3-[[3-(dimethylamino)propyl]amino]propiononitrile

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 07 June 2016 and 06 July 2016

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)

Version / remarks:

April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

Principles of method if other than guideline:

N/A - study to guideline

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

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 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:

yes

Details on test solutions:

Reconstituted water (ISO medium) used for both the range-finding and definitive tests is defined as follows:
Reconstituted Water – ISO Medium
Ingredient Final Concentration
(mg/L)
CaCl2.2H2O 294
MgSO4.7H2O 123
NaHCO3 65
KCl 5.8

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.

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 150 mL glass beakers containing Elendt M7 medium (see below) 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.

Reconstituted Water – Elendt M7 Medium
Ingredient Final Concentration (mg/L)
H3BO3 0.715
MnCl2.4H2O 0.090
LiCl 0.077
RbCl 0.018
SrCl2.6H2O 0.038
NaBr 0.004
Na2MoO4.2H2O 0.016
CuCl2.2H2O 0.004
ZnCl2 0.013
CoCl2.6H2O 0.010
KI 0.0033
Na2SeO3 0.0022
NH4VO3 0.00058
Na2EDTA.2H2O 0.625
FeSO4.7H2O 0.249
CaCl2.2H2O 293.8
NaHCO3 64.8
MgSO4.7H2O 123.3
Na2SiO3.9H2O 10
KCl 5.8
NaNO3 0.274
K2HPO4 0.184
KH2PO4 0.143
Thiamine hydrochloride 0.075
Cyanocobalamine (vitamin B12) 0.0010
D(+) biotin (vitamin H) 0.00075

The pH of the prepared media was 7.9 ± 0.3 and stored at approximately 21 ºC.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

N/A

Post exposure observation period:

None

Hardness:

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

Test temperature:

18 to 22 ± 1”C

pH:

7.9 ± 0.3

Salinity:

N/A

Conductivity:

N/A

Nominal and measured concentrations:

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.

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.
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.
In the range-finding test 10 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. 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 the 10 and 100 mg/L test concentrations 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.
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.
The prepared test concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0 and 48 hours (see Annex 4).

Exposure Conditions
As in the range-finding test, 150 mL glass beakers 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 48 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
The appearance of the test media and 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.

Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
•No more than 10% of the control control daphnids show immobilization or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water).
•The dissolved oxygen concentration at the end of the test is equal to or greater than 3 mg/L in the control and test vessels.

Reference substance (positive control):

yes

Remarks:

A positive control (Envigo Study Number MS29MC) 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.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Immobilization

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Immobilization

Details on results:

Range-finding test
No immobilization was observed in any of the concentrations tested.
After 24 hours exposure 1 daphnid in each of the 1.0 and 10 mg/L test concentrations and 2 daphnia in the 100 mg/L test concentration were observed to be trapped at the surface. After 48 hours exposure in the 100 mg/L test concentration, 9 daphnia were observed with reduced mobility and 1 daphnid was observed to be trapped at the surface.
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/EC Test Guidelines 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 concentrations. Analysis of the 48-Hour samples showed the 10 mg/L test concentration to be near nominal, however the 100 mg/L test concentration gave a measured test concentration of 224 mg/L. This high result was considered to be anomalous and hence the test item was considered to be stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 and 48 hours (see Annex 4) showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations.

Immobilization Data
Exposure of 20 daphnids to a test concentration of 100 mg/L for a period of 48 hours resulted in immobilization of one daphnia. This level of immobilization was considered not to be significant. Exposure of Daphnia magna to the test item gave EC50 values 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.

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 none 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
Temperature was maintained at approximately 22 C throughout the test, while there were no treatment related differences for oxygen concentration, the pH of the 100 mg/L test concentration was significantly higher than the control.
Throughout the test the light intensity was observed to be in the range 501 to 526 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):

Analysis of the immobilization data by the probit analysis using the linear maximum likelihood regression method at 24 and 48 hours using the ToxRat Professional computer software package based on the nominal test concentrations gave the following results:
Time Point
(Hours) EC50 (mg/L) 95% Confidence Limits (mg/L) NOEC (mg/L) LOEC (mg/L)
24 0.83 0.70 - 0.98 0.56 1.0
48 0.64 Not possible to determine 0.56 1.0

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.

Reported statistics and error estimates:

N/A

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 No Observed Effect Concentration 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, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a concentration of 100 mg/L for 48 hours at a temperature of approximately 22 C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 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 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 Aradur 1019 to daphnia magna was determined according to OECD test guideline 202.

The result is given as EC50 (48h) >100 mg/l

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L