N-isopropylacrylamide

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 12 September 2017 and 07 November 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

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

Analytical monitoring:

yes

Details on sampling:

Range-Finding Test
The analytical methodology was not validated prior to the analysis of range-finding samples; full validation was later conducted at a suitable concentration.

Definitive Test
Validation of the Analytical Method

Specificity
The biological control samples and an analyzed analytical blank showed no significant interfering response at the retention time of the test item. The standard solutions contained a peak specific for the test item whose area changed accordingly with known concentration, hence the specificity of the method by retention time was confirmed.

Linearity
The data was found to have a linear correlation within the calibration range of 0 to 119 mg/L. The R² fit of the calibration curve to the data was 0.9983, and was considered to be acceptable.

Accuracy (Recovery) and Precision
A set of recovery samples accurately fortified at a relevant concentration of test item was prepared five-fold and analyzed. The method was considered to be sufficiently accurate and precise for the purposes of this test. The test sample results were not corrected for recovery.

Limit of Quantification
The limit of quantification (LOQ) was determined by calculating the sample concentration that gave a peak equivalent to ten times the baseline noise. Using this method the LOQ was determined to be 0.26 mg/L.

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 0.10, 1.0, 10 and 100 mg/L.
A nominal amount of test item (50 mg) was dissolved in test water and the volume adjusted to 500 mL to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 0.10, 1.0 and 10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
A nominal amount of test item (100 mg) was dissolved in test water and the volume adjusted to 1 liter to give the 100 mg/L test concentration.

Test organisms (species):

Daphnia magna

Details on test organisms:

Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium (see Annex 2) in a temperature controlled room maintaining the water temperature at 18 to 22 °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 mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. 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:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

49 h

Remarks on exposure duration:

Due to a technical oversight, organism immobilization observations and analytical samples were taken at 49 hours instead of 48.

Test temperature:

18 to 22 °C with a maximum deviation of ± 1 °C

pH:

7.8-7.9

Dissolved oxygen:

8.9-9.0 mg O2/L

Nominal and measured concentrations:

Nominal concentration: 100 mg/mL

Details on test conditions:

Range-finding Test
In the range finding test 5 daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °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. Two replicate test and control vessels were prepared. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.
The control group was maintained under identical conditions but not exposed to the test item.
A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

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/EC Test Guidelines, no immobilization or adverse reactions to exposure were observed.

The concentration and stability of the test item in the test preparation was verified by chemical analysis at 0 and 49 hours.

Exposure Conditions
As in the range finding test 150 mL glass jars containing approximately 100 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 maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 Lux) 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 item.
The test preparations were not renewed during the exposure period.

Assessments
Test Organism Observations
Any immobilization or adverse reactions to exposure were recorded at 24 and 49 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

Water Quality Criteria
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
The light intensity during the light period was measured using an ATP Instrumentation Lux meter.
The appearance of the test media was recorded daily.

Verification of Test Concentrations
Samples were taken from the control and the 100 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 49 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 49 hours, and stored frozen for further analysis if necessary.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

49 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

49 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
No immobilization was observed throughout the test.
Sub lethal effects of exposure were observed in the 100 mg/L test concentration. This response was reduced mobility.
Based on this information, a single test concentration of 4 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 no immobilization or adverse reactions to exposure were observed.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to be near nominal. There was no significant change in the measured concentrations at 48 hours indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 and 49 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations.

Immobilization Data
There was no immobilization in 20 daphnids exposed to a test concentration of 100 mg/L for a period of 49 hours.
The No Observed Effect Concentration after 24 and 49 hours exposure was 100 mg/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Sub-Lethal Effects
No sub lethal effects of exposure were observed throughout the test.

Validation Criteria
The test was considered to be valid given that no more than 10% of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Water Quality Criteria
The results of the water quality measurements are given in Table 3.
Temperature was maintained at approximately 21 °C to 22 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Throughout the test the light intensity was observed to be in the range 715 to 846 Lux.

Observations on Test Item Solubility
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.
Analysis of the immobilization data was carried out using the Trimmed Spearman Karber method at 24 hours and the Binomial Distribution method at 48 hours.
The No Observed Effect Concentration is based upon equal to or less than 10 % immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.

 Immobilization Data

Inspection of the immobilization data gave the following results:

Time (Hour)	EC50 (mg/L)
24	>100
49	>100

Positive Control

All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)			NOEC (mg/L)	LOEC (mg/L)
24	0.79	0.73	-	0.86	0.56	1.0
48	0.75	0.56	-	1.0	0.56	1.0

NOEC     =      No Observed Effect Concentration

LOEC      =      Lowest Observed Effect Concentration

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48 Hour EC50 value of greater than 100 mg/L. The NOEC was 100 mg/L.

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphniasp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods

Following a preliminary range‑finding test, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a nominal concentration of 100 mg/L for 48 hours at a temperature of 21 to 22 °C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 49 hours. The guideline states that immobilization is recorded at both 24 and 48 hours, however, due to a technical oversight, immobilization was recorded at 24 and 49 hours. 

Results

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations.

Exposure of Daphnia magna to the test item gave EC₅₀ values of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) 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 acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48‑Hour EC₅₀ value of greater than 100 mg/L. The NOEC was 100 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L