4-(2-methylpropyl)benzyl propyl ether

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:

The study was conducted between 20 March 2018 and 23 May 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study conducted according to OECD TG 202 in compliance with GLP, without deviations that influence the quality of the results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

The changes were considered to have had no impact on the outcome of the study as the overall conduct of the study remains the same.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 11-0539
Physical state/Appearance: clear colorless liquid
Storage Conditions: room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. Samples were taken for immediate analysis and duplicate sets of samples were frozen.

Vehicle:

no

Details on test solutions:

Preliminary Media Preparation Trial
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.

Preparation
A nominal amount of test item (1100 mg) was dispersed, in duplicate, in 11 liters of deionized reverse osmosis water with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre treatments:
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 1 liter discarded in order to pre condition the filter)
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 2 liters discarded in order to pre condition the filter)

Results
Stirring Period and Treatment Measured Concentration (mg/L)
24 Hours Centrifuged 10000 g 3.10
24 Hours Centrifuged 40000 g 2.30
24 Hours Filtered ~ 1 liter discarded 0.129
24 Hours Filtered ~ 2 liters discarded 2.00
48 Hours Centrifuged 10000 g 2.49
48 Hours Centrifuged 40000 g 2.55
48 Hours Filtered ~ 1 liter discarded 1.76
48 Hours Filtered ~ 2 liters discarded 2.03

Discussion
It was evident from these results that there was no significant increase in the dissolved test item concentration obtained when the preparation period was extended beyond 24 hours.
Based on this information the test item 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 item by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded) to give a nominal test concentration of approximately 2.0 mg/L.
A reduction in the initial loading rate to 50 mg/L would still ensure saturation of the test media.

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

Post exposure observation period:

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.

Test temperature:

18 to 22 °C with a maximum deviation of ±1 °C

pH:

pH 7.7-8.1

Dissolved oxygen:

9.0 - 9.3 mg O2/L

Nominal and measured concentrations:

Range-finding test: of 0.10, 1.0, 10 and 100% v/v saturated solution
Initial experiment: 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution
Definitive test: 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution

Details on test conditions:

Positive control:
A positive control (Envigo study number LK67NP) used potassium dichromate as the reference item. The positive control was conducted between 23 January 2018 to 25 January 2018.

Test water:
Reconstituted Water – Elendt M7 Medium
Ingredient Final Concentration (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
D(+) biotin (vitamin H) 0.00075

The pH of the prepared media was 7.9 ±0.3 and stored at approximately 21 ºC

Experimental Design and Study Conduct
Range-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 2.0 mg/L could be obtained in reverse osmosis water 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 (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 (the 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.
In the range finding test five 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 and 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 was completely filled and sealed with a ground glass stopper. 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.
A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. Samples were taken for immediate analysis and duplicate sets of samples were frozen.

Initial Experiment
Based on the results of the range finding test the following test concentrations were assigned to the initial experiment: 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution.
This initial experiment passed the validation criteria but had to be repeated as analytical validation ran alongside the test sample analyses failed. An amended method of analysis was developed to ensure successful analysis of the definitive test preparations. This meant that the analytical method used to analyze the samples from the definitive test differed from that used in the range-finding test. The test item concentrations in the range-finding test samples were determined by high-performance liquid chromatography (HPLC) which was not validated. The test item concentrations in the definitive test samples were determined by an amended method of analysis using gas chromatography (GC). The use of two methods was considered to have had no impact on the outcome of the study as the GC method passed validation.

Definitive Test
Based on the results of the range finding test the following test concentrations were assigned to the definitive test: 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution.
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 (the 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 the required test concentrations of 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0, 24 and 48 hours.

Exposure Conditions
As in the range finding test 150 mL conical flasks completely filled and sealed with ground glass stoppers were used. At the start of the test five daphnids were placed in each test and control vessel at random, containing the test preparations. Four replicate test and control vessels were prepared. The test vessels were then 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.

