3-{2-[2-(3-azaniumylpropoxy)ethoxy]ethoxy}propan-1-aminium di[(2Z)-3-carboxyprop-2-enoate]

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:

30 May 2018 to 25 Jul 2018

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 (incl. QA statement)

Specific details on test material used for the study:

- Identification: Bis-Aminopropyl Diglycol Dimaleate
- Appearance: Clear to transparent yellow liquid
- Purity/Composition: Not indicated
- Test item storage: At room temperature

Analytical monitoring:

yes

Details on sampling:

Sampling for Analysis of Test Concentrations
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below:
- Frequency at t=0 h and t=48 h
- Volume 2.0 mL from the approximate centre of the test vessels
- Storage Samples were taken in silanized amber-coloured glass vials (4 mL) and stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples of 2.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

The batch of Bis-Aminopropyl Diglycol Dimaleate tested was a clear to transparent yellow liquid of which the exact purity was unknown. The test item had a water content of 74% and was completely soluble in test medium at the concentrations tested. A correction was made for the water content of the test item. A correction factor of 3.85 was used and all further concentrations reported are expressed as the active ingredient (a.i.).

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20% , presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: Daphnia, less than 24 hours old, from parental daphnids of more than two weeks old.

Breeding:
Start of each batch: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation, half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Culture medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

The following salts and vitamins were added to freshly prepared test medium (see test procedure and conditions) to reach the following concentrations:

Salts: H3BO3 0.73 mg/L
FeSO4.7H2O 0.25 mg/L
MnCl2.4H2O 0.092 mg/L
LiCl 0.078 mg/L
RbCl 0.018 mg/L
SrCl2.6H2O 0.039 mg/L
Na2MoO4.2H2O 0.016 mg/L
NaBr 0.0041 mg/L
CuCl2.2H2O 0.0043 mg/L
ZnCl2 0.013 mg/L
CoCl2.6H2O 0.010 mg/L
KI 0.0033 mg/L
Na2SeO3 0.0022 mg/L
NH4VO3 0.00059 mg/L
Na2EDTA.2H2O 0.64 mg/L
Na2SiO3.5H2O 7.6 mg/L
NaNO3 0.28 mg/L
KH2PO4 0.15 mg/L
K2HPO4 0.19 mg/L

Vitamins: Thiamine hydrochloride 75.0 µg/L
B12 1.0 µg/L
Biotin 0.75 µg/L

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

Hardness of test medium expressed as CaCO3: 180 mg/L

Test temperature:

18-22 °C, constant within ±1°C

pH:

6-9

Dissolved oxygen:

≥3 mg/L at the end of the test

Nominal and measured concentrations:

Bis-Aminopropyl Diglycol Dimaleate Nominal conc. (mg a.i./L): Control (0.0), 2.2, 4.6, 10, 22, 46, 100

Details on test conditions:

The project started with a combined limit/range-finding test. Twenty daphnids per concentration (four replicates, 5 daphnids per vessel) were exposed to a control and a concentration of 100 mg a.i./L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to 0.10, 1.0 and 10 mg a.i./L in the combined range-finding test.
• At the end of the test, dissolved oxygen concentrations were only measured in the control and the highest test concentration.

Test Procedure and Conditions:

Test duration: 48 hours
Test type: Static
Test vessels: 60 mL, all-glass, silanized.
Test medium The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):

CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

The hardness of test medium expressed as CaCO3: 180 mg/L with a pH between 6 and 9.

Number of daphnids 20 per concentration
Loading 5 per vessel containing 50 mL of test solution.
Light A daily photoperiod of 16 hours.
Feeding No feeding
Aeration No aeration of the test solutions was applied.
Introduction of daphnids 27 minutes after preparation of the test solutions.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 80 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 80 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

Immobility
At the end of the test, biologically relevant immobility of 15% was observed at the highest concentration tested. Since the four highest actual exposure concentrations of the anionic constituent remained stable throughout the test, the effect parameters were expressed in terms of initially measured
concentrations. The responses recorded in this test allowed for reliable determination of an EC50.

Results with reference substance (positive control):

The 48h-EC50 was 0.59 mg/L with a 95% confidence interval between 0.53 and 0.72 mg/L.

