N-isopropylmethacrylamide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 18 June 2012, Experimental Completion Date: 30 July 2012

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 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

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 ai/L.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 (fresh media), 24 and 96 hours (old media).

- Sampling method:
Water samples were taken from the control and 100 mg ai/L test vessel at 0 (fresh media), 24 and 96 hours (old media) for quantitative analysis. The 0 and 24 hour samples were stored at approximately -20°C prior to analysis.

Duplicate samples, and samples at 24 (fresh media), 48 and 72 hours (old and fresh media), 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 fish were exposed to a nominal test concentration of 100 mg active ingredient (ai)/L. A single test concentration was used as information provided gave an EC50 value for Daphnia magna at greater than 100 mg ai/L. It was therefore considered that toxicity would not be expected at the concentration used in the test. The test item was dissolved directly in dechlorinated tap water with the aid of high shear mixing. The test item contained 8.7% water therefore all test concentrations were corrected for this.

An amount of test item (1095 mg) was dissolved in dechlorinated tap water, with the aid of high shear mixing at approximately 7500 rpm for approximately 20 minutes, and the volume adjusted to 1 liter to give a 1000 mg ai/L stock solution. The entire volume of this stock solution was diluted in a final volume of 10 liters of dechlorinated tap water, and stirred using a flat bladed mixer for approximately 1 minute, to give the 100 mg ai/L test concentration.

The stock solution was inverted several times to ensure adequate mixing and homogeneity.

In the range-finding test 3 fish were added to each 10 liter test and control vessel and maintained at approximately 14°C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours under static test conditions.

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

Each vessel was covered to reduce evaporation. After 3, 6, 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish.

A sample of the 100 mg ai/L test concentration was taken for chemical analysis at 0 and 24 hours in order to determine the stability of the test item under test conditions. All samples were stored at approximately -20°C prior to analysis.

DEFINITIVE TEST:
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg ai/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no mortalities or sub-lethal effects of exposure were observed.

EXPERIMENTAL PREPARATION:
For the purpose of the definitive test the test item was dissolved directly in dechlorinated tap water with the aid of high shear mixing. The test item contained 8.7% water therefore all test concentrations were corrected for this.

An amount of test item (2191 mg) was dissolved in dechlorinated tap water, with the aid of high shear mixing at 0 and 24 hours, for approximately 20 minutes, and the volume adjusted to 1 liter to give a 2000 mg ai/L stock solution. At 48 and 72 hours no high shear mixing was used to dissolve the test item. The entire volume of this stock solution was diluted in a final volume of 20 liters of dechlorinated tap water, and stirred using a flat bladed mixer for approximately 1 minute, to give the 100 mg ai/L test concentration.

The stock solution was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, UK and maintained in-house since 10 July 2012. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 11 July 2012 to 23 July 2012. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

The water temperature was controlled at approximately 14°C with a dissolved oxygen content of greater than or equal to 9.1 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There was less than 1% mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.2 cm (sd = 0.2) and a mean weight of 1.32 g (sd = 0.21) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.46 g bodyweight/liter.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Not applicable.

Hardness:

Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3.

Test temperature:

The test vessels were maintained at approximately 14°C.
Water temperature was recorded daily throughout the test.

pH:

The pH of test solutions ranged from 7.8 - 8.4.
pH was recorded daily throughout the test.

Dissolved oxygen:

Dissolved oxygen concentration expressed as a percentage of Air Saturation Value (ASV) ranged from 91 - 98% for test solutions.
Dissolved oxygen concentrations were recorded daily throughout the test.

Nominal and measured concentrations:

Definitive test:
Nominal concentration: 100 mg ai/L.
Measured concentration: 94 to 100% of nominal.

Details on test conditions:

EXPOSURE CONDITIONS:
In the definitive test, 20 liter glass exposure vessels were used for each test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at approximately 14°C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.

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

A semi-static test regime was employed in the test involving a daily renewal of the test preparations to ensure that the concentrations of the test item remained near nominal and to prevent the build up of nitrogenous waste products.

TEST WATER:
The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.

Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

PHYSICO-CHEMICAL MEASUREMENTS
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

RANGE-FINDING TEST
Cumulative mortality data from the exposure of rainbow trout to the test item during the range-finding test are given in Table 1. There were no sub-lethal effects of exposure during the range-finding test.

The results showed no mortalities at the test concentration of 100 mg ai/L.

Based on this information, a single test concentration of 100 mg ai/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 mortalities or sub-lethal effects of exposure were observed.

Chemical analysis of the test preparations at 0 and 24 hours (see Appendix 3 - Analytical Investigations, attached background material) showed measured concentrations of 95% of nominal indicating that the test item was stable under test conditions.

DEFINITIVE TEST
MORTALITY DATA:
Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 2.
There were no mortalities in 7 fish exposed to a test concentration of 100 mg ai/L for a period of 96 hours. Inspection of the mortality data gave the following results:
3 h LC50: >100 mg ai/L
6 h LC50: >100 mg ai/L
24 h LC50: >100 mg ai/L
48 h LC50: >100 mg ai/L
72 h LC50: >100 mg ai/L
96 h LC50: >100 mg ai/L

The results of the definitive test showed the highest test concentration resulting in 0% mortality to be greater than or equal to 100 mg ai/L, the lowest test concentration resulting in 100% mortality to be greater than 100 mg ai/L. The No Observed Effect Concentration (NOEC) was 100 mg ai/L.

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

SUB-LETHAL EFFECTS:
There were no sub-lethal effects of exposure observed in 7 fish exposed to a test concentration of 100 mg ai/L for a period of 96 hours.

OBSERVATIONS ON TEST ITEM 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 4 (see attached background material). Temperature was maintained at approximately 14°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, 24 and 96 hours (see Appendix 3 - Analytical Investigations, attached background material) showed measured test concentrations to range from 94% to 100% of nominal and so it was considered justifiable to estimate the LC50 values in terms of the nominal test concentrations only.

VALIDITY CRITERIA:
There were no mortalities or signs of stress in the control fish during the 96 hours. The dissolved oxygen concentration was ≥60% of ASV in the control and test vessels throughout the duration of the test.

Sublethal observations / clinical signs:

Table 1: Cumulative Mortality Data in the Range-finding Test

Nominal Concentration (mg ai*/L)	Cumulative Mortality (Initial Population = 3)
	3 hours	6 hours	24 hours	48 hours	72 hours	96 hours
Control	0	0	0	0	0	0
100	0	0	0	0	0	0

 

* ai = Active ingredient

Table 2: Cumulative Mortality Data in the Definitive Test

Nominal Concentration (mg ai*/L)	Cumulative Mortality (Initial Population = 7)						% Mortality
	3 hours	6 hours	24 hours	48 hours	72 hours	96 hours	96 hours
Control	0	0	0	0	0	0	0
100	0	0	0	0	0	0	0

 

* ai = Active ingredient

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 of greater than 100 mg ai/L. The No Observed Effect Concentration was 100 mg ai/L.

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Methods

Following a preliminary range-finding test, seven fish were exposed to an aqueous solution of the test item, at a single concentration of 100 mg active ingredient (ai)/L for a period of 96 hours at a temperature of approximately 14ºC under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results

The 96-Hour LC₅₀ based on nominal test concentrations was greater than 100 mg ai/L. The No Observed Effect Concentration was 100 mg ai/L.

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

Analysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to range from 94% to 100% of nominal and so the results are based on nominal test concentrations only.

Description of key information

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 of greater than 100 mg ai/L. The No Observed Effect Concentration was 100 mg ai/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

Introduction

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Methods

Following a preliminary range-finding test, seven fish were exposed to an aqueous solution of the test item, at a single concentration of 100 mg active ingredient (ai)/L for a period of 96 hours at a temperature of approximately 14ºC under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results

The 96-Hour LC₅₀ based on nominal test concentrations was greater than 100 mg ai/L. The No Observed Effect Concentration was 100 mg ai/L.

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

Analysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to range from 94% to 100% of nominal and so the results are based on nominal test concentrations only.