.
Chemical analysis of the 100 mg/L test preparations at 0 and 72 hours showed near nominal concentrations were obtained indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 and 72 hours (see Annex 4) showed measured test concentrations to range from 110 % to 121 % of nominal and so the results are based on nominal test concentrations only.

Growth Data
It is clear that the growth rate (r) and yield (y) of Pseudokirchneriella 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.
Accordingly the following results were determined from the data:

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 %.
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 (Sokal and Rohlf, 1981). 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 was 100 mg/L.

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 %.
Statistical analysis of the yield data was carried out as in Section 6.2.3. 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 yield was 100 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 229 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Nominal cell density of control at 0 hours: 5.00 x 103 cells per mL
Mean cell density of control at 72 hours :1.15 x 106 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 7 % and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35 %.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 1 % and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7 %.

Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours.

Results with reference substance (positive control):

A positive control 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 the positive control 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.

Water Quality Criteria

The pH values of the control and each test preparation are given in Table 2. Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures was observed to increase from pH 7.5 at 0 hours to pH 8.5 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 Guidelines.

Observations on Test Item Solubility

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions.

Cell Densities 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	5.68E+03	5.95E+05	-	-
	R2	5.36E+03	6.64E+05
	Mean	5.52E+03	6.29E+05
0.10	R1	5.46E+03	6.68E+05	0	[2]
	R2	5.66E+03	6.18E+05
	Mean	5.56E+03	6.43E+05
1.0	R1	5.73E+03	3.80E+05	9	33
	R2	5.95E+03	4.70E+05
	Mean	5.84E+03	4.25E+05
10	R1	5.58E+03	5.55E+05	[2]	[8]
	R2	5.65E+03	8.00E+05
	Mean	5.62E+03	6.77E+05
100	R1	5.20E+03	6.03E+05	2	11
	R2	5.55E+03	5.22E+05
	Mean	5.38E+03	5.63E+05

*    Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁and R₂= Replicates 1 and 2

Cell Densities and pH Values in the Definitive Test

Nominal Concentration (mg/L)		pH	Cell Densities*(cells per mL)			pH
		0 h	24 h	48 h	72 h	72 h
Control	R1	7.5	2.88E+04	2.07E+05	1.11E+06	8.5
	R2		3.60E+04	1.97E+05	1.09E+06
	R3		3.41E+04	2.11E+05	1.23E+06
	R4		3.47E+04	2.27E+05	1.19E+06
	R5		3.41E+04	2.18E+05	1.16E+06
	R6		3.48E+04	2.05E+05	1.10E+06
	Mean		3.38E+04	2.11E+05	1.15E+06
100	R1	7.4	3.09E+04	1.99E+05	1.08E+06	8.7
	R2		3.27E+04	1.94E+05	1.11E+06
	R3		4.10E+04	2.43E+05	1.33E+06
	R4		3.85E+04	2.29E+05	1.23E+06
	R5		3.63E+04	2.27E+05	1.20E+06
	R6		3.25E+04	2.24E+05	1.27E+06
	Mean		3.53E+04	2.19E+05	1.20E+06

 

Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.073	0.082	0.070
	R2	0.082	0.071	0.071
	R3	0.080	0.076	0.073
	R4	0.081	0.078	0.069
	R5	0.080	0.077	0.070
	R6	0.081	0.074	0.070
	Mean	0.080	0.076	0.071

 

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	0.075		1.10E+06
	R2	0.075		1.09E+06
	R3	0.076		1.22E+06
	R4	0.076	-	1.19E+06	-
	R5	0.076		1.16E+06
	R6	0.075		1.09E+06
	Mean	0.076		1.14E+06
	SD	0.001		5.63E+04
100	R1	0.075	1	1.08E+06
	R2	0.075	1	1.11E+06
	R3	0.078	[3]	1.33E+06
	R4	0.076	0	1.22E+06
	R5	0.076	0	1.19E+06
	R6	0.077	[1]	1.26E+06
	Mean	0.076	[0]	1.20E+06	[5]
	SD	0.001		9.41E+04

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EC50 values of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

Executive summary:

Introduction

A study was perford to assess the effect of the test item on the growth of the green alga Pseudokirchneriellasubcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Methods.

Following a preliminary range-finding test,Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at a concentration of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

Results.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 110 % to 121 % of nominal and so the results are based on nominal test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC₅₀values of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

 

Description of key information

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EC₅₀ values of greater than 100 mg/L. The No Observed Effect Concentration was 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