3,3'-ethylenedioxybis(propylamine)

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:

2017-06-27 - 2017-10-26

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:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD No. 23

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss GLP Monitoring Authorities, Date of decission 2015-11-16

Analytical monitoring:

yes

Details on sampling:

- Sampled concentrations: 6.25, 12.5, 25, 50, and 100 mg/L
- Sampling method: duplicate samples were taken from each test concentration and the control at
the start and at the end of the test (after 48 hours). For the 48-hour stability samples, the contents
of the respective replicates were combined prior to sampling.
- Sample storage conditions before analysis: all samples were frozen (at -20 ± 5 °C)

- Analysed samples: The concentrations of JEFFAMINE® EDR-176 were analyzed in one of the duplicate test medium
samples from the control and the nominal concentrations of 50 and 100 mg/L from both sampling
times (0 and 48 hours). The samples of the lower test concentrations (6.25, 12.5 and 25 mg/L)
were not analyzed since these concentrations were below the 48 hour NOEC determined in this
test and were therefore not relevant for the interpretation of the biological results.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving
101 µL of the test item completely in 1000 mL of test water. This volume is equivalent to a loading rate of
100 mg/L, considering the relative density of the test item of 0.99. The test item was dissolved by intense
stirring for 15 minutes at room temperature. During this stirring period the pH was adjusted from pH 10 to
7.5 with 0.1 M hydrochloric acid solution.
- Eluate:The highest test concentration of 100 mg/L was further diluted with test water to prepare the test
media with the lower test concentrations. The test medium was prepared just before the introduction of
the daphnids (i.e., start of the test).
- Evidence of undissolved material (e.g. precipitate, surface film, etc.):After the dissolving procedure the highest
test concentration of 100 mg/L was a clear solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Daphnia magna Straus
- Source: Daphnia Collection of the University of Basel/Switzerland in 2015
- Age of parental stock: 3-4 Weeks. At the start of the test, the organisms used
in the test were 6 to 24 hours old and were not first brood progeny.
- Feeding during test: no


ACCLIMATION
- Acclimation period: no acclimatisation was necessary, since the bred of the daphnia
was under identical conditions to those of the test.
- Type and amount of food during the breeding: Algal suspension of the green algae
Desmodesmus subspicatus supplied by the Collection of Algal Cultures (SAG, Institute for
Plant Physiology, University of Göttingen, 37073 Göttingen / Germany) and cultivated at
IES Ltd Laboratories under standardized conditions or a mixture of this algal suspension and a
commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-GmbH, 49324 Melle / Germany).
- Feeding frequency: three times a week
- Health during acclimation (any mortality observed): The daphnia were healthy, no mortality was observed.

Test type:

static

Water media type:

other: ISO Test water (Medium according to the guideline)

Limit test:

no

Total exposure duration:

48 h

Hardness:

2.5 mmol/L = 250 mg/L as CaCO3

Test temperature:

22°C

pH:

7.5-7.9

Dissolved oxygen:

at the start of the test at all concentrations and the control: 8.5 mg/L oxygen
at the end of the test at all concentrations and the control: 8.2-8.3 mg/L oxygen

Salinity:

n.d.

Conductivity:

n.d.

Nominal and measured concentrations:

nominal concentrations (measured concentrations 0/48h): 6.25, 12.5, 25, 50 (49/48) and
100 (96/96) mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Beakers
- Type (delete if not applicable): Open (The test vessels were coverd with glass plates to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions.)
- Material, size, headspace, fill volume: glas, 100 mL, open, 50 mL
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): no, static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates):4
- No. of vessels per control (replicates):4
- No. of vessels per vehicle control (replicates):no
- Biomass loading rate: 1 Daphnia / 2 mL test media

