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Diss Factsheets

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date: 15 August 2016 Experimental completion date: 03 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
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
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethoxy-4-(hydroxymethyl)phenol
EC Number:
674-192-5
Cas Number:
4912-58-7
Molecular formula:
C9H12O3
IUPAC Name:
2-ethoxy-4-(hydroxymethyl)phenol
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Information as provided by the Sponsor. A Certificate of Analysis provided by the sponsor is presented as.

Identification: EVA
CAS Number: 4912-58-7
CAS Name: Benzenemethanol, 3-ethoxy-4-hydroxy-
Chemical Name: 2-Ethoxy-4-(hydroxymethyl)phenol
Physical state/Appearance: Pale yellow powder
Batch: 4K0001
Purity: 100%
Expiry Date: 31 December 2017
Storage Conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Remarks:
The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard.
Details on sampling:
- Concentrations: 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 mg/L.
- Sampling method: Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Definitive Test
A nominal amount of test item (200 mg) was dissolved in test water and the volume adjusted to 2 liters to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 10, 18, 32 and 56 mg/L.
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 were verified by chemical analysis at 0 and 48 hours.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnia 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 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.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
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.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h

Test conditions

Hardness:
250 mg/L as CaCO3
Test temperature:
22 Deg C
pH:
7.7 - 7.9
Dissolved oxygen:
8.3 - 8.4 mg
Salinity:
Not applicable.
Conductivity:
Not specified.
Nominal and measured concentrations:
Nominal: 10, 18, 32, 56 and 100 mg/L
Details on test conditions:
TEST SYSTEM
Exposure Conditions
As in the range-finding test 150 mL glass beakers containing approximately 100 mL of test preparation were used. At the start of the test 5 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 then covered to reduce evaporation 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.
The test preparations were not renewed during the exposure period.
Temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

TEST MEDIUM / WATER PARAMETERS
Reconstituted water (ISO medium) was used for both the range-finding and definitive tests.

Ingredient Final Concentration (mg/L)
CaCl2.2H2O 294
MgSO4.7H2O 123
NaHCO3 65
KCl 5.8

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

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.
- Intervals of water quality measurement: The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter. No adjustment required.
- Photoperiod and light intensity: 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition. The light intensity during the light period was measured using an ATP Instrumentation Lux meter.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 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.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 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 mg/L.
A nominal amount of test item (50 mg) was dissolved in test water and the volume adjusted to 500 mL to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 0.10, 1.0 and 10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding test 10 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. 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.
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. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
A positive control (Envigo Study Number MS29MC) used potassium dichromate as the reference item. The positive control was conducted between 29 February 2016 and 02 March 2016.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Range-finding Test
No immobilization was observed at the test concentrations of 0.10, 1.0 and 10 mg/L, however, immobilization was observed at 100 mg/L.
Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 95% to 99% of nominal concentrations. There was no significant change in the measured concentrations at 48 hours indicating that the test item was stable under test conditions.
Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 97% to 105% of nominal and so the results are based on nominal test concentrations.
Sub-Lethal Effects
Sub-lethal effects of exposure were observed in the 18, 32 and 56 mg/L test concentrations. These responses were trapping at the surface of the water and reduced mobility.
Immobilization Data
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test can be found in the section "Any other information on results, inl. table", below.
The relationship between percentage immobilization and concentration at 48 hours is given in Figure 1 attached below.
Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression based on the nominal test concentrations gave the following results:

Time (h) EC50 (mg/L) 95% Confidence limits (mg/L)
24 42 37 – 48
48 24 22 - 26

The No Observed Effect Concentrations after 24 and 48 hours exposure were 32 and 18 mg/L respectively. The Lowest Observed Effect Concentrations after 24 and 48 hours exposure were 56 and 32 mg/L respectively
The slopes and their standard errors of the response curves at 24 and 48 hours were 14 (SE = 0.024) and 13 (SE = 0.019) respectively.

Results with reference substance (positive control):
A positive control (Envigo Study Number MS29MC) 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 by the probit analysis using the linear maximum likelihood regression method at 24 and 48 hours using the ToxRat Professional computer software package based on the nominal test concentrations gave the following results:

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)
24 0.83 0.70 - 0.98 0.56 1.0
48 0.64 Not possible to determine 0.56 1.0

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 item.

Any other information on results incl. tables

Cumulative Immobilization Data and Observations in the Range-finding Test

Nominal Concentration (mg/L)

Observations (Initial Population: 10 per replicate)

24 hours

48 hours

Cumulative Immobilized Daphnia

Observations

Cumulative Immobilized Daphnia

Observations

Control

0

10 N

0

10 N

0.10

0

10 N

0

10 N

1.0

0

10 N

0

10 N

10

0

10 N

0

10 N

100

10

A/I

10

A/I

Immobilization Data

Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in the following table:

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal 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

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

18

0

0

0

0

0

0

3 T 2 N

1 T 4 N

5 N

5 N

32

1

0

0

0

1

5

4 N

5 N

5 N

2 T 3 N

56

5

5

5

4

19

95

A/I

A/I

A/I

A/I

100

5

5

5

5

20

100

A/I

A/I

A/I

A/I

Nominal 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

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

18

0

0

0

0

0

0

2 T 3 N

1 T 4 N

5 N

5 N

32

5

5

5

5

20

100

A/I

A/I

A/I

A/I

56

5

5

5

5

20

100

A/I

A/I

A/I

A/I

100

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

T = Trapping at the surface

R = Reduced mobility

A/I = All daphnia immobilized

Validation Criteria

The test was considered to be valid given that none 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 21 °C to 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 394 to 413 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.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Exposure of freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results:

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 24 22 – 26 18 32
Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. The number of immobilized daphnia were recorded after 24 and 48 hours.

Results

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations.

Exposure of Daphnia magna to the test item gave the following results:

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

24

22 – 26

18

32