1-ethoxy-2-(2-methoxyethoxy)ethane

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of experimental phase: 08 September 2021
End of experimental phase: 30 September 2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

Concerning: Date of Draft Report According to the Study Plan: Not later than 05 November 2021 Deviation: 19 November 2021 Reason for deviation: Unscheduled delay Presumed Effect on the Study: None

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Item name: 1-ethoxy-2-(2-methoxyethoxy)ethane
Lot number: 09511
Active ingredient content: ˃ 99.9%
Appearance: water clear, liquid
Expiry date: March 28, 2023
Storage: at room temperature, protected from light

The test item of a suitable chemical purity was supplied by the Sponsor. All precautions required in the handling and disposal of the test item were outlined by the Sponsor. These documents are part of the raw data. Identification of the test item was performed in the Laboratory of TOXI-COOP ZRT. on the basis of the information included in the test item documentation supplied by the Sponsor.

Analytical monitoring:

yes

Remarks:

Test item concentrations were determined at the start and at the end of one renewal period weekly. Sampling of control was also performed at each analytical occasion. The samples were diluted and analysed by GC-MS.

Details on sampling:

Five samples were taken from the test solutions and one sample was taken from the control solution at each analytical occasion.
Samples were twofold diluted with acetonitrile.

Stock Solution: 1 mg/mL: approximately 10 mg of the test item was dissolved in acetonitrile to a total volume of 10 mL.
Calibration series: Stock solution was properly diluted with acetonitrile/water (1/1) mixture.
Nominal concentrations: 0.1, 0.2, 0.5, 1, 2, 5 and 8 µg/mL.

At the start and at the end of four renewal periods of the test five replicate samples were taken from the test solutions and one sample was taken from the control solution. Mean of the measured concentrations are presented in "Any other information on materials and methods".

Vehicle:

no

Details on test solutions:

The test solutions used in the test were prepared by mechanical dispersion without using any solubilising agent, as the substance is well water soluble.
For each water renewal period, a stock solution of 50 mg/L nominal concentration was first prepared by dissolving an amount of 0.025 g test item in 500 mL dilution water. Test solutions of chosen test concentrations were prepared by appropriate dilution of this stock solution. (see "Any other information on materials and methods".). After the formulation procedure the test animals were immediately introduced into the test solutions, at each water renewal period.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species and strain: Daphnia magna (Straus)
Age of animals: Less than 24 hours old at the beginning of the test
Sex: Female
Origin: Laboratory of Hydrobiology (Central Agricultural Office,
Directorate of Plant-, and Soil Protection)
2100 Gödöllő, Kotlán S. u. 3. Hungary
Breeding Conditions: The Daphnia are bred in the Laboratory of TOXI-COOP ZRT.
Parent Animals: The animals present at the start of the test and their reproductive output is the object of this study. The animals used for the test were not the first brood progeny; this was ensured by holding the stock animals individually and monitoring the production of their first brood