Water Quality Criteria
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.

Verification of Test Concentrations
Samples were taken from the control and each test group from the bulk fresh test preparation at 0 and 24 hours and from the old pooled replicates at 24 and 48 hours for quantitative analysis. All samples were provided to the Department of Analytical Services (Envigo – Shardlow) for quantitative analysis at the time of sampling. Duplicate sets of samples were taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.92 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL 0.60 – 63 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL 0.19 – 0.24 mg/L

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.
No sub-lethal effects of exposure were observed throughout the test.
Based on this information test concentrations of 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution were selected for the definitive test.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) determined to be 0.0043 mg/L and 4.7 mg/L. There was a significant decline in the measured concentrations at 48 hours indicating that the test item was not stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the freshly prepared test media at 0 and 24 hours showed measured test concentrations to range from 0.032 to 0.91 mg/L. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to range from 0.028 and 0.72 mg/L (between 69 and 99% of their starting concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data.

Immobilization Data
Analysis of the immobilization data by Probit analysis using Linear Maximum Likelihood regression at 24 and 48 hours based on the geometric mean measured test concentrations gave the following results:
Time (hour) EC50 (mg/L) 95% Confidence limits(mg/L)
24 0.92 0.60 – 63
48 0.21 0.19 – 0.24
The NOEC after 24 and 48 hours exposure were 0.55 and 0.14 mg/L respectively. The LOEC after 48 hours exposure was 0.17 mg/L.
The slopes and their standard errors of the response curves at 24 and 48 hours were 3.6 (standard error = 0.16) and 9.3 (standard error = 0.025) respectively.

Sub-Lethal Effects
Sub lethal effects of exposure were observed in test concentrations of 0.14, 0.17, 0.29 and 0.55 mg/L. These responses were pale and reduced mobility.

Validation Criteria
The test was considered to be valid given that no more than 10% of the control daphnids showed immobilization 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 20 °C to 21 °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 712 to 759 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.

Results with reference substance (positive control):

A positive control (Envigo study number LK67NP) used potassium dichromate as the reference item 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 Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber 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:
Time Point (Hours) EC50 (mg/L) 95% Confidence Limits (mg/L) NOEC (mg/L) LOEC(mg/L)
24 0.79 0.73 - 0.86 0.56 1.0
48 0.75 0.56 - 1.0 0.56 1.0

The NOEC 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 item*.

NOEC = No observed effect concentration
LOEC = Lowest observed effect concentration
*The results of the inter laboratory tests and a Technical Corrigendum to ISO 6341 give a 24 Hour EC50 for potassium dichromate within the range 0.6 mg/L to 2.1 mg/L

Verification of Test Concentrations

The geometric mean measured test concentrations were determined to be:

Nominal Test Concentration (% v/v Saturated Solution)	Geometric Mean Measured Test Concentration (mg/L)
1.0	0.051
1.8	0.14
3.2	0.17
5.6	0.29
10	0.55

Cumulative Immobilization Data and Observations in the Range‑finding Test

Nominal Concentration (mg/L)	Observations (Initial Population: 5 Per Replicate)
	24 Hours				48 Hours
	Cumulative Immobilized Daphnia		Observations		Cumulative Immobilized Daphnia		Observations
	Rep1	Rep2	Rep1	Rep2	Rep1	Rep2	Rep1	Rep2
Control	0	0	5N	5 N	0	0	5 N	5 N
0.10	0	0	5 N	5 N	0	0	5 N	5 N
1.0	0	0	5 N	5 N	0	0	5 N	5 N
10	1	1	4 N	4 N	5	5	AI	AI
100	5	5	AI	AI	5	5	AI	AI