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Medium
Ingredients / Properties [mmol/L] / Concentration [mg/L]
CaCl2 × 2H2O / 2.0 / 294
MgSO4 × 7H2O / 0.5 / 123
NaHCO3 / 0.75 / 65
KCl / 0.075 / 5.8
- Alkalinity: 0.8 mmol/L
- Water Hardness: 2.5 mmol/L = 250 mg/L as CaCO3

OTHER TEST CONDITIONS
- Adjustment of pH: 7.5 with 0.1M hydrochloric acid
- Photoperiod:16-hour light to 8-hour dark cycle with a 30-minute transition period
- Light intensity:19 and 20 µE m-2 s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility of the daphnids and other non-lethal effects (e.g. abnormal behavior or appearance) after 24 and 48 hours of exposure.

VEHICLE CONTROL PERFORMED: no, not needed


RANGE-FINDING STUDY without pH adjustment:
- Test concentrations: 1.0 / 10 / 100 mg/L
- Results used to determine the conditions for the definitive study:
48-hour immobilization: 1.0 mg/L / 0%; 10 mg/L / 0%; 100 mg/L / 60%

RANGE-FINDING STUDY with pH adjustment: The pH was adjusted in the 100 mg/L stock solution from pH10 to pH 7.5
- Test concentrations: 1.0 / 10 / 100 mg/L
- Results used to determine the conditions for the definitive study:
48-hour immobilization: 1.0 mg/L / 0%; 10 mg/L / 0%; 100 mg/L / 60%

Reference substance (positive control):

yes

Remarks:

Potassium dichromate / Separate reference studies under GLP twice a year.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: no abnormalities in the control and up and including the concentration 50 mg/L, at the highest concentration 100 mg/L adverse effect was noted. All surviving daphnids showed slower swimming activity compared to the control
- Mortality of control: no
- Other adverse effects control: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no


Thus, the correct dosage of the test item JEFFAMINE® EDR-176 was confirmed. The test item was stable in the test media over the test period of 48 hours. Therefore, the endpoint values were related on the nominal concentrations of the test item. The samples from the nominal test concentrations of 6.25 to 25 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test and thus not relevant for the interpretation of the biological results.


During the first 24 hours of the test, no immobilized test organisms were determined in the control and up to and including the highest test item concentration of 100 mg/L. Only an adverse effect was noted at the highest concentration. Two daphnids showed slower swimming activity compared to the control animals. Thus, the 24-hour EC50 was clearly higher than 100 mg/L.

After 48 hours of exposure, no immobilized test organisms were determined and no adverse effects were noted in the control and up to and including the test item concentration of 50 mg/L.
At the highest concentration of 100 mg/L, 20 % of the daphnids were immobile. In addition, adverse effect was noted at this concentration. All surviving daphnids showed slower swimming activity compared to the control animals.

Therefore, the 48-hour EC50 of the test item could not be calculated, because none of the responses exceeded 50%. Thus the EC50 was determined to be >100 mg/L.
The 48-hour EC0 and NOEC of JEFFAMINE® EDR-176 were both 50 mg/L, since no toxic effect was observed up to and including this test concentration.

The EC100 could not be determined due to the low toxicity of the test item. However, concentrations above 100 mg/L were not tested, in accordance with the test guidelines.
No remarkable observations were made concerning the appearance of the test medium. The test media were clear solution throughout the entire test duration.

At the beginning and end of the test period, the dissolved oxygen concentration in the test medium and the control was at least 8.2 mg/L. The pH values were between 7.5 and 7.9. The water temperature during the test was maintained at 22 °C.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 0.19-3.0 mg/L
- Dose-response test: Yes
- ECx: 24-hour EC50: 1.2 mg/L

For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control twice a year. The result of the latest positive control test in March 2017 (24-hour EC50: 1.2 mg/L, IES Laboratories Study 20170076) showed that the sensitivity of the test organisms was within the range given by the guideline (24-hour EC50: 0.60-2.1 mg/L).

