Dioctyl maleate, branched

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish embryo acute toxicity (FET)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 January 2018-3 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 236 (Fish embryo acute toxicity (FET) test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Name: Bernel Ester DCM
Other names: bis(1-methylheptyl) maleate
Physical state: Clear liquid
CAS number: 85566-63-8
EC number 287-673-6
Molecular weight: 340.5 g/mol
Purity: 92.78%
Arrival date: 4 September 2017
Storage conditions: Room temperature (15 to 30°C)

Analytical monitoring:

yes

Details on sampling:

Freshly prepared media were sampled at the start of the test (0 hours) and at 72 hours, ca 20 mL samples of freshly prepared test media were taken from control and each test concentration test media preparation flasks for chemical analysis.
Corresponding old test media were sampled at 24 and 96 hours, replicate test vessels at each treatment level were pooled and sampled. Samples (ca 20 mL) of each were taken for chemical analysis.
At each sampling occasion, duplicate samples were taken. One sample for the initial analysis and one stored as “back-up”.

Vehicle:

no

Details on test solutions:

Based on the results of the range-finding test, which is not fully reported, and in agreement with the Sponsor the definitive test was conducted at nominal concentrations of 4.3, 9.5, 21, 45.5 and 100% saturated solution.
At the start of the test, an amount of test substance (100.09 mg) was added to 1000 mL of treated mains water. The preparation was slow stirred for ca 24 hours, with a vortex no deeper than ca 1 cm. Stirring was then stopped and the media was allowed to settle for 40 minutes at 0 hours, 1 hours and 5 minutes at 24 hours, 1 hour and 10 minutes at 48 hours and 1 hour and 15 minutes at 72 hours, these settle peiords deviate form the stated 1 hour settle period, however, This was not considered to have an impact on the study as the final media were observed to be colourless solutions throughout the test, 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. Final media were then syphoned from the mid-section (aqueous phase) of the vessel, serial dilutions were then performed to give the remaining test substance concentrations. A control treatment was prepared by adding treated mains water only to the control vessels. At 24, 48 and 72 hours media were prepared using the same method, 99.96 mg of test substance was weighed for use at 24 hours, 100.12 mg for use at 48 hours, and 100.05 mg for use at 72 hours.

A positive control was also included using 3,4-dichloroaniline at a single concentration of 4.0 mg/L, this was prepared by adding the following weights of substance to 500 mL of treated mains water, 2.03 mg for 0 hour preparation, 2.04 mg for 24 hour preparation and 2.06 mg for 48 hour preparation and 2.08 mg for the preparation made at 72 hours.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

test Organism
The Danio rerio (zebra fish) eggs used in this study were obtained from an in-house laboratory breeding system.
Mating and spawning was conducted in plastic mating tanks with a plastic mesh at the bottom through which eggs could fall and be collected. Males and females were separated using a plastic divider in the tank which was removed when the lights were on to initiate spawning/mating.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Total Ca: 24.1 mg/l

Test temperature:

24.3 °C to 26.4 °C

pH:

7.14 - 7.86

Dissolved oxygen:

The dissolved oxygen concentration was maintained above 80% of the air saturation value (ASV).

Salinity:

not applicable

Nominal and measured concentrations:

% saturated solution: 4.3, 9.5, 21, 45.5 100 (nominal)

Details on test conditions:

Dilution Water
The dilution water used for conducting the culturing, breeding and tests was treated mains water that had been passed through activated carbon filters. The typical constituents of the water are presented in Appendix 1.

Range-finding Test
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).
The range-finding test was conducted at nominal concentrations of 1.0, 10 and 100% saturated solution under static test conditions with media preparation at 0 hours only. Control and positive control groups were also included. Ten individual eggs were exposed for the control and each test group along with four internal controls. Based on nominal concentrations, the results of the range-finding test suggested that the 96 hour LC50 value could be greater than 100% saturated solution. However, as the range-finding test was conducted static it was decided to run the definitive test with a full range of concentrations to take into account the possibility of toxicity caused by the concentrations being maintained at higher levels due to the definitive test being run semi-static.

Media Trial
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 96 hours. Old media samples were then taken in duplicate to assess stability.

