Bis[(3,4-epoxycyclohexyl)methyl] adipate

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:

04 December 2017 to 05 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)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Samples were taken from the control and each test group from the bulk test preparation at 0 and 24 hours and from the pooled replicates at 24 and 48 hours for quantitative analysis.
- All samples were stored frozen prior to analysis.
- Duplicate samples were taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary.
- Only samples at the NOEC were provided for analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Preliminary solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. A test concentration of 34 mg/L was obtained using a preliminary solution in reverse osmosis water.
- Based on this information the test material was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document, therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.
- As a result of the trial the test material was prepared using a saturated solution method of preparation at an initial loading rate of 50 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test material by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded) to give a nominal test concentration of approximately 34 mg/L.
- A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 10, 18, 32 and 56% v/v saturated solution. The saturated solution preparation for media renewal at 24 hours was stirred for 25 hours due to a technical oversight. However, this was deemed not to have had an impact on the outcome of the study as dissolved concentrations were not affected and were similar to those in the preparations at 0 hours.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: In house
- Age: First instar; gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
- Feeding during test: No

ACCLIMATION
- Acclimation conditions: Adult daphnids were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a cycle of 16 hours of light and 8 hours of darkness with 20 minute dawn and dusk transition periods. Culture conditions ensured that reproduction was by parthenogenesis.
- Type and amount of food: Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension.
- Feeding frequency: Daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

21 - 23 °C

pH:

7.9 - 8.1

Dissolved oxygen:

7.8 - 8.9 mg O2/L

Nominal and measured concentrations:

Nominal: 10, 18, 32, 56 and 100 % v/v saturated solutions
Measured (100 % saturated solution): 35 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass beakers
- Type: The test vessels were covered to reduce evaporation
- Material, size, headspace, fill volume: 100 mL of test preparation
- Aeration: no
- Renewal of test solution: Semi static test conditions were employed in the test in an effort to maintain dissolved test material concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24 Hour old test media into the fresh test media.
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water,Elendt M7, contained the following substances in mg/L: H3BO3 0.715, MnCl2.4H2O 0.090, LiCl 0.077, RbCl 0.018, SrCl2.6H2O 0.038, NaBr 0.004, Na2MoO4.2H2O 0.016, CuCl2.2H2O 0.004, ZnCl2 0.013, CoCl2.6H2O 0.010, KI 0.0033, Na2SeO3 0.0022, NH4VO3 0.00058, Na2EDTA.2H2O 0.625, FeSO4.7H2O 0.249, CaCl2.2H2O 293.8, NaHCO3 64.8, MgSO4.7H2O 123.3, Na2SiO3.9H2O 10, KCl 5.8, NaNO3 0.274, K2HPO4 0.184, KH2PO4 0.143, Thiamine hydrochloride 0.075, Cyanocobalamine (vitamin B12) 0.0010 and D(+) biotin (vitamin H) 0.00075.
- Water quality assessments: The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal and on termination of the test after 48 hours represent those of the used or 24 hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The light intensity during the light period was measured using an ATP Instrumentation Lux meter. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8 hours of darkness with 20 minute dawn and dusk transition periods
- Light intensity: 200 to 1200 Lux

EFFECT PARAMETERS MEASURED:
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilised if they were unable to swim within 15 seconds after gentle agitation.

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100 % v/v saturated solution.
- Results used to determine the conditions for the definitive study: Yes. Based on the results of the range finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 % v/v saturated solution. No immobilisation was observed in the range finding test, however, as the definitive test had a semi-static design and given that there was a sub lethal effect seen in the 100 % v/v saturated solution in the range-finding test, it was decided that a full range would be suitable.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 35 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

35 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 35 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

35 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

RANGE-FINDING TEST
- No immobilisation was observed at all test concentrations.
- A sub lethal effect of exposure was observed in the 100 % v/v saturated solution. This was reduced mobility. Based on this information test concentrations of 10, 18, 32, 56 and 100% v/v saturated solution were selected for the definitive test.
- Chemical analysis of the 100 % v/v saturated solution test preparation at 0 hours showed a measured test concentration of 39 mg/L. There was a significant decline in the measured concentration at 48 hours indicating that the test material was not stable under test conditions.


DEFINITIVE TEST
Verification of Test Concentrations
- Analysis of the 100 % v/v saturated solution fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 35 to 36 mg/L. There was no significant change in the measured concentrations obtained from the old or expired test preparations at 24 and 48 hours and so the results are based on the average fresh measured test concentration only.
- Results from the analysis of the 48-Hour control sample showed a measured concentration of 0.045 mg/L. As analysis of the corresponding 24-Hour fresh preparation showed a measured concentration of less than the LOQ, this was considered to be due to post sampling contamination and as such had no adverse effect on the outcome of the test.

Immobilisation Data
- There was no significant immobilisation in 20 daphnids exposed to a test concentration of 36 mg/L for a period of 48 hours. Inspection of the immobilisation data gave the following results:
24 and 48 h EC50: > 35 mg/L
24 and 48 h NOEC: 35 mg/L

Sub-Lethal Effects
- A sub lethal effect of exposure was observed in the 18 % v/v saturated solution test concentration. This response was reduced mobility.

Validation Criteria
- The test was considered to be valid given that no more than 10 % of the control daphnids showed immobilisation or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Water Quality Criteria
- Temperature was maintained at 21 to 23 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
- Throughout the test the light intensity was observed to be in the range 774 to 984 Lux.

Test Material Solubility
- At the start and throughout the test all control and test solutions were observed to be clear colourless solutions.

Results with reference substance (positive control):

- A positive control study used potassium dichromate as the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.
Analysis of the immobilization data was carried out using the Trimmed Spearman-Karber method at 24 hours and the Binomial Distribution method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:
- 24h: EC50: 0.79 mg/L (0.73 - 0.86 mg/L), NOEC: 0.56 mg/L and LOEC: 1.0 mg/L.
- 48h: EC50: 0.75 mg/L (0.56 - 1.0 mg/L), NOEC: 0.56 mg/L and LOEC: 1.0 mg/L.
- The No Observed Effect Concentration is based upon equal to or less than 10% immobilization at this concentration.
- The results from the positive control with potassium dichromate were within the normal range for this reference material.

Reported statistics and error estimates:

An estimate of the EC50 values was given by inspection of the immobilisation data.

Table 1: Cumulative Immobilisation Data and Observations in the Definitive Test after 24 Hours

Nominal Concentration (% v/v Saturated Solution)	24 Hours
	Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
10	0	0	0	0	0	0	5 N	5 N	5 N	5 N
18	0	0	0	0	0	0	5 N	5 N	5 N	5 N
32	0	0	0	0	0	0	5 N	5 N	5 N	5 N
56	0	0	0	0	0	0	5 N	5 N	5 N	5 N
100	0	0	0	0	0	0	5 N	5 N	5 N	5 N

R = Replicate

N = Normal

Table 2: Cumulative Immobilisation Data and Observations in the Definitive Test after 48 Hours

Nominal Concentration (% v/v Saturated Solution)	48 Hours
	Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	1	0	0	0	0	5	4 N	5 N	5 N	5 N
10	0	0	0	0	0	0	5 N	5 N	5 N	5 N
18	0	0	0	0	0	0	5 N	4 N 1 R	5 N	5 N
32	0	0	1	0	1	5	5 N	5 N	4 N	5 N
56	0	0	0	0	0	0	5 N	5 N	5 N	5 N
100	0	0	0	0	0	0	5 N	5 N	5 N	5 N

R = Replicate

N = Normal

T = Trapped at surface

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study the acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and based on the average fresh measured test concentration gave a 48 hour EC50 value of greater than 35 mg/L. The NOEC was 35 mg/L. This study showed that there were no toxic effects at saturation.

Executive summary:

The acute toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions using an acute daphnia immobilisation study.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 34 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range finding test, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at nominal concentrations of 10, 18, 32, 56 and 100 % v/v saturated solution for 48 hours at a temperature of 21 to 23 °C under semi-static test conditions. The test material solutions were prepared by stirring an excess (50 mg/L) of test material in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre condition the filter) to produce a 100 % v/v saturated solution of the test material. This saturated solution was then further diluted as necessary, to provide the remaining test concentrations. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the freshly prepared 100 % v/v saturated solution test preparations at 0 and 24 hours showed measured test concentrations to range from 35 to 36 mg/L. There was no significant change in the measured concentrations obtained from the old or expired test preparations at 24 and 48 hours and so the results are based on the average fresh measured test concentration only.

Results from the analysis of the 48-Hour control sample showed a measured concentration of 0.045 mg/L. As analysis of the corresponding 24-Hour fresh preparation showed a measured concentration of less than the LOQ, this was considered to be due to post sampling contamination and as such had no adverse effect on the outcome of the test.

This study showed that there were no toxic effects at saturation.

Under the conditions of this study the acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and based on the average fresh measured test concentration gave a 48 hour EC50 value of greater than 35 mg/L. The NOEC was 35 mg/L.

Description of key information

The acute toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions using an acute daphnia immobilisation study. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 34 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range finding test, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at nominal concentrations of 10, 18, 32, 56 and 100 % v/v saturated solution for 48 hours at a temperature of 21 to 23 °C under semi-static test conditions. The test material solutions were prepared by stirring an excess (50 mg/L) of test material in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre condition the filter) to produce a 100 % v/v saturated solution of the test material. This saturated solution was then further diluted as necessary, to provide the remaining test concentrations. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the freshly prepared 100 % v/v saturated solution test preparations at 0 and 24 hours showed measured test concentrations to range from 35 to 36 mg/L. There was no significant change in the measured concentrations obtained from the old or expired test preparations at 24 and 48 hours and so the results are based on the average fresh measured test concentration only. Results from the analysis of the 48-Hour control sample showed a measured concentration of 0.045 mg/L. As analysis of the corresponding 24-Hour fresh preparation showed a measured concentration of less than the LOQ, this was considered to be due to post sampling contamination and as such had no adverse effect on the outcome of the test.

This study showed that there were no toxic effects at saturation.

Under the conditions of this study, the acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and based on the average fresh measured test concentration gave a 48 hour EC50 value of greater than 35 mg/L. The NOEC was 35 mg/L.