[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 05 October 2016 and 15 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study conducted according to OECD TG 202 in compliance with GLP, without deviations that influence the quality of the results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

EC No 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

EC Number:

289-348-4

EC Name:

[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

Cas Number:

87788-32-7

Molecular formula:

C10H22B2F6N2

IUPAC Name:

[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

impurity 1

Reference substance name:

{3-[(amino-N)methyl]-3,5,5-trimethylcyclohexanamine}(trifluoro)boron

Molecular formula:

C10H22BF3N2

IUPAC Name:

{3-[(amino-N)methyl]-3,5,5-trimethylcyclohexanamine}(trifluoro)boron

additive 1

Reference substance name:

Methanol

EC Number:

200-659-6

EC Name:

Methanol

Cas Number:

67-56-1

Molecular formula:

CH4O

IUPAC Name:

methanol

Test material form:

liquid

Specific details on test material used for the study:

Identification: (μ(5-amino-1,3,3-trimethylcycloihexylamine-N,N')hexafluorodiboron
Physical state/Appearance: Clear colorless liquid
Batch: AEF0009100
Purity: Not provided
Expiry Date: 29 August 2017
Storage Conditions: Room temperature in darkness

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

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 concentration to be used for the initial experiment was analyzed

Test solutions

Vehicle:

yes

Remarks:

Reconstituted water (ISO medium)

Details on test solutions:

Range-finding Test
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 10, 1.0 and 0.10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity
Definitive Test
Based on the results of the initial experiment, the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/L.
A nominal amount of test item (200 mg) was dissolved in test water and the volume adjusted to 2 liters to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 50, 25, 12.5 and 6.25 mg/L.#Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

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 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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

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

Test temperature:

18-22 ±1”C

pH:

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl

Dissolved oxygen:

aerated until the dissolved oxygen concentration was approximately air-saturation value

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Chemical analysis of the 100 mg/L test preparation at 0 hours showed a measured test concentration of 103% of the nominal concentration. There was no significant change in the measured concentration at 48 hours indicating that the test item was stable under test conditions.

Details on test conditions:

Experimental Design and Study Conduct

Range-finding Test
The test concentration used in the initial experiment 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 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 maintaining the water temperature at 18 °C 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 was 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 concentration to be used for the initial experiment was analyzed.
Initial Experiment
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 concentration was inverted several times to ensure adequate mixing and homogeneity.
Due to high immobilization in the 100 mg/L test concentration, the definitive test was performed with a range of five test concentrations.

Definitive Test
Based on the results of the initial experiment, the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/L.
A nominal amount of test item (200 mg) was dissolved in test water and the volume adjusted to 2 liters to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 50, 25, 12.5 and 6.25 mg/L.

Each prepared concentration was 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 48 hours (see Annex 5).

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 °C 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
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.

Data Evaluation
Statistical Analysis
The EC50 value and associated confidence limits at 24 and 48 hours, with the slope of the response curve and its standard error, were calculated by Probit analysis using Linear Maximum-Likelihood regression. The Lowest Observed Effect Concentration and the No Observed Effect Concentration at 24 and 48 hours were calculated using the Step-down Cochran-Armitage Test Procedure. All results were calculated using the ToxRat Professional computer software package (ToxRat 2015).

alidation Criteria
The results of the test are considered valid if the following performance criteria are met:
• No more than 10% of the 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:

Dissiminated (delete when editing) add what is needed

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

62 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding Test
No immobilization was observed at the test concentrations of 0.10, 1.0, 10 and 100 mg/L.
Based on this information, a single test concentration of four replicates, of 100 mg/L was selected for the initial experiment. 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 100 mg/L test preparation at 0 hours showed a measured test concentration of 103% of the nominal concentration. There was no significant change in the measured concentration at 48 hours indicating that the test item was stable under test conditions.

Initial Experiment
The initial experiment failed validity criteria for control immobilization and sub-lethal effects at 48 hours. Additionally, high immobilization (90%) was observed in the 100 mg/L test group. Therefore, the definitive test was performed with a range of five test concentrations.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 and 48 hours (see Table 1, above) showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations.

Immobilization Data
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.
Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression at 24 and 48 hours based on the nominal test concentrations gave the following results:
Time (h) EC50 (mg/L) 95% Confidence limits (mg/L)
24 > 100 Not determined
48 62 Not determined

The No Observed Effect Concentrations after 24 and 48 hours exposure were greater than 100 and 25 mg/L, respectively. The Lowest Observed Effect Concentration after 48 hours exposure was 50 mg/L.
The slopes and their standard errors of the response curves at 24 and 48 hours were 1.8 (SE = 0.26) and 2.9 (SE = 0.063) respectively.

Sub-Lethal Effects
Sub-lethal effects of exposure were observed in the 50 and 100 mg/L test concentrations. This response was reduced mobility

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 21”C to 22”C throughout the test. While there were no treatment related differences for oxygen concentration, concentration dependent differences in pH were observed at 0 hours.
Throughout the test the light intensity was observed to be in the range 416 to 569 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 (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.
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) No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (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.

Any other information on results incl. tables

Table 2: Cumulative Immobilization Data and Observations in the Definitive Test

24 Hours

Nominal Conc. (mg/L)	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	TOTAL	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5N	5N	5N	5N
6.25	0	0	0	0	0	0	5N	5N	5N	5N
12.5	0	0	0	0	0	0	5N	5N	5N	5N
25	0	0	0	0	0	0	5N	5N	5N	5N
50	1	3	0	2	6	30	4N	2N	5N	3N
100	1	1	1	0	3	15	3N, 1R	4N	3N, 1R	5N

48 Hours

Nominal Conc. (mg/L)	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	TOTAL	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5N	5N	5N	5N
6.25	0	0	0	1	1	5	5N	5N	5N	4N
12.5	0	0	0	0	0	0	5N	5N	5N	5N
25	0	0	0	0	0	0	5N	5N	5N	5N
50	2	3	1	2	8	40	3N	2N	4N	3N
100	4	3	4	5	16	80	1R	1N, 1R	1R	A/I

R1 – R4 = Replicates 1 to 4

N = No sub-lethal effects observed

R = Reduced mobility

A/I = All daphnia immobilized

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

The 48 h EC50 (immobilisation) value with Daphnia magna is 62 mg/I.
The 48 h NOEC (immobilisation) value with Daphnia magna is 25 mg/I.
The 48 h LOEC (immobilisation) value with Daphnia magna is 50 mg/I.

Executive summary:

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 and an initial experiment, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 6.25, 12.5, 25, 50 and 100 mg/L for 48 hours at a temperature of 21C to 22C under static test conditions.  The number of immobilized daphnia was 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 the following results:

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
4	62	Not determined	25	50