Bicyclo[2.2.1]heptanebis(methylamine)

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:

31 March 2016 to 13 October 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)

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:

Samples were taken from the control and each 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 at 0 and 48 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A nominal amount of test material (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 56, 32, 18 and 10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Adult daphnia
- Feeding during test: None

ACCLIMATION
- Type and amount of food: Each culture was fed with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension.
- Feeding frequency: Daily
- Health during acclimation: No mortality specified.

QUARANTINE
- Not applicable; the test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium 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. 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:

no

Total exposure duration:

48 h

Test temperature:

21 - 22 °C

pH:

7.2 - 10.3

Dissolved oxygen:

8.7 - 8.9 mg/L

Nominal and measured concentrations:

Range-Finding Test
0.1, 1.0, 10 and 100 mg/L (nominal)


Definitive Test
10, 18, 32, 56, 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL conical flasks
- Open/closed: The test vessels were sealed with ground glass stoppers.
- Aeration: No
- No. of organisms per vessel: Five
- No. of vessels per concentration: Four replicates
- No. of vessels per control: Four replicates


TEST MEDIUM / WATER PARAMETERS
Reconstituted Water – ISO Medium
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.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Light intensity: 200 - 1200 Lux. The light intensity during the light period was measured using an ATP Instrumentation Lux meter.
- 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.


EFFECT PARAMETERS MEASURED
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.


RANGE-FINDING STUDY
The test concentrations to be used in the definitive test were 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 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 250 mL test and control vessel was completely filled with test media and sealed with a ground glass stopper to reduce possible losses through volatilization. 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.
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

54 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % confidence limits of 47 - 63 mg/L.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % confidence limits of 27 - 37 mg/L.

Details on results:

RANGE-FINDING TEST
No immobilisation was observed at the test concentrations of 0.10, 1.0 and 10 mg/L, however, 100 % immobilisation was observed at 100 mg/L.
Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.
Chemical analysis of the 10 and 100 mg/L test preparations at 0 and 48 hours showed near nominal concentrations were obtained indicating that the test material was stable under test conditions.

DEFINITIVE TEST
The No Observed Effect Concentrations after 24 and 48 hours exposure were 32 and 10 mg/L respectively.
The Lowest Observed Effect Concentrations after 24 and 48 hours exposure were 56 and 18 mg/L respectively.
The slopes and their standard errors of the response curves at 24 and 48 hours were 7.9 (SE = 0.030) and 6.7 (SE = 0.032) respectively.

- Verification of Test Concentrations
Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 92 % to 104 % of nominal concentration and so the results are based on nominal test concentrations.

- Immobilization Data
Sub-Lethal Effects
After 48 hours sub-lethal effects of exposure were observed in the 32 mg/L test concentration. This response was reduced mobility.

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

Post Study Confirmatory Test
Complete immobilisation was observed to have occurred indicating that the effects seen in the definitive test were not related to the alkaline nature of the test material but to some other intrinsic property.

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 immobilisation 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:

24 hour EC50 = 0.83 mg/L with 95 % confidence limits of 0.7 - 0.98 mg/L.
48 hour EC50 = 0.64 mg/L. 95 % confidence limits were not determined.
The NOEC was 0.56 mg/L and the LOEC was 1.0 mg/L.

The No Observed Effect Concentration is based upon equal to or less than 10 % immobilisation 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:

The EC50 values and associated confidence limits at 24 and 48 hours and 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. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Validation Criteria

The test was considered to be valid given that none of the control daphnids showed immobilisation 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 or pH.

Throughout the test the light intensity was observed to be in the range 581 to 682 Lux.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 48 hour EC50 was determined to be 31 mg/L.

Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 202 and EU Method C.2.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. The number of immobilised daphnia were recorded after 24 and 48 hours.

Due to the potentially volatile nature of the test material, testing was conducted in completely filled and sealed test vessels.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 92 % to 104 % of nominal concentration and so the results are based on nominal test concentrations.

Exposure of Daphnia magna to the test item gave the following results:

48 h EC50: 31 mg/L, with 95 % confidence limits of 27 – 37 mg/L. 

The NOEC was 10 mg/L and the LOEC was 18 mg/L.

It was noted during the definitive test that the test material was highly alkaline in nature, particularly at 100 mg/L. In order to confirm whether the immobilisation observed during the definitive test was due to the alkaline nature of the test material in solution, or some other intrinsic property, a post study confirmatory test was conducted. A single nominal concentration of 100 mg/L was prepared and the pH adjusted to pH 8.0 prior to the addition of the daphnids. Immobilisation and any other adverse effects to exposure were noted after 24 and 48 hours. The results obtained showed that 100% immobilisation occurred suggesting that some other intrinsic property of the test material was causing the effects seen.

Under the conditions of the study the 48 hour EC50 was determined to be 31 mg/L.

Description of key information

48-hour EC50 31.0 mg/L (Daphnia magna), OECD 202, EU Method C.2, H Vryenhoef (2016).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

31 mg/L

Additional information

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 202 and EU Method C.2.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. The number of immobilised daphnia were recorded after 24 and 48 hours.

Due to the potentially volatile nature of the test material, testing was conducted in completely filled and sealed test vessels.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 92 % to 104 % of nominal concentration and so the results are based on nominal test concentrations.

Exposure of Daphnia magna to the test item gave the following results:

48 h EC50: 31 mg/L, with 95 % confidence limits of 27 – 37 mg/L. 

The NOEC was 10 mg/L and the LOEC was 18 mg/L.

It was noted during the definitive test that the test material was highly alkaline in nature, particularly at 100 mg/L. In order to confirm whether the immobilisation observed during the definitive test was due to the alkaline nature of the test material in solution, or some other intrinsic property, a post study confirmatory test was conducted. A single nominal concentration of 100 mg/L was prepared and the pH adjusted to pH 8.0 prior to the addition of the daphnids. Immobilisation and any other adverse effects to exposure were noted after 24 and 48 hours. The results obtained showed that 100 % immobilisation occurred suggesting that some other intrinsic property of the test material was causing the effects seen.

Under the conditions of the study the 48 hour EC50 was determined to be 31 mg/L.