3,6-dioxaoctamethylenediamine

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:

March 29, 2016 - May 14, 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

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: Jeffamine EDR 148
- Source and lot/batch No.of test material: 4M413
- Expiration date of the lot/batch: 31 December 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: Stable
- purity: 99%

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, 10, 18, 32, 56, and 100 mg/L
- Sampling method: Samples taken from the bulk preparations at 0 hours and from the pooled replicates at 48 hours for future analysis.
- Sample storage conditions before analysis: frozen

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Progressive dilution of stock solution
*Range-finding:
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.
During this test, the pH of the 100 mg/L test concentration was measured to be outside the guideline criteria of 6-9, therefore the range finding test was repeated with the inclusion of a pH adjusted preparation in order to determine whether the increased pH had an effect on the toxicity of the test item. In the second range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L, along with a 100 mg/L test concentration without adjustment of the pH. 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 pH of this 100 mg/L stock solution was measured to be 10.0 and was adjusted to 7.8 using dilute hydrochloric acid. A series of dilutions was performed from this 100 mg/L test concentration to give further test concentrations of 0.10, 1.0 and 10 mg/L. An aliquot of the 100 mg/L stock solution was also tested alongside prior to the adjustment of the pH.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding tests 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 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 contained 200 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 samples from the second range finding test of the concentrations within the range to be used for the definitive test were analyzed.
*Definitive test:
Based on the results of the range-finding tests the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 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 10, 18, 32 and 56 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: Water flea
- Source: 1st instar Daphnia magna derived from in-house laboratory cultures.
- Age of parental stock (mean and range, SD)
- Feeding during test: no
ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not): 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.
- Type and amount of food: mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension.
- Feeding frequency: daily
METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Gravid adults were isolated the day before testing, and the young produced overnight were used in testing.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

250 mg/L as CaCO3

Test temperature:

21-22 degrees C

pH:

8.0-9.9 across all concentrations

Dissolved oxygen:

8.4-8.6 mg/L

Salinity:

not applicable

Conductivity:

not measured

Nominal and measured concentrations:

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. Based on the results of the range-finding tests the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass
- Type (delete if not applicable): covered to reduce evaporation
- Material, size, headspace, fill volume: 150 mL glass, filled with 100 mL test solution
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Culture medium:
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 Water:
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.

- Culture medium different from test medium: yes
- Intervals of water quality measurement: 0, 24, and 48 h

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light, 8 h dark, 20 min dawn and dusk transitions
- Light intensity: 200-1200 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): immobility, 24 and 48 h 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.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations:
First range-finding- Control, 0.10, 1.0, 10, and 100 mg/L.
Second range-finding-Control, 0.01, 1.0, 10, 100, and 100 (pH adjusted) mg/L.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 66-85 mg/L 95% CL

Details on results:

- Behavioural abnormalities: Sub-lethal effects of exposure were observed in the 100 mg/L test concentration at 24h. This response was reduced movement.
* Range-finding
In the initial range finding test, no immobilization was observed at the test concentrations of 0.10, 1.0 and 10 mg/L, however, immobilization was observed at 100 mg/L. A single daphnia was observed to be immobilized in the control group, however, this was considered to be due to natural causes given that no more than 10% immobilization was observed.
In the second range finding test, no immobilization was observed at the test concentrations of 0.10, 1.0 and 10 mg/L, however, immobilization was observed at 100 mg/L, both with and without pH adjustment. In the 0.10 mg/L test concentration, 4 out of the 10 daphnia were observed to be trapped at the surface at the end of the test, however, given that this was not a concentration dependant response, this was considered not to have had an impact on the outcome of the test.
- Mortality of control: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Other: 48-h LOEC = 100 mg/L; 48-h NOEC = 56 mg/L

Results with reference substance (positive control):

- Results with reference substance valid? Yes
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

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 Tets Procedure. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Validity criteria fulfilled:

yes

Conclusions:

For the test material, the 48-h EC50 to Daphnia magna was determined in an OECD 202 study. The 48-h EC50 was 75 mg/L, with a 95% Confidence limit of 66-85 mg/L.

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.

Following preliminary range-finding tests, 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 immobilized daphnia were recorded after 24 and 48 hours.

Analysis of the 56 and 100 mg/L 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)
48	75	66 -85	56	100

Description of key information

A study was performed to assess the acute toxicity of the test item to Daphnia magna.

Following preliminary range-finding tests, 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 immobilized daphnia were recorded after 24 and 48 hours.

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)
48	75	66 -85	56	100

The test item is no toxic on Daphnia magna.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

75 mg/L

Additional information

The test item is no toxic on invertebrate.