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

Test Vessel Preparation
The test vessels were 24-well microtiter plates. A single plate was used for the control, each test concentration and the positive control. Twenty wells on each plate were filled with 2 mL of the appropriate control or test media. The remaining four wells in each plate were filled with treated mains water to act as internal controls. It was stated on page 2 of the protocol that at daily renewal 90% of the old solution would be removed and immediately replaced with new solution, taking care to avoid contact with eggs. The volume removed was not recorded in the data in error. However, the chemical analysis shows renewal of media through increase and decrease in test concentrations. This deviation was not considered to impact study integrity as it is clear media were renewed each day, only the volume removed cannot be confirmed, which is not considered to have any impact on the study.

Egg Addition and Observations
Following collection, approximately 600 fertilised eggs were placed in crystallising dishes containing treated mains water no later than 60 minutes post-fertilization and acclimatised to laboratory conditions. Fertilized eggs were separated from non-fertilised eggs and transferred to the corresponding well plates containing the test solutions within 1.5 hours of fertilization.
At this point the eggs were observed to be between the 2 and 8 cell stage. A single fertilised egg was added to each well of the 24-well microtiter plates.
After ca 24, 48, 72 and 96 hours, observations were performed on the eggs for coagulation of the egg, absence of somite formation, non-detachment of the tail and lack of heart beat, where appropriate. The egg was considered to be dead if a positive response to any of these lethal endpoints was observed. The total number of dead eggs was recorded.
It was stated on page 5 of the protocol that the magnification on the microscope used to verify absence of heart beat will be at least 80X. However, this was not recorded in the data in error. This minor deviation was not considered to impact the study as any assessment to heart beat would have to use sufficient magnification to see the heart beating or not beating. It is therefore considered that an appropriate magnification was used.

Water Quality and Environmental Conditions
The test was conducted in a temperature and light controlled incubator (16 hour light:8 hour dark). A light timer was set up in the incubator, the biologist running the test confirmed the timer had moved to 8 hours (dark period) each day but this cannot be confirmed as it was not recorded in the data in error. The incubator was confirmed as set up to run a 16 hour light/8 hour dark cycle. It was also confirmed between the 07 February 2018 and 23 February 2018 (on a total of 6 occasions) after running the test that the incubator was running the correct light cycle, this was therefore not considered to impact on study integrity.
At the start and end of the test and at each media renewal the pH, dissolved oxygen (% air saturation value and mg/L) and temperatures were carried out in the controls and the test concentration.
The temperature was recorded continuously using a max/min thermometer.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.156 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: No toxicity wa oberved at the solubility level of the test material

Details on results:

Range-finding Test
The results of the 96-hour range-finding test are summarised in the table below:
The below results suggested that the LC50 value would be greater than 100% saturated solution.

Media Trial Work
Media preparation work indicated that the test substance declined in concentration over the 96 hour test period. However, the batch failed as concentrations were below the lowest calibration point of the batch. Therefore, the batch was marked as data not used and the results have not been reported, this was used for information purposes only.

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) was 0.005 mg/L.
Chemical analysis of the test concentrations and control was conducted on fresh media at 0 and 72 hours, analysis was conducted on the corresponding old media at 24 and 96 hours. Analysis of the test samples at 0, 24, 72 and 96 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, the time-weighted mean was calculated for the 100% saturated solution test group only as there were no observed effects during the test making this the only critical level. This was calculated to be 0.156 mg/L.

Test Media Descriptions
The test preparations were observed to be colourless solutions throughout the duration of the test.

Water Quality
The water quality determinations taken during the definitive test are presented in Table 2. All values were considered to be within acceptable limits except the temperatures detailed below.
At 0 hours the following temperatures were recorded. In the 9.5% saturated solution test group a temperature of 24.3°C was recorded, in the 21% saturated solution test group a temperature of 24.3°C was recorded. These temperatures deviated from the study requirement of 26 ± 1°C by 0.7°C. These temperature deviations were not considered to impact the study integrity as all other validity criteria were met and the small deviations of 0.7°C were not considered to have a detrimental effect to the embryos. Other minor fluctuations in temperature were recorded, however, these rounded up to 25°C and were therefore not considered to be deviations.


