N-isopropylmethacrylamide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 23 October 2007 and 22 November 2007.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
A "Limit test" was conducted at a concentration of 100 mg/l.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 48 hours

- Sampling method:
Water samples were taken from the control (replicates R1 - R4 pooled) and the 100 mg/l test group (replicates Rl - R2 and R3 -R4 pooled) at 0 and 48 hours for quantitative analysis.

Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

RANGE-FINDING TEST:
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.1 0, 1.0, 10, and 100 mg/l. The test material was dissolved directly in water.

An amount of test material (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give the 100 mg/l test concentration from which serial dilutions were performed in reconstituted water to give the remainder of the test series of 10, 1.0 and 0.10 mg/l.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 21.2°C to 22.0°C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Whilst the temperature was measured to be slightly in excess of the 20 ± 1°C given in the protocol, this was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and the temperatures were within the test guideline specification. Each 250 ml test and control vessel contained 200 ml of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.

The control group was maintained under identical conditions but not exposed to the test material.

DEFINITIVE TEST:
Based on the results 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/EEC Test Guidelines, no immobilisation or adverse reactions to exposure were observed.

EXPERIMENTAL PREPARATION:
For the purpose of the definitive test the test material was dissolved directly in reconstituted water.

An amount of test material (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give the 100 mg/l test concentration.

The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a suspension of algae (Chlorella sp.). Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable.

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

The test vessels were maintained in a temperature controlled room at approximately 20°C.
Water temperature was recorded daily throughout the test.

pH:

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCI.
pH was recorded at the start and termination of the test.

Dissolved oxygen:

The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Dissolved oxygen concentrations were recorded at the start and termination of test.

Nominal and measured concentrations:

Definitive test:
Nominal concentration: 100 mg/l.
Measured concentration: 95% to 96% of nominal value.

Details on test conditions:

EXPSOURE CONDITIONS:
As in the range-finding test 250 ml glass jars containing approximately 200 ml of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at approximately 20°C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test material.

The test preparations were not renewed during the exposure period.


TEST MEDIUM / WATER PARAMETERS
The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals and is defined below:

Reconstituted Water
i)   Stock Solutions
a)  CaCl2.2H2O: 11.76 g/l
b)  MgSO4.7H2O: 4.93 g/l
c)  NaHCO3:  2.59 g/l
d)  KCl :  0.23 g/l
ii)  Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

An estimation of hte EC50 value was given by inspection of the immobilisation data.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

RANGE-FINDING TEST:
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in Table 1.

No significant immobilisation was observed at the test concentrations of 0.1 0, 1.0 and 10 mg/l. However, a single daphnid at 100 mg/l was observed to be immobilised after 48 hours exposure. This was considered to be due to handling stress and/or natural causes and not due to test material toxicity as no other immobilisation was observed throughout the test.

Based on this information, a single test concentration of four 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/EEC Test Guidelines, no immobilisation or adverse reactions to exposure were observed.

DEFINITIVE TEST:
Immobilisation data:
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2.

There was no immobilisation in 20 daphnids exposed to a test concentration of 100 mg/l for a period of 48 hours. Inspection of the immobilisation data gave the following results:
24 h EC50: >100 mg/l
48 h EC50: >100 mg/l

The No Observed Effect Concentration after 24 and 48 hours exposure was 100 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

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

OBSERVATIONS ON TEST MATERIAL SOLUBILITY:
The test preparations were observed to be clear colourless solutions throughout the duration of the test.

PHYSICO-CHEMICAL MEASUREMENTS:
The results of the physico-chemical measurements are given in Appendix 3. Temperature was maintained at approximately 20°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

VERIFICATION OF TEST CONCENTRATIONS:
Analysis of the test preparations at 0 and 48 hours (see Appendix 2 – attached background material) showed measured test concentrations to range from 95% to 96% of nominal value and so it was considered justifiable to estimate the EC50 values in terms of the nominal test concentrations only.

