Bis(2-ethylhexyl) malate

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:

9 Feb 2018 to 18 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

23 November 2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Council Regulation 440/2008, 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) malate

EC Number:

260-070-5

EC Name:

Bis(2-ethylhexyl) malate

Cas Number:

56235-92-8

Molecular formula:

C20H38O5

IUPAC Name:

1,4-bis(2-ethylhexyl) 2-hydroxybutanedioate

Test material form:

liquid

Details on test material:

Identification: Dermol DOM
Appearance: Clear colourless to pale yellow liquid
Batch: P7677
Purity/Composition: >95%
Test item storage: At room temperature
Stable under storage conditions until: 31 December 2018 (retest date)
Additional information
Test Facility test item number: 209081/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Molecular structure: Not indicated
Molecular formula: C20H38O5
Molecular weight: 358.5

Specific details on test material used for the study:

Identification: Dermol DOM
Appearance: Clear colourless to pale yellow liquid
Batch: P7677
Purity/Composition: >95%
Test item storage: At room temperature
Stable under storage conditions until: 31 December 2018 (retest date)

Additional information
Test Facility test item number: 209081/A
Purity/Composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: Diethylhexyl Malate
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Solubility in water: Insoluble
Stability in water: Stable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

During the limit test samples for possible analysis were taken from the limit concentration and the control according to the schedule below. In addition, the glass wool containing the undissolved residue was kept for possible analysis. The method of analysis is described in the appended Analytical Report (Appendix 2).
Frequency 1. At the start of the test and after 24 hours from the freshly prepared solutions.
2. At the first renewal (t=24h) and the end of the test from the 24-hour old solutions.
Volume 2.0 mL from the approximate centre of the test vessels
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of each refreshment 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.
4.9. Measurements and Recordings
Immobility (including mortality) At 24 hours and at 48 hours.
pH and dissolved oxygen At the beginning, after 24 hours of exposure (in the old and freshly prepared test solutions) and at the end of the test, for all concentrations and the control.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of Dermol DOM tested was a clear colourless to pale yellow liquid with a purity >95% which was not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying a three day period (first combined limit/range-finding test) or two-day period (second combined limit/range-finding test and limit test) of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of one hour (first combined limit/range-finding test) or overnight (second combined limit/range-finding test and limit test). Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning through glass wool and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
Any residual volumes were discarded.

Test organisms

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 For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.
4.7. Breeding
Start of each batch With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them 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.
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).

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

unknown

Test temperature:

Control 19.2 °C (0 hours) to 18.6 °C (48 hours)
100 19.4 °C (0 hours) to 18.6 °C (48 hours)

pH:

7.9-9.0

Dissolved oxygen:

9.0

Nominal and measured concentrations:

Dermol DOM A SS prepared at a loading rate of 100 mg/L, the regulatory limit concentration.
Controls Test medium without test item or other additives.

Details on test conditions:

- Water characteristics:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Acidity / Alkalinity:
- Culture medium for organisms different from test medium:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobilisation

VEHICLE CONTROL PERFORMED: yes/no

Reference substance (positive control):

yes

Remarks:

[State positive control substance]

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Table 1 shows the responses recorded during the first combined limit/range-finding test. Biological relevant immobility of 90 and 95% was observed in the undiluted SS after 24 and 48 hours of exposure, respectively.
Based on these results, samples taken from the undiluted SS were analysed. The actual exposure concentration was 22 mg/L at the start of the test, and decreased to 10 mg/L at the end of the test (see also Table 2 of the appended Analytical Report). The measured concentrations exceeded the water solubility of the test item (i.e. 0.22 mg/L; Test Facility Study No. 20136808) during the entire test period. It can be consequently stated that the SS prepared at 100 mg/L was over-saturated. In facts, after 24 hours of exposure, a floating layer was observed in the undiluted SS, indicating precipitation of the test item over time. Microscopic observation revealed that the test item was attached onto immobilised daphnids. In order to differentiate between physical and toxicological effects, a second combined limit/range-finding test was performed with a slightly different procedure for preparation of test solutions (see paragraph 4.5).
All test conditions were maintained within the limits prescribed by the study plan.
Table 1
Number of Introduced Daphnids and Incidence of Immobility in the First Combined Limit/Range-Finding Test
Time (h) Replicate Dermol DOM
%SS prep. at a loading rate of 100 mg/L
Control 1.0 10 100
0 A 5 5 5 5
B 5 5 5 5
C 5 5
D 5 5
Total introduced 20 10 10 20

