Dioctyl maleate, branched

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:

6 November 2017 - 12 February 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

Specific details on test material used for the study:

Test Substance Name Bernel Ester DCM
CAS Number: 85566-63-8
Purity: 92.78%
Receipt Date 04 September 2017
Storage on Receipt: Room temperature (15 – 30°)

Analytical monitoring:

yes

Details on sampling:

At the start of the test (0 hours), ca 20 mL samples of freshly prepared test media were taken from the control and 100% saturated solution for chemical analysis.
At 24 hours, ca 20 mL samples were taken for chemical analysis from both freshly prepared media and the pooled old test media from the control and 100% saturated solution test group.
At 48 hours, ca 20 mL samples were taken for chemical analysis from the pooled old test media from the control and 100% saturated solution test group.
In each case duplicate samples were taken, one for chemical analysis and one as a ‘back-up’ should further analysis be required.

Vehicle:

no

Details on test solutions:

The biological phase of the first range-finding test was conducted between 7 November 2017 and 8 November 2017 at test concentrations of 1.0, 10 and 100% saturated solution. A control group was also included. However, the test was repeated as the Daphnia were observed stuck at the media surface in test substance globules during the 24-hour observations. Therefore, it was considered inappropriate to filter the media as test substance was passing through the filters. Media were then prepared in later tests using a slow stir, settle period, and syphon method to avoid globules of test substance in the final media.

Based on the results of the first range-finding test, the second range-finding test was conducted at nominal test substance concentrations of 1.0, 10 and 100% saturated solution under static test conditions. During the first range-finding test media were prepared using a 24-hour stir and filtering method however, during range-finding test 1 test substance globules were observed to have passed through the filter. Therefore, the test was repeated using a 24-hour stir, settle and syphon preparation method. A
control group was also included. Duplicate test vessels were prepared for the control and each test group. Five juvenile daphnia (<24 hours old) were added to each test vessel.

Based on the results of the range-finding test and media preparation work, for which the key results only have been reported, the definitive limit test was conducted at a single test concentration of 100% saturated solution. The test was conducted with a semi-static test system design, media was prepared at 0 and 24 hours.

The test vessels were 100 mL glass beakers (filled to approximately 50 mL). Four replicate test vessels were prepared for the control and 100% saturated solution test group.
At the start of the test, an amount of test substance (99.95 mg) was added to 1000 mL of Elendt M4 medium. The preparation was stirred for ca 24 hours (with a vortex no deeper than ca 1 cm), media were then allowed to settle for ca 1 hour, final media were then syphoned from the mid-section (aqueous phase) of the preparation. A control treatment was prepared by adding Elendt M4 medium only to the control vessels. At 24 hours media were prepared using the same method, 99.99 mg of test substance was weighed for use at 24 hours. Daphnia were then transferred to the new media prepared at 24 hours.

The settle period for the test media preparation was supposed to be an hour, however, the settle period was only carried out for 45 minutes in error. This was not considered to have an impact on the study as the final media were observed to be colourless solutions, indicating that no particulate was syphoned up. This would suggest that the settle period was sufficiently long to avoid syphoning up test substance particulate which is the reason a settle period was used.

Test organisms (species):

Daphnia magna

Details on test organisms:

Juvenile Daphnia magna (Straus), less than 24 hours old, were taken from healthy parental laboratory cultures, held and maintained at the test facility. The culture was originally obtained from Microbiotest, Belgium.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

CaCl2.2H2O 283,800 mg/l

Test temperature:

19.2-22.0

pH:

7.58-7.90

Dissolved oxygen:

>88%

Salinity:

not applicable

Conductivity:

not reported

Nominal and measured concentrations:

Range finding test 1: 1.0, 10.0 & 100.0% saturated solution (nominal)
Range finding test 2: 1.0, 10.0 & 100.0% saturated solution (nominal)
Definitive test: 100.0% saturated solution (nominal)
Definitive test: 0.106 mg/l (measured time weighted mean)

Details on test conditions:

Preparation of Media
The dilution water used in the study was Elendt M4 medium. Full preparation details are presented in Appendix 1.