Reported statistics and error estimates:

No statistics needed.
The 24-hour and 48-hour NOEC, EC0 and EC100 were determined directly from the raw data. The
24-hour and 48-hour EC50 of the test item could not be calculated, because none of the responses
exceeded 50%. The EC50 was, therefore, also determined directly from the raw data.

Analytical results

Sampling Day/ Sample Age	Nominal Concentration of Test Item cnom	Measured Concentration  of Test Item x	Sample Preparation Factor F	Determined Concentration of Test Item c	% of Nominal Concentration
[d/h]	[mg/L]	[mg/L]		[mg/L]	[%]
0/0	Control	n.d.	1	< LOQ	n.a.
(fresh)	50	9.85	5	49.2	98
	100	9.61	10	96.1	96
2/48	Control	n.d.	1	< LOQ	n.a.
(aged)	50	9.59	5	47.9	96
	100	9.64	10	96.4	96

n.d.        = no test item detected

n.a.        = not applicable

LOQ:     3.31 mg/L

Biological Results: Effect of JEFFAMINE® EDR-176 on the Mobility of Daphnia magna

 

Nominal Test Item Concentration	No. of Daphnids Tested	Immobilized Daphnids after 24 Hours	Immobilized Daphnids after 24 Hours	Immobilized Daphnids after 48 Hours	Immobilized Daphnids after 48 Hours
[mg/L]		No.	[%]	No.	[%]
Control	20	0	0	0	0
6.25	20	0	0	0	0
12.5	20	0	0	0	0
25	20	0	0	0	0
50	20	0	0	0	0
100	20	0(2A)	0	4(16A)	20

A: number of mobile test animals with slower swimming activity compared to the control animals

 

 

Validity criteria fulfilled:

yes

Conclusions:

In a valid, reliable and conclusive study according to OECD 202 (2004), the effect of the test substance on the mobility of young daphnids of the species Daphnia magna after 48 hours was determined. The 48-hour EC50 of the test item could not be calculated, because none of the responses exceeded 50%. Thus the EC50 was determined to be >100 mg/L.
The 48-hour EC0 and NOEC of the test substance were both 50 mg/L, since no toxic effect was observed up to and including this test concentration.

Executive summary:

The acute toxicity of the test item JEFFAMINE^®EDR-176 on Daphnia magna was determined in a 48‑hour static test according to the OECD Guideline for Testing of Chemicals, No. 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2.

The nominal test item concentrations tested were 6.25, 12.5, 25, 50 and 100 mg/L. Additionally, a control (test water without test item) was tested in parallel.

The measured concentrations of JEFFAMINE^®EDR-176 in the test media of the test concentrations of 50 and 100 mg/L were 98 and 96 % of the nominal values, respectively at the start of the test and 96 % for both concentrations at the end of the test.

Thus, the correct dosage of the test item JEFFAMINE^®EDR-176 was confirmed. The test item was stable in the test media over the test period of 48 hours. Therefore, the endpoint values were related on the nominal concentrations of the test item. The samples from the nominal test concentrations of 6.25 to 25 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test and thus not relevant for the interpretation of the biological results.

The biological test results (based on nominal concentration) were as follows:

24-hour EC₅₀ > 100 mg/L

24-hour EC₀ ≥ 100 mg/L

24-hour EC₁₀₀ > 100 mg/L

48-hour EC₅₀ > 100 mg/L

48-hour EC₀ and  48-hour NOEC: 50 mg/L

48-hour EC₁₀₀ > 100 mg/L

 

Description of key information

The acute toxicity of the test item on Daphnia magna was determined in a 48‑hour static test according to the OECD Guideline for Testing of Chemicals, No. 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2.

The biological test results (based on nominal concentration) were as follows:

24-hour EC₅₀> 100 mg/L

24-hour EC₀≥100 mg/L

24-hour EC₁₀₀> 100 mg/L

48-hour EC₅₀> 100 mg/L

48-hour EC₀and  48-hourNOEC: 50 mg/L

48-hour EC₁₀₀> 100 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

The test item is not toxic for invertebrate.