(3-methoxy-2-methylpropyl)benzene

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2015-07-14 to 2015-07-16

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

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected on June 03-05, 2013 / signed on November 05, 2013

Specific details on test material used for the study:

- Density: 0.925 (d20/20) (according to FIRMENICH, 2011)
- Vapour pressure: 34 Pa at 25 °C (according to FIRMENICH, 2010)
- Partition coefficient: 3.32, shake flask method (determined at DR.U.NOACK LABORATORIEN; Study ID: 141007FC / COS16265)
- Water solubility: 195 mg/L (20 °C, pH 6.4) in double distilled water, 193 mg/L (20 °C, pH 7.8) in daphnia M4 medium (determined at DR.U.NOACK LABORATORIEN; Study ID: 141007FC / CWF16265)

Analytical monitoring:

yes

Details on sampling:

- Determination of the test item: All concentration levels and the control were analytically verified via HPLC-UV at the start (0 hours) and at the end of the exposure (48 hours).
- Sampling for the analytical monitoring: At the start of the exposure (0 hours), sampling was carried out after preparation of all test item concentrations. At the end of the exposure intervals (48 hours), samples were taken directly from the test vessels.
- Quality criteria for the analytical monitoring: Recoveries of the test item should be within ± 20 % of the nominal or initially measured concentrations. The effect levels can be given based on the nominal concentrations, if this quality criteria is fulfilled.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The stock solution (81.0 mg/L of the test item were weighed out) was freshly prepared with dilution water (ISO test water) before the start of the exposure (at 0 hours).
The stock solution was stirred for 2 hours with approximately 1100 rpm at room temperature and used as highest test concentration.
Application: The test vessels were filled up with the test solutions. The daphnids were inserted with a small amount of dilution water by pipette. Thereafter, the test vessels were closed immediately with screw caps.
- Controls: Dilution water without test item tested under the same conditions as the test groups.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna STRAUS (Clone 5)
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), D-14195 Berlin, Germany.
- Breeder: DR.U.NOACK-LABORATORIEN, Käthe-Paulus-Str. 1, D-31157 Sarstedt, Germany.
- Age at study initiation: 2 to 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels latest 22 hours before the start of the exposure and discarded. The juveniles born within this period of max. 22 hours preceding the exposure were used for the test after an acclimatisation phase of at least 2 hours in the dilution water. No first brood progeny was used for the test.
- Culture: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2°C, in an incubator, 16 h illumination, light intensity of max. 20 µE/m2/s
- Culture feeding: The culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 10^6 cells/mL. The algae are cultured at the test facility.
- Origin of the food algae: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, Germany.
- Feeding during test: The daphnids were not fed during the study.

ACCLIMATION
- Acclimation period: At least 2 h in dilution water

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

none

Post exposure observation period:

None

Hardness:

272 mg/L as CaCo3

Test temperature:

19 – 20 °C

pH:

7.85 (0 h)
7.32-8.76 (48 h)

Dissolved oxygen:

9.75 mg/L (0 h)
7.26-7.80 (48 h)

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: 16, 24, 36, 54 and 81 mg/L.

Details on test conditions:

TEST METHOD:
A static test design in glass flasks sealed with screw caps (made from polypropylene) under diffuse light conditions was chosen to reduce contact with air and losses of the test item by evaporation. The glass flasks were filled up with the test solutions having nearly no headspace. The test item was expected to evaporate. To reduce losses of the test item, the study was conducted in a closed system.

TEST SYSTEM
- Test vessels/volume: Sealed glass flasks (4.5 (ID) x 9.5 (H) cm, ca. 130 mL) with screw caps (made from polypropylene) were used.
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- No. of organisms per vessel: 5 (control and test material groups)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Dilution water: ISO Test water, according to OECD 202
- Culture medium: Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L, is used.
- Alkalinity: 272 mg CaCO3/L
- Conductivity: 651 µs/cm

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/8 h dark cycle
- Light intensity: Diffuse light; light intensity of max. 20 µE/m2/s (1340 lx)

EFFECT PARAMETERS MEASURED:
Biological parameters:
- Immobilisation and other observations: Immobilisation was determined in all groups after 24 and 48 h. An animal was considered to be immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Other relevant observations were not made.