Appearance of Stock and Test Media
The appearance, colour and behaviour of the test substance in the test media were recorded.

Test Procedures
Based on the low solubility of the compound, which was stated as “insoluble in water” on the MSDS (material safety data sheet), a saturated solution preparation was considered most suitable (OECD 23).

Media Preparation Work
After the range-finder test was conducted without chemical analysis a media trial was conducted to assess test substance solubility and stability in the test system. Duplicate media were prepared using a saturated solution preparation method. New media samples were taken in duplicate at 0 hours, media were then stored in test conditions for ca 48 hours. Old media samples were then taken in duplicate to assess stability.

Definitive Test
Based on the results of the range-finding test and media preparation work, for which the key results only have been reported, the definitive limit test was conducted at a single test concentration of 100% saturated solution. The test was conducted with a semi-static test system design, media was prepared at 0 and 24 hours.
The test vessels were 100 mL glass beakers (filled to approximately 50 mL). Four replicate test vessels were prepared for the control and 100% saturated solution test group.
At the start of the test, an amount of test substance (99.95 mg) was added to 1000 mL of Elendt M4 medium. The preparation was stirred for ca 24 hours (with a vortex no deeper than ca 1 cm), media were then allowed to settle for ca 1 hour, final media were then syphoned from the mid-section (aqueous phase) of the preparation. A control treatment was prepared by adding Elendt M4 medium only to the control vessels. At 24 hours media were prepared using the same method, 99.99 mg of test substance was weighed for use at 24 hours. Daphnia were then transferred to the new media prepared at 24 hours.

The settle period for the test media preparation was supposed to be an hour, however, the settle period was only carried out for 45 minutes in error. This was not considered to have an impact on the study as the final media were observed to be colourless solutions, indicating that no particulate was syphoned up. This would suggest that the settle period was sufficiently long to avoid syphoning up test substance particulate which is the reason a settle period was used.

Five juvenile Daphnia magna, less than 24 hours old, were added to each test and control vessel, using a wide bore glass pipette to avoid damaging the animals during transfer. The Daphnia magna were not fed during the test.
After 24 and 48 hours, the Daphnia magna in each test vessel were observed for evidence of immobility.
The observations differentiated between mobile and immobile daphnids. An individual was considered immobile if, when the contents of the test vessel were briefly agitated, it did not swim during a 15-second period of observation. In addition, Daphnia magna submerged in the body of the test media and those that were held at the surface of the test media were also recorded.

Water Quality and Environmental Conditions
The test was conducted with a 16-hour light: 8-hour dark photoperiod.
The pH, dissolved oxygen concentration (% air saturation value (ASV) and mg/L) and temperatures were determined in freshly prepared test media at the start of the test and at media renewal at 24 hours and in the old media at 24 and 48 hours. Continuous temperatures were measured using a digital (min/max) thermometer in an additional vessel maintained in the test area.
The freshly prepared media water quality measurements were performed on media sampled directly from preparation vessels. The old media water quality measurements were conducted using pooled replicate samples of old test media.

Statistical Analysis
As no significant toxicity to Daphnia was observed during the definitive test statistical analysis was not performed, results were determined empirically through observation of the data.

Calculation of Time Weighted Mean Measured Concentrations
Time-weighted mean measured concentrations were calculated as follows;

TWM = Total area / Total days

Where;
TWM = time-weighted mean
Area = area under the exponential curve for each renewal period
Area is calculated as follows;

Area = ((C0 - C1) / ln(C0) - ln(C1)) x days

Where;
C0 = measured concentration at start of renewal period
C1 = measured concentration at end of renewal period
days = number of days in renewal period

Mathematical Rounding Statement
Throughout the results, numerical data may have been rounded for presentation purposes. Therefore, manual recalculation of the data may result in slightly different values to those shown.