Toxicity to Danio rerio embryos
Embryo mortality recorded during the definitive test is presented in Table 3 and hatching is presented in Table 5. The same parameters for the internal controls are presented in Table 4 and Table 6.
A summary of the percentage survival after 96-hours during the definitive test is presented below.
Based on time-weighted mean measured concentrations, the 96-hour LC50 value was estimated to be >0.156 mg/L.

Validity Criteria
The following validity criteria were all achieved and therefore the test is considered valid:
• The fertility rate was 92%, which was greater than the validity criterion of ≥70%.
• The hatching success in the control was 100% at the end of the test, which was greater than the validity criteria of ≥80%.
• The overall survival in the control was 95%, which was equal to the validity criterion of >90%.
• For the reference substance, 3,4-dichloroaniline, the observed mortality was 100% which was in excess of the minimum 30% validity criteria.
• The dissolved oxygen concentration was maintained above 80% of the air saturation value (ASV).
• The water temperature was maintained at 26 ± 1°C during the test. The temperature ranged from 25.3 to 26.4°C, excluding the deviation documented on page 19, which were not considered to impact on study integrity.

Reported statistics and error estimates:

Statistical Analysis
Statistical analysis was performed using the CETIS program v 1.8.6.1.
The LC50 are defined as the concentrations that result in a 50% mean toxicity, relative to the control.
Linear interpolation (ICPIN) analysis was performed in order to estimate LC50 values. Where possible, 95% confidence limits were also calculated.

Sublethal observations / clinical signs:

Results of range finding test.

Nominal concentration (% saturated solution)	96-hour survival (%)
Control	90
Positive Control	0
1	100
10	70
100	80

Chemical analysis of test samples.

Nominal Concentration (% saturated solution)		Measured Concentration (mg/L)
		0 hours (New)	24 hours (Old)	72 hours (New)	96 hours (Old)
Control		-	-	-	-
4.3		0.0061	<LOQ	0.0229	<LOQ
9.5		0.0124	<LOQ	0.0303	<LOQ
21*		0.0276	<LOQ	0.074	0.0184
45.5		0.0818	0.0066	0.175	0.03
100		0.183	0.0298	0.328	0.149
-	None found above LOQ (0.005 mg/L)
*	Time weighted mean not calculated for 21% saturated solution test group as it was excluded from the results due to the mortality observed within the internal control wells, based on guidance from the study guideline, no impact to study as no other toxicity was observed in the other internal controls that would require the full plate to be excluded.

% survival of embryos.

Nominal concentration (% saturated solution)	96-hour survival (%)
Control	95
Positive Control	0
4.3	85
9.5	95
21*	55
45.5	85
100	75
*21% sat sol test group excluded from results due to mortality observed in the internal controls.

LC50 values.

Parameter	Toxicity value (mg/L)
	24 hours	48 hours	72 hours	96 hours
LC50*	>0.156	>0.156	>0.156	>0.156
*95% confidence limits were not calculated
Results calculated using Linear Interpolation (ICPIN)

Validity criteria fulfilled:

yes

Conclusions:

The effects of the test substance on Danio rerio embryos by comparison of lethal endpoints with the controls were determined over a 96-hour period. The study was conducted in accordance with the known requirements of OECD Chemicals Testing Guideline No. 236: Fish Embryo Acute Toxicity (FET) Test (adopted 26 July 2013).
Based on time-weighted mean measured concentrations, the 96-hour LC50 value was calculated to be >0.156 mg/L.

No toxicity was observed at the solubility level of the compound in test media.
All validity criteria were met during the test.

Executive summary:

The effects of the test substance on Danio rerio embryos by comparison of lethal endpoints with the controls were determined over a 96-hour period. The study was conducted in accordance with the known requirements of OECD Chemicals Testing Guideline No. 236: Fish Embryo Acute Toxicity (FET) Test (adopted 26 July 2013).

Based on time-weighted mean measured concentrations, the 96-hourLC₅₀ value was calculated to be >0.156 mg/L.

 

No toxicity was observed at the solubility level of the compound in test media. All validity criteria were met during the test.

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 fish

Fresh water fish

Effect concentration:

0.156 mg/L