Rep  = Replicate

N      = Normal

AI     = All Daphnia immobilized

Cumulative Immobilization Data and Observations in the Definitive Test

Geometric Mean Measured Concentration (mg/L)	24 Hours
	Cumulative ImmobilizedDaphnia (Initial Population: 5 Per Replicate)						Observations
	Rep1	Rep2	Rep3	Rep4	Total	%	Rep1	Rep2	Rep3	Rep4
Control	0	0	0	0	0	0	5N	5 N	5 N	5 N
0.051	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.14	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.17	0	0	0	0	0	0	3 N 2 P	1 N 4 P	2 N 3 P	2 N 3 P
0.29	1	0	0	0	1	5	4 P	5 P	2 N 3 P	3 N 2 P
0.55	1	1	1	1	4	20	1 R 3 P	4 P	1 R 3 P	4 P
Geometric Mean Measured Concentration (mg/L)	48 Hours
	Cumulative ImmobilizedDaphnia (Initial Population: 5 Per Replicate)						Observations
	Rep1	Rep2	Rep3	Rep4	Total	%	Rep1	Rep2	Rep3	Rep4
Control	0	0	0	1	1	5	5 N	5 N	5 N	4 N
0.051	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.14	1	1	0	0	2	10	1 R 3 N	1 R 3 N	5 N	5 N
0.17	0	1	0	3	4	20	5 P	1 R 3 P	5 P	2 P
0.29	5	5	4	4	18	90	AI	AI	1 R	1 R
0.55	5	5	5	5	20	100	AI	AI	AI	AI

Rep  = Replicate

N      = Normal

P     = Pale

R     = Reduced mobility

AI     = All Daphnia immobilized

Water Quality Measurements

Geometric Mean Measured Concentration (mg/L)		0 Hours (Fresh Media)			24 Hours (Old Media)
		pH	mg O2/L	Temperature °C	pH	mg O2/L	Temperature °C
Control	Rep1	8.1	9.3	20	7.9	8.7	21
0.051	Rep1	8.0	9.2	21	7.8	8.2	21
0.14	Rep1	8.0	9.2	21	7.7	8.3	21
0.17	Rep1	8.0	9.2	21	7.7	8.2	21
0.29	Rep1	8.0	9.1	21	7.7	8.2	21
0.55	Rep1	8.0	9.1	21	7.7	8.0	21
Geometric Mean Measured Concentration (mg/L)		24 Hours (Fresh Media)			48 Hours (Old Media)
		pH	mg O2/L	Temperature °C	pH	mg O2/L	Temperature °C
Control	Rep1	7.8	9.1	21	7.7	7.7	21
0.051	Rep1	7.8	9.0	21	7.6	7.9	21
0.14	Rep1	7.8	9.0	21	7.7	8.0	21
0.17	Rep1	7.7	9.0	21	7.7	8.0	21
0.29	Rep1	7.8	9.0	21	7.7	7.9	21
0.55	Rep1	7.7	9.0	21	7.7	8.1	21

Rep= Replicate

Validity criteria fulfilled:

yes

Conclusions:

The 48h EC50 (immobilisation) value with Daphnia magna is 0.21 mg/L.

Executive summary:

A study was performed to assess the acute toxicity of FRET 11-0539 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 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution concentrations of the substance dispersed in test water. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours and the following values determined: 0.92 mg/L and 0.21 mg/L, respectively. All results are expressed in terms of geometric mean measured test concentrations. Analysis of the freshly prepared test media at 0 and 24 hours showed measured test concentrations to range from 0.032 to 0.91 mg/L. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to range from 0.028 and 0.72 mg/L (between 69 and 99% of their starting concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration 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 FRET 11-0539 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 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution concentrations of the substance dispersed in test water. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours and the following values determined: 0.92 mg/L and 0.21 mg/L, respectively. All results are expressed in terms of geometric mean measured test concentrations. Analysis of the freshly prepared test media at 0 and 24 hours showed measured test concentrations to range from 0.032 to 0.91 mg/L. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to range from 0.028 and 0.72 mg/L (between 69 and 99% of their starting concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration 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:

0.21 mg/L