Water Quality Parameters:
Dilution water: Prior to the start of the exposure (0 hours), the water quality parameters (i.e. pH value, dissolved oxygen concentration, temperature, conductivity and total hardness) of the dilution water were measured.
Test media: At the start of the exposure (0 hours), the water quality parameters of the fresh media (i.e. pH value, dissolved oxygen concentration) were measured in one additional replicate per concentration level and control. At the end of the exposure (48 hours), the water parameters of the old media were measured in all replicates per concentration level and control.
Temperature: The incubator temperature was recorded throughout the period of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Factor 1.5
- Range finding study: Test concentrations were selected based on the results of a non GLP preliminary range finding test. This test was conducted under static conditions with three concentrations of the test item of 1, 10 and 100 mg/L and two replicates per concentration level with 10 daphnids each were tested in a closed system. The percentage immobility was determined in all concentrations and control groups after 24 and 48 h of exposure.
- Results used to determine the conditions for the definitive study: After 24 and 48 h of exposure the total rate of immobilisation of daphnia was 0, 0 and 100% at 1, 10 and 100 mg/L, respectively.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

30.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% Cl: 28.0 – 32.1 mg/L

Details on results:

- Biological data: Immobilization of daphnids for the definitive test was 0, 0, 0, 30, 75 and 100 % (24 h); 0, 0, 5, 90, 100 and 100 % (48 h) at 16, 24, 36, 54 and 81 mg/L, respectively.

- Measured Exposure Concentrations during the Definitive Test: The measured concentrations of the test item at the start of the exposure (0 hours) were in the range of 91 to 98 % of the nominal values. At the end of the exposure (48 hours), the measured concentrations of the test item were in the range of 83 to 91 % of the nominal values. The measured test item concentrations were all within ± 20 % of the nominal concentrations. This indicates that the test item concentrations were successfully maintained for the duration of the test.

- Water Quality Parameters: The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 h) and at the end of the exposure intervals (24 and 48 h), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

Results with reference substance (positive control):

The percentage immobility for the reference item was determined after 24 h. The EC50-value with 95 % confidence limits (CI) was calculated by sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was:
EC50: 1.88 mg/L (Cl 1.62 - 2.19 mg/L)
The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

Reported statistics and error estimates:

EC50-values (after 24 and 48 hours) were calculated by sigmoidal dose-response regression. The 95 % confidence limits were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism5. The concentration-effect relationships are shown graphically. The EC50-values are given based on the nominal concentrations of the test item, because the measured concentrations were within ± 20 % of the nominal concentrations.
The EC50-value for the reference item and its 95 % confidence limits were calculated accordingly.

Table 6.1.3/1:  Immobilisation Rates after 24 and 48 h of Exposure in the Definitive Test

 

	IMMOBILISATION [%]
Nominal test item concentration [mg/L]	24 h					48 h
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
81.0	100	100	100	100	100	100	100	100	100	100
54.0	80	60	80	80	75	100	100	100	100	100
36.0	20	40	40	20	30	100	100	80	80	90
24.0	0	0	0	0	0	20	0	0	0	5
16.0	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

n = 20, divided into 4 replicates with 5 daphnids each

Table 6.1.3/2: Measured Concentrations of the Test Item during the Definitive Test

Sampling date	2015-07-14 Start of the exposure, 0 hours		2015-07-16 End of the exposure, 48 hours
Date of analysis	2015-07-14		2015-07-16
Nominal test item concentration [mg/L]	Test item
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
81.0	75.6	93	69.0	85
54.0	52.9	98	49.0	91
36.0	33.5	93	29.8	83
24.0	22.7	95	21.3	89
16.0	14.6	91	13.6	85
Control	< LOQ		< LOQ

Meas. conc. = measured concentration of the test item, dilution factor taken into account

% = percent of the nominal concentration of the test item

LOQ = limit of quantification (1.00 mg/L of the test item)

Validity criteria:

- In the control group, no daphnids were immobilised or showed any signs of disease or stress, e.g. discoloration or unusual behaviour such as trapping on the surface of the water, during the 48-hour test period (required: not more than 10 percent of the daphnids in the control).