Reported statistics and error estimates:

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Combined Limit / Range-Finding Test

At the end of the test, 5% immobility was observed in the control. Biologically relevant immobility and entrapment of the daphnids at the surface (i.e. >10%) was observed at all test concentrations throughout the test duration. However, the results did not show a clear dose‑response effect. Consequently, obtained results were considered unreliable. It should be noted that 10% immobility is allowed in the control and thus effects below 10% are considered to be not biologically relevant.

Based on the biological results, samples taken from nominally 0.10 and 100 mg a.i./L were analysed. In each sample, two test item related peaks were recorded that were identified as a cationic and an anionic constituent.

Actual exposure concentrations based on the separate responses of both the cationic and anionic constituent were at the level of nominal throughout the test (i.e. 97 – 111% relative to nominal.

All test conditions were maintained within the limits prescribed by the study plan.Since the range of the toxicity of the test item is unknown based on these results, a broad range of 6 test item concentrations were tested in the final test.

Table 1          
Number of Introduced Daphnids and Incidence of Immobility in the Combined Limit / Range-Finding Test

Time (h)	Replicate	Bis-Aminopropyl Diglycol Dimaleate Nominal conc. (mg a.i./L)
		Control	0.10	1.0	10	100
0	A	5	5	5	5	5
	B	5	5	5	5	5
	C	5				5
	D	5				5
	Total introduced	20	10	10	10	20
24	A	1	1 (4)	2 (2)	0 (3)	4 (2)
	B	0	2 (3)	1 (4)	0 (3)	4 (4)
	C	0				5 (5)
	D	0				5 (3)
	Total immobilised	1	3	3	0	18
	Effect %	5	30	30	0	90
48	A	1	2	2	1	5
	B	0	2	1	2	5
	C	0				4
	D	0				5
	Total immobilised	1	4	3	3	19
	Effect %	5	40	30	30	95

( ) between brackets: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

Final Test:

Measured Concentrations

Samples taken from all test concentrations and the control were analysed. In each sample, two test item related peaks were recorded that were identified as a cationic and an anionic constituent. Similar to the combined limit/range-finding test, actual test item concentrations were expressed using either the measured cationic or anionic responses.

The actual exposure concentrations based on the responses of the cationic constituent were at the level of nominal throughout the test (i.e. were at 96 – 108% relative to nominal).

At the start of the test, the actual exposure concentrations based on the responses of the anionic constituent were close to the level of nominal (i.e. 72 – 84% relative to nominal). Contrary to the cationic constituent, the actual test item concentrations based on the anionic constituent remained stable for the three highest test groups, while a significant concentration dependent decrease was observed towards the lower test groups.

Similar to the combined limit/range-finding test, the pH of the test solutions decreased with increasing test item concentrations (see Table 4). Hence, it is most likely that the test item affected the pH of the test medium. In addition, stability of the anionic constituent increased with increasing test item concentrations (see appended Analytical Report) and seemed to correlate to the pH. However, this contradicts the results of the combined limit/range-finding test in which the lowest and highest test concentrations were at the level of nominal. Therefore, existence of this correlation cannot be proven and the exact reason for the difference in stability of the anion is unknown.

It should be noted that a small response of both constituents was observed in the control which was, however, negligible compared to the responses recorded at any test concentration.

It is unknown which constituent caused toxicity towards daphnia. Therefore, effect parameters were based on the actual exposure concentrations determined from measurements of the anionic constituent as the worst-case scenario.

     

Immobility

Since the four highest actual exposure concentrations of the anionic constituent remained stable throughout the test, the effect parameters were expressed in terms of initially measured concentrations.

The responses recorded in this test allowed for reliable determination of an EC₅₀.