24 A 0 0 0 4(4)*
B 0 0 0 5(5)*
C 0 5(5)*
D 0 4(5)*#
Total immobilised 0 0 0 18
Effect % 0 0 0 90

48 A 0 0 0 4
B 0 0 0 5
C 0 5
D 0 5
Total immobilised 0 0 0 19
Effect % 0 0 0 95
( ) - between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms
were reimmersed into the respective solutions before recording of mobility.
* - a layer of test item was observed at the surface of the test solution.
# - microscopic observation revealed that test item was attached to the daphnids.Limit/Range-Finding Test
Time (h) Replicate Dermol DOM
%SS prep. at a loading rate of 100 mg/L
Control 1.0 10 100
0 A 5 5 5 5
B 5 5 5 5
C 5 5
D 5 5
Total introduced 20 10 10 20

24 A 0 0 0 4(4)*
B 0 0 0 5(5)*
C 0 5(5)*
D 0 4(5)*#
Total immobilised 0 0 0 18
Effect % 0 0 0 90

48 A 0 0 0 4
B 0 0 0 5
C 0 5
D 0 5
Total immobilised 0 0 0 19
Effect % 0 0 0 95
( ) - between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms
were reimmersed into the respective solutions before recording of mobility.
* - a layer of test item was observed at the surface of the test solution.
# - microscopic observation revealed that test item was attached to the daphnids.
Table 2 shows the responses recorded during the second combined limit/range-finding test. No immobility was observed in the control or at any of the test concentrations throughout the test.
Based on these results, samples taken from the undiluted SS prepared at a loading rate of 100 mg/L were analysed. The measured concentration was 0.65 mg/L at the start of the test, and decreased to 1.8% of initial at the end of the test (see also Table 3 of the appended Analytical Report). Results of the analysis for determination of exposure concentrations showed a significant decrease in exposure concentrations during the test period. Consequently, it was decided to perform a limit test with renewal of the test solutions after 24 hours.
All test conditions were maintained within the limits prescribed by the study plan.

Any other information on results incl. tables

Limit/Range-Finding Test

Time (h)	Replicate	Dermol DOM %SS prep. at a loading rate of 100 mg/L
		Control	1.0	10	100
0	A	5	5	5	5
	B	5	5	5	5
	C	5			5
	D	5			5
	Total introduced	20	10	10	20
24	A	0	0	0	4(4)*
	B	0	0	0	5(5)*
	C	0			5(5)*
	D	0			4(5)*^#
	Total immobilised	0	0	0	18
	Effect %	0	0	0	90
48	A	0	0	0	4
	B	0	0	0	5
	C	0			5
	D	0			5
	Total immobilised	0	0	0	19
	Effect %	0	0	0	95

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

* - a layer of test item was observed at the surface of the test solution.

^#- microscopic observation revealed that test item was attached to the daphnids.

Table2          
Number of Introduced Daphnids and Incidence of Immobility in the Second Combined Limit/Range-Finding Test

Time (h)	Replicate	Dermol DOM %SS prep. at a loading rate of 100 mg/L
		Control	1.0	10	100
0	A	5	5	5	5
	B	5	5	5	5
	C	5			5
	D	5			5
	Total introduced	20	10	10	20
24	A	0	0	0	0
	B	0	0	0	0
	C	0			0
	D	0			0
	Total immobilised	0	0	0	0
	Effect %	0	0	0	0
48	A	0	0	0	0
	B	0	0	0	0
	C	0			0
	D	0			0
	Total immobilised	0	0	0	0
	Effect %	0	0	0	0

1.1.                       

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 48h-EC50 for Daphnia magna exposed to Dermol DOM exceeded the solubility limit in test medium with an average exposure concentration of 0.15 mg/L.

Executive summary:

In conclusion, the 48h-EC₅₀forDaphnia magna exposed to Dermol DOM exceeded the solubility limit in test medium with an average exposure concentration of 0.15 mg/L.