- The dissolved O₂ concentration at the end of the exposure intervals was ≥ 7.26 mg/L (required: ≥ 3 mg/L at the test end of the exposure intervals) in the test vessels of all tested concentration levels and the control.

Validity criteria fulfilled:

yes

Conclusions:

Based on the analytically confirmed nominal concentrations of the test item, the 48 hour-EC50 for Daphnia magna was 30.3 mg/L (95 % confidence limits: 28.0 – 32.1 mg/L).

Executive summary:

Study was performed according to OECD Guideline 202 with GLP statement, to assess the 48 h-acute toxicity of the of the test substance to Daphnia magna, under static conditions.

Test item was exposed to daphnids at the concentrations of 16, 24, 36, 54 and 81 mg/L for 48 hours. The no-treatment control daphnids were exposed to dilution water only. There were four replicates per treatment with five daphnids per replicate, which provided a total of twenty daphnids per each treatment and control group at test initiation. All test solutions were prepared in dilution water. The test temperature was 19-20 °C. The test item was expected to evaporate. To reduce losses of the test item, the study was conducted in a closed system. The tested solutions were clear throughout the exposure period. Before definitive test, a 48-hour static range-finding test was conducted and the percent immobilization was 0, 0 and 100% at 1, 10 and 100 mg/L, respectively. 

 

The concentrations of the test item were analytically verified via HPLC-UV at the start of the exposure (0 hours) and at the end of the exposure (48 hours) in all concentration levels and the control. The measured concentrations of the test item at the start of the exposure (0 hours) were in the range of 91 to 98 % of the nominal values. At the end of the exposure (48 hours), the measured concentrations of the test item were in the range of 83 to 91 % of the nominal values. The measured test item concentrations were all within ± 20 % of the nominal concentrations. This indicates that the test item concentrations were successfully maintained for the duration of the test.The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 h) and at the end of the exposure (48 h), were within the acceptable limits. The validity criteria of the test guidelines were fulfilled.

 

Immobilization of daphnids for the definitive test was 0, 0, 0, 30, 75 and 100 % (24 h); 0, 0, 5, 90, 100 and 100 % (48 h) at 16, 24, 36, 54 and 81 mg/L, respectively. 24 and 48 h EC50 values were 43.3 mg/L (CI: 41.0 – 45.8) and 30.3 mg/L (CI: 28.0-32.1). The EC50 -value of the reference item, potassium dichromate at 1.88 mg/L (Cl 1.62 - 2.19 mg/L) after 24 h was within the prescribed concentration range of 0.6 - 2.1 mg/L.

 

Based on the analytically confirmed nominal concentrations of the test item, the 48 hour-EC50 for Daphnia magna was 30.3 mg/L (95 % confidence limits: 28.0 – 32.1 mg/L).

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

From 2015-09-08 to 2015-09-09

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
iSafeRat® HA-QSAR toolbox v1.1

2. MODEL (incl. version number)
iSafeRat® holistic HA-QSAR v1.4

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
c1cccc(CC(C)COC)c1
The water solubility given as input was experimentally measured: 195 mg/L

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF

5. APPLICABILITY DOMAIN
See attached QPRF

6. ADEQUACY OF THE RESULT
See attached QPRF

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

not applicable

Remarks:

(QSAR model)

Principles of method if other than guideline:

The acute toxicity to aquatic invertebrates was determined using a validated QSAR for the Mode of Action in question, based on validated data derived from 48-hour EC50 tests on daphnia, for which the concentrations of the test item had been determined by chemical analyses over the test period. The QSAR is a simple linear regression of sub-cooled liquid solubility versus 48-h EC50 for daphnia and is valid within the applicability domain defined in the QMRF.

GLP compliance:

no

Specific details on test material used for the study:

- Water solubility 195 mg/L (from experimental study)

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

Not applicable

Test organisms (species):

Daphnia sp.

Details on test organisms:

Not applicable

Test type:

other: QSAR

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

none

Post exposure observation period:

Not applicable

Hardness:

The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.

