[No public or meaningful name is available]

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:

Experimental start date: 18 July 2017 Experimental completion date: 28 September 2017

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: FRET 13-0460
Chemical (IUPAC) Name: bis(cyclohexylmethyl) ether
Appearance: Clear colorless liquid
Storage Conditions: Approximately 4 °C in the dark

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 and stored frozen for further analysis if necessary.
Only samples at the No Observed Effect Concentration and above were analyzed.

.

Vehicle:

no

Details on test solutions:

Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions

Range-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.43 mg/L could be obtained using a saturated solution method of preparation.
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
A nominal amount of test item (1100 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 0.10, 1.0 and 10% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
A nominal amount of test item (1100 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). A series of dilutions was made from this saturated solution to give further test concentrations of 1.0, 3.2, 10 and 32% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnids were maintained in 150 mL glass beakers containing 100 mL 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 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. 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. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

22 ºC

pH:

7.8 - 8.0

Dissolved oxygen:

8.0 - 8.7 mg O2/L

Nominal and measured concentrations:

Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to range from less than the Limit of Quantification (LOQ), determined to be 0.0091 mg/L, to 0.23 mg/L. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined in some instances, to between less than the LOQ and 0.12 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.
The geometric mean measured test concentrations were determined to be:
Nominal test concentration (% v/v saturated solution): 1.0, 3.2, 10, 32 and 100
Geometric mean measured test concentration (mg/L): -, 0.0046, 0.017, 0.056 and 0.14

Details on test conditions:

Range-finding Test
In the range-finding test 5 daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Two replicate test and control vessels were prepared. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.
The control group was maintained under identical conditions but not exposed to the test item.

Definitive Test
Exposure Conditions
In the definitive test 100 mL completely filled glass vessels were used. At the start of the test five daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were sealed as an extra precaution and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test item.
Semi-static test conditions were employed in the test in an effort to maintain dissolved test item 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.

Assessments
Test Organism Observations
Any immobilization 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 immobilized if they were unable to swim within 15 seconds after gentle agitation.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.085 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL 0.057 - 0.15 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.014 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL Not determined due to the nature of the data

Details on results:

Range-finding Test
No immobilization was observed at the test concentrations of 0.10 and 1.0% v/v saturated solution, however, immobilization was observed at 10 and 100% v/v saturated solution.
Sub-lethal effects of exposure were observed in the control and the 0.10, 10 and 100% v/v saturated solution test concentrations. These responses were trapping at the surface and reduced movement.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution were selected for the definitive test.
Chemical analysis of the 1.0, 10 and 100% v/v saturated solution test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ), determined to be 0.0091 mg/L, and 0.28 mg/L. There was a significant decline in the measured concentrations at 48 hours to between less than the LOQ and 0.010 mg/L indicating that the test item was not stable under test conditions.
Measured concentrations in the range finding test were significantly lower than those observed in the media preparation trial. This was considered to be due to the decreased solubility of the test item in the media used for testing when compared to the deionized reverse osmosis water used in the media preparation trial and was considered not to have had an impact on the outcome of the test.

Definitive Test
Immobilization Data

Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression method at 24 and 48 hours based on the geometric mean measured test concentrations gave the following results:

Time (h) EC50 (mg/L) 95% Confidence limits (mg/L)
24 0.085 0.057 – 0.15
48 0.014 Not determined due to the nature of the data

The No Observed Effect Concentration after 24 and 48 hours exposure was 0.0046 mg/L. The Lowest Observed Effect Concentration after 24 and 48 hours exposure was 0.017 mg/L.
The slopes and their standard errors of the response curves at 24 and 48 hours were 2.1 (SE = 0.094) and 9.9 (SE = 0.39) respectively.

Sub-Lethal Effects
Sub-lethal effects of exposure were observed in the 0.017, 0.056 and 0.14 mg/L test concentrations.

Results with reference substance (positive control):

Time Point EC50 95% Confidence Limits No Observed Effect Concentration (NOEC) Lowest Observed Effect Concentration (LOEC)
(Hours) (mg/L) (mg/L) (mg/L) (mg/L)
24 1.3 1.0 - 1.8 1.0 1.8
48 1.2 1.1 - 1.3 0.56 1.0

The No Observed Effect Concentration is based upon equal to or less than 10% immobilization at this concentration.

Water Quality Criteria

Temperature was maintained at 22 °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 410 to 598 Lux.