Reference substance (positive control):

yes

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 0.016 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

0.106 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

24 h

Dose descriptor:

LOEC

Effect conc.:

> 0.106 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.016 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

NOEC

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: loading rate

Duration:

48 h

Dose descriptor:

LOEC

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: loading rate

Details on results:

Range-finding Test 2
The results of the range-finding test are summarised below.
Chemical analysis was not conducted during the range-finding test.
The results of the range-finding test suggested that toxicity would not be observed at the highest concentration of 100% saturated solution or the limit of solubility of the test substance.

Definitive Test
Chemical Analysis
The results of the chemical analysis are presented in Table 1.
Example chromatograms of test samples, standard solution and a typical calibration line are presented in Appendix 2.
The limit of quantification (LOQ) for Bernel Ester DCM in Elendt M4 medium using this method was 0.002 mg/L.
Analysis of the test samples at 0, 24 and 48 hours are summarised in the table below.
Chemical analysis showed concentrations of Bernel Ester DCM declined over each 24 hour renewal period. Therefore, results were based on time-weighted mean measured concentrations. This was calculated to be 0.106 mg/L

Water Quality
Water quality determinations for pH, temperature and dissolved oxygen (% ASV and mg/L) during the definitive test are presented in Table 2.
During the definitive test the test vessel temperatures varied by more than 1°C from the initial vessel temperatures, 20.9°C was recorded at 0 hours, 19.5°C was then recorded at 24 hours, meaning a maximum deviation of 1.4°C, this temperature deviations of 0.4°C over the acceptable variation was not considered to impact the integrity of the study as all established validity criteria were met and there was no significant toxicity observed during the study.

Test Media Descriptions
The test preparations were observed to be colourless solutions throughout the duration of the test.
Toxicity to Daphnia magna
The numbers of immobilised Daphnia magna at 24 and 48 hours, following exposure are presented in Table 3 and Table 4. The percentage immobility data is summarised below.
No significant toxicity (>10%) was observed during the test. Due to the decline in test substance concentration observed over each renewal period results were based on time weighted mean measured concentration. This was calculated to be 0.106 mg/L.
The EC50 values, the corresponding NOEC and LOEC values are presented in the table below.
Based on time-weighted mean measured concentrations, the 48-hour EC50 was estimated to be >0.106 mg/L.
The corresponding NOEC value was estimated to be 0.106 mg/L.

Range finding test 2 results

Nominal concentration (% saturated solution)		Cumulative immobilised Daphnia magna*
		24 hours		48 hours		% Immobility
		R1	R2	R1	R2
Control		0	0	0	0	0
1		0	0	0	0	0
10		0	0	0	0	0
100		0	0	0	0	0
*	Initial population of Daphnia magna: five per replicate
R	Replicate

Definitive test - Chemical analysis

Nominal Concentration (% saturated solution)	Measured Concentration (mg/L)
	0 hours (New)	24 hours (Old)	24 hours (New)	48 hours (Old)
Control	-	-	-	-
100	0.103	0.0343	0.205	0.106
-	None found above LOQ (0.002 mg/L)

Definitive test - Toxicity to Daphnia magna

Nominal concentration (% saturated solution)	Cumulative immobilised Daphnia magna (initial population: 5 Daphnia per replicate)
	24 hours					48 hours*
	R1	R2	R3	R4	% Immobility	R1	R2	R3	R4	% Immobility
Control	0	1	0	0	5	0	0	0	0	5
100	1	0	1	0	10	0	0	0	0	10
R	Replicate
*	All daphnia in replicate 2, 3 and 4 of the 100% saturated solution test group were observed to be at the surface of the test media at 48 hours.