Test temperature:

The temperatures varied from approximately 20 to 23 °C depending on the species used to construct the algorithm. This small difference is not expected to contribute to the variability of the EC50 values found in experimental data.

pH:

The QSAR is based on data from studies performed at acceptable pH between 6.0 - 9.0.

Dissolved oxygen:

The QSAR is based on data from studies performed at acceptable oxygen concentrations (generally >60%).

Salinity:

Not applicable.

Nominal and measured concentrations:

The QSAR is based on data from studies performed using measured concentrations or with acceptable stability.

Details on test conditions:

Not applicable

Reference substance (positive control):

no

Remarks:

QSAR model

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

9.2 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 8.4-10 mg/L

Details on results:

The water solubility value given as input to the Ecotox module of the iSafeRat® Holistic HA-QSAR falls within the descriptor domain of the model between log water solubility (in log (mol/L)) of -4.70 to 0.87. Moreover the test substance is attributed to the class of non-polar narcotic compounds (MOA 1).

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

95% CL: 8.4-10 mg/L
QSAR statistical parameters are given in the QMRF and the QPRF

No additional information

Validity criteria fulfilled:

yes

Remarks:

The substance falls into the applicability domains of the QSAR model.

Conclusions:

The 48-h EC50 value based on mobility and measured concentrations was determined to be 9.2 mg/L with 95%-Confidence Limit between 8.4 and 10 mg/L.

Executive summary:

A QSAR prediction was performed to assess the acute toxicity of the test substance to daphnid. This QSAR has been validated to be compliant with the OECD recommendations for QSAR modelling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following OECD Guideline 202. The criterion predicted was the EC50 (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.

 

The immobility of the daphnids was determined using a validated QSAR for the Mode of Action in question, (MOA 1, non-polar narcosis). The QSAR is based on validated data for a training set of 58 chemicals derived from 48-hour tests on daphnids, for which the concentrations of the test substance had been determined by chemical analyses over the test period. The water solubility of the substance given as input was experimentally measured at 195 mg/L. The substance falls within the applicability domain of the model as demonstrated in the QPRF.

The 48-h EC50 value based on mobility and measured concentrations was determined to be 9.2 mg/L with 95%-Confidence Limit between 8.4 and 10 mg/L.

Description of key information

OECD Guideline 202, GLP, key study, validity 1:

48h-EC50 (Daphnia magna) = 30.3 mg/L (95% CL: 28.0 - 32.1 mg/L) based on analytically confirmed nominal concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

30.3 mg/L

Additional information

To assess the short-term toxicity of the registered substance to aquatic invertebrates, two data are available.

The first study (NOACK, 2015) was assessed as the key study and was performed on the registered substance according to OECD Guideline 202 with GLP statement. In this study, the test substance was exposed to Daphnia magna at the concentrations of 0 (control with dilution water only), 16, 24, 36, 54 and 81 mg/L for 48 hours, under closed system and static conditions (four replicates per treatment; five daphnids per replicate). According to the results of this study, the 48h-EC50 was determined at 30.3 mg/L (95% CL: 28.0 - 32.1 mg/L), based on analytically confirmed nominal concentrations.

The second data (KREATiS, 2015), assessed as a supporting data, is a QSAR. This QSAR prediction (iSafeRat holistic approach v1.4) was performed on the registered substance, to assess the acute toxicity of the substance to daphnids. This QSAR has been validated to be compliant with the OECD recommendations for QSAR modelling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following OECD Guideline 202. The immobility of the daphnids was determined using a validated QSAR for the Mode of Action in question, (MOA 1, non-polar narcosis). This QSAR is based on validated data for a training set of 58 chemicals derived from 48-hour tests on daphnids, for which the concentrations of the test substance had been determined by chemical analyses over the test period. The water solubility of the substance given as input was experimentally measured (195 mg/L). The substance falls within the applicability domain of the model. The 48h-EC50 value based on mobility and measured concentrations was determined to be 9.2 mg/L (95% CL: 8.4 - 10 mg/L). This QSAR result supports the key study performed on the same substance with an EC50 value more conservative validating the model for use with this substance.

In conclusion, according to the key experimental study (NOACK, 2015), the 48h-EC50 value on Daphnia magna is 30.3 mg/L (95% CL: 28.0 - 32.1 mg/L).