Results with reference substance (positive control):

Postive Control:
Cumulative immobilisation data from the exposure of Daphnia magna to the reference material the positive control are given in Table 3.

Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the trimmed Spearman-Karber method at 24 and 48 hours based on the nominal test concentrations gave the following results:

3 h EC50: >3.2 mg/l
24 h EC50: 0.65 mg/l (95% Confidence limits 0.58 - 0.72 mg/l)
48 h EC50: 0.47 mg/l (95% Confidence limits 0.543 - 0.53 mg/l)

The No Observed Effect Concentration after 24 and 48 hours was 0.32 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

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

Table 1 Cumulative Immobilisation Data in the Range-Finding Test

 

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 per Replicate)
	24 Hours	48 Hours
Control	0	0
0.10	0	0
1.0	0	0
10	0	0
100	0	1*

 

*One daphnid at 100 mg/I was observed to be immobilised after 48 hours exposure. This was considered to be due to handling stress and/or natural causes and not due to test material toxicity.

Table 2 Cumulative Immobilisation Data in the Definitive Test

 

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)
	24 Hours			48 Hours
	No. Per Replicate	Total	%	No. Per Replicate	Total	%
Control
R1	0	0	0	0	0	0
R2	0			0
R3	0			0
R4	0			0
100
R1	0	0	0	0	0	0
R2	0			0
R3	0			0
R4	0			0

R₁ - R₄ = Replicates 1 to 4

Table 3 Cumulative Immobilisation Data in the Positive Control

 

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	3 Hours				24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0	0	0	0	0
0.56	0	0	0	0	2	3	5	25	9	7	16	80
1.0	0	0	0	0	10	10	20	100	10	10	20	100
1.8	0	0	0	0	10	10	20	100	10	10	20	100
3.2	1	1	2	10	10	10	20	100	10	10	20	100

R₁ - R₂ = Replicates 1 and 2

Appendix 3 Physico-Chemical Measurements

 

Nominal Concentration (mg/l)	0 Hours				24 Hours	48 Hours
	pH	mg O2/l	%ASV*	T°C	T°C	pH	mg O2/l	%ASV*	T°C
Control
R1	7.9	8.8	97	20	20	7.9	8.7	96	20
R2	7.9	8.9	98	20	20	7.9	8.7	96	20
R3	7.9	8.9	98	20	20	7.9	8.7	96	20
R4	7.9	8.9	98	20	20	7.9	8.7	96	20
100
R1	7.8	8.8	97	20	20	7.9	8.7	96	20
R2	7.9	8.9	98	20	20	7.9	8.7	96	20
R3	7.8	8.8	97	20	20	7.9	8.7	96	20
R4	7.9	8.9	98	20	20	7.9	8.7	96	20

*ASV = Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

R₁ - R₄ = Replicates 1 to 4

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.

Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202,"Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a concentration of 100 mg/l for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results.

The 48-Hour EC₅₀ for the test material to Daphnia magna based on nominal test concentrations was greater than 100 mg/l and correspondingly 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.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 95% to 96% of nominal value and so the results are based on nominal test concentrations only.

The 48-Hour EC₅₀ for the reference material to Daphnia magna based on nominal concentrations was 0.47 mg/l with 95% confidence limits of 0.43 - 0.53 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

Description of key information

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

Introduction.

A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202,"Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a concentration of 100 mg/l for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results.

The 48-Hour EC₅₀ for the test material to Daphnia magna based on nominal test concentrations was greater than 100 mg/l and correspondingly 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.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 95% to 96% of nominal value and so the results are based on nominal test concentrations only.

The 48-Hour EC₅₀ for the reference material to Daphnia magna based on nominal concentrations was 0.47 mg/l with 95% confidence limits of 0.43 - 0.53 mg/l. The No Observed Effect Concentration was 0.32 mg/l.