Table 2          
Number of Introduced Daphnids and Incidence of Immobility in the Final Test

 

Time (h)	Replicate	Bis-Aminopropyl Diglycol Dimaleate Nominal conc.(mg a.i./L)
		Control	2.2	4.6	10	22	46	100
0	A	5	5	5	5	5	5	5
	B	5	5	5	5	5	5	5
	C	5	5	5	5	5	5	5
	D	5	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20	20
24	A	0	0 (1)	0	0	1	0	1
	B	0	0 (1)	0	0	0	0	2 (1)
	C	0	0 (1)	0	0	0	0	2
	D	0	0	0	0	0	0	2
	Total immobilised	0	0	0	0	0	0	7
	Effect %	0	0	0	0	0	0	35
48	A	0	0 (1)	0	0 (1)	0	0	1
	B	0 (1)	0	0 (2)	0	0	0	0 (1)
	C	0	0	0	0	0 (1)	0	1 (1)
	D	0	0	0	0	0	0	1 (1)
	Total immobilised	0	0	0	0	0	0	3
	Effect %	0	0	0	0	0	0	15

( ) between brackets: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

Determination of Effect Concentrations

Table 3

 
Effect Parameters

Parameter	Bis-Aminopropyl Diglycol Dimaleate Measured conc. (mg a.i./L)
24, 48h-EC50	>80

Experimental Conditions

These test conditions remained within the limits prescribed by the study plan (pH: 6‑9, not varying by more than 1.5 units; oxygen:³3 mg/L at the end of the test).

The temperature continuously measured in a temperature control vessel varied between 19 and 20°C during the test, and complied with the requirements as laid down in the study plan (18‑22°C, constant within ±1°C).

 

Table 4          
pH and Oxygen Concentrations (mg/L) During the Final Test

Bis-Aminopropyl Diglycol Dimaleate Nominal conc.(mg a.i./L)	Start (t=0 h)		End (t=48 h)
	O2	pH	O2	pH
Control	8.6	8.2	8.5	8.1
2.2	8.7	8.1	8.2	8.0
4.6	8.7	8.0	8.3	8.0
10	8.7	7.8	8.5	7.9
22	8.8	7.3	8.5	7.7
46	8.7	6.7	8.3	7.3
100	8.8	6.1	8.5	6.3

 

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 48h-EC50 for Daphnia magna exposed to Bis-Aminopropyl Diglycol Dimaleate was beyond the range of concentrations tested, i.e. exceeded a measured concentration of 80 mg a.i./L based on the measured anionic constituent as a worst-case approach.

Executive summary:

The objective of the study was to evaluate Bis-Aminopropyl Diglycol Dimaleate for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC50 at 24 and 48 hours of exposure. The study procedures described in this report were based on the OECD guideline No. 202, and performed under GLP conditions.

 

The batch of Bis-Aminopropyl Diglycol Dimaleate tested was a clear to transparent yellow liquid of which the exact purity was unknown. The test item had a water content of 74% and was completely soluble in test medium at the concentrations tested. A correction was made for the water content of the test item. A correction factor of 3.85 was used and all further concentrations reported are expressed as the active ingredient (a.i.).

 

A final test was performed based on the results of a preceding combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to nominally 2.2, 4.6, 10, 22, 46 and 100 mg a.i./L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

 

The actual exposure concentrations based on the responses of the cationic constituent were at the level of nominal throughout the test (i.e. were at 96 – 108% relative to nominal).

 

At the start of the test, the actual exposure concentrations based on the responses of the anionic constituent were close to the level of nominal (i.e. 72 – 84% relative to nominal). Contrary to the cationic constituent, the actual test item concentrations based on the anionic constituent remained stable for the three highest test groups, while a significant concentration dependent decrease was observed towards the lower test groups.

 

At the end of the test, biologically relevant immobility of 15% was observed at the highest concentration tested.

 

The study met the acceptability criteria prescribed by the study plan and was considered valid.

 

In conclusion, the 48h-EC50 for Daphnia magna exposed to Bis-Aminopropyl Diglycol Dimaleate was beyond the range of concentrations tested, i.e. exceeded a measured concentration of 80 mg a.i./L based on the measured anionic constituent as a worst-case approach.

Description of key information

In conclusion, the 48h-EC50 for Daphnia magna exposed to Bis-Aminopropyl Diglycol Dimaleate was beyond the range of concentrations tested, i.e. exceeded a measured concentration of 80 mg a.i./L based on the measured anionic constituent as a worst-case approach.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

80 mg/L