Definitive test - Dose descriptor values

	Time weighted mean measured concentration (mg/L)		Statistical Test
	24-hour	48-hour
EC50	>0.106 mg/L	>0.106 mg/L	Derived Empirically
NOEC	0.106 mg/L	0.106 mg/L
LOEC	>0.106 mg/L	>0.106 mg/L

Validity criteria fulfilled:

yes

Remarks:

The validity criteria for control immobility (≤10%) and dissolved oxygen (≥3 mg/L) were both satisfied. The test is therefore considered valid.

Conclusions:

The 48-hour acute toxicity of Bernel Ester DCM to the freshwater planktonic crustacean, Daphnia magna, was determined in accordance with the requirements of OECD Chemicals Testing Guideline No. 202 Daphnia sp. Acute Immobilisation Test (adopted April 13 2004).
The validity criteria for control immobility (≤10%) and dissolved oxygen (≥3 mg/L) were both satisfied. The test is therefore considered valid.
Based on time-weighted mean measured concentration, the 48-hour EC50 value was determined to be >0.106 mg/L.
The corresponding No Observed Effect Concentration (NOEC) was considered to be 0.106 mg/L.

Executive summary:

The objective of the study was to determine the 48-hour acute toxicity of Bernel Ester DCM against the mobility of the freshwater planktonic crustacean, Daphnia magna.

The study was conducted in accordance with the requirements of OECD Chemicals Testing Guideline No. 202 Daphnia sp. Acute Immobilisation Test (adopted April 13 2004).

Based on the results of two range-finding tests, for which the key results only have been reported, an initial definitive test was conducted at a single concertation of 100% saturated solution. A control group was also included. The test was conducted under semi-static test conditions with media preparation at 0 and 24 hours. However, the test was repeated as the 0 hour chemistry result was above the validated range for the method.

Based on the results of two range-finding tests and an initial definitive test, for which the key results only have been reported, a definitive limit test was conducted at a single concertation of 100% saturated solution. A control group was also included. The test was conducted under semi-static test conditions with media preparation at 0 and 24 hours.

At the start of the test, five juvenileDaphnia magnawere added to each test vessel. TheDaphnia magnain each test vessel were observed at 24 and 48 hours. TheDaphnia magnawere not fed during the test.

Chemical analysis of the 100% saturated solution test concentration and control was conducted on fresh media at 0 and 24 hours, analysis was conducted on the corresponding old media at 24 and 48 hours.

Analysis of the test samples at 0, 24 and 48 hours are summarised in the table below.

Nominal Concentration (% saturated solution)		Time Weighted Mean Measured Concentration (mg/L)
		0 hours (New)	24 hours (Old)	24 hours (New)	48 hours (Old)
Control		-	-	-	-
100		0.103	0.0343	0.205	0.106
-	None found above LOQ (0.002 mg/L)

Chemical analysis showed concentrations of Bernel Ester DCM declined in concentration over the 24-hour renewal period. Therefore, results were based on time weighted mean measured concentrations. This was calculated to be 0.106 mg/L.

The test results, including NOEC, LOEC and EC₅₀values are summarised in the table on the following page.

The[1]EC₅₀values, the corresponding[2]NOEC and[3]LOEC values are presented in the following table.

	Time weighted mean measured concentration (mg/L)		Statistical Test
	24-hour	48-hour
EC50	>0.106 mg/L	>0.106 mg/L	Derived Empirically
NOEC	0.106 mg/L	0.106 mg/L
LOEC	>0.106 mg/L	>0.106 mg/L

Based on time weighted mean measured concentrations, the 48-hourEC₅₀was estimated to be >0.106 mg/L.

The corresponding NOEC values were estimated to be 0.106 mg/L.

 

The validity criteria for control immobility (≤10%) and dissolved oxygen (≥3 mg/L) were both satisfied. The test is therefore considered valid.

[1]Concentration resulting in 50% immobility of exposedDaphnia magna

[2]No observed effect concentration

[3]Lowest observed effect concentration

Description of key information

The key study was conducted according to internationally recognised testing guidelines and with GLP certification

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.106 mg/L