 Observations on Test Item Solubility

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Cumulative Immobilization Data and Observations in the Range-finding Test

Nominal Loading Rate (mg/L)	Observations (Initial Population: 5 Per Replicate)
	24 Hours				48 Hours
	Cumulative Immobilized Daphnia		Observations		Cumulative Immobilized Daphnia		Observations
	R1	R2	R1	R2	R1	R2	R1	R2
Control	0	0	5 N	1 T 4 N	0	0	5 N	5 N
0.10	0	0	1 T 4 N	5 N	0	0	5 N	5 N
1.0	0	0	5 N	5 N	0	0	5 N	5 N
10	2	1	3 R	4 R	2	1	3 R	4 R
100	2	3	3 R	2 R	5	3	A/I	2 R

  R₁– R₂= Replicates 1 and 2

N = No sub-lethal effects observed

T = Trapped at surface

R = Reduced movement

A/I = All daphnia immobilized

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)	Geometric Mean Measured Concentration (mg/L)	24 Hours
		Cumulative Immobilized 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
1.0	-	0	0	0	0	0	0	5 N	5 N	5 N	5 N
3.2	0.0046	0	0	0	0	0	0	5 N	5 N	5 N	5 N
10	0.017	0	0	1	1	2	10	5 R	5 R	4 R	4 R
32	0.056	1	3	2	0	6	30	4 R	2 R	3 R	5 R
100	0.14	1	5	4	4	14	70	4 R	A/I	1 R	1 R

 

Nominal Concentration (% v/v Saturated Solution)	Geometric Mean Measured Concentration (mg/L)	48 Hours
		Cumulative Immobilized 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
1.0	-	0	0	0	0	0	0	5 N	5 N	5 N	5 N
3.2	0.0046	0	0	0	0	0	0	5 N	5 N	5 N	5 N
10	0.017	4	4	4	4	16	80	1 R	1 R	1 R	1 R
32	0.056	5	5	5	5	20	100	A/I	A/I	A/I	A/I
100	0.14	5	5	5	5	20	100	A/I	A/I	A/I	A/I

R₁– R₄= Replicates 1 to 4

N = No sub-lethal effects observed

R = Reduced movement

A/I = All daphnia immobilized

Water Quality Measurements

Nominal Concentration (% v/v Saturated Solution)		0 Hours (Fresh Media)			24 Hours (Old Media)
		pH	mg O2/L	T°C	pH	mg O2/L	T°C
Control	R1	8.0	8.7	22	7.8	8.2	22
1.0	R1	8.0	8.7	22	7.9	8.2	22
3.2	R1	8.0	8.7	22	7.9	8.2	22
10	R1	8.0	8.7	22	7.9	8.2	22
32	R1	8.0	8.7	22	8.0	8.2	22
100	R1	8.0	8.7	22	8.0	8.3	22

 

Nominal Concentration (% v/v Saturated Solution)		24 Hours (Fresh Media)			48 Hours (Old Media)
		pH	mg O2/L	T°C	pH	mg O2/L	T°C
Control	R1	7.9	8.5	22	7.8	8.3	22
1.0	R1	7.9	8.5	22	7.9	8.3	22
3.2	R1	8.0	8.5	22	7.9	8.2	22
10	R1	8.0	8.5	22	7.9	8.4	22
32	R1	8.0	8.4	22	7.9	8.1	22
100	R1	8.0	8.3	22	7.9	8.3	22

 

R₁= Replicate 1

Validity criteria fulfilled:

yes

Conclusions:

The 48 h EC50 (immobilisation) value with Daphnia magna is 0.014 mg/I.

Executive summary:

A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202. Groups of twenty, 1st instar Daphnia (less than 24 hours old) were exposed for 48 hours to an aqueous solution of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution.

Exposure of Daphnia magna to the test item gave the following results based on the geometric mean measured test concentrations:

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
48	0.014	Not determined	0.0046	0.017

Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to range from less than the Limit of Quantification (LOQ), determined to be 0.0091 mg/L, to 0.23 mg/L. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined in some instances, to between less than the LOQ and 0.12 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data..

Description of key information

 A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202. Groups of twenty, 1st instar Daphnia (less than 24 hours old) were exposed for 48 hours to an aqueous solution of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution.

 

Exposure of Daphnia magna to the test item gave the following results based on the geometric mean measured test concentrations:

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
48	0.014	Not determined	0.0046	0.017

Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to range from less than the Limit of Quantification (LOQ), determined to be 0.0091 mg/L, to 0.23 mg/L. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined in some instances, to between less than the LOQ and 0.12 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data..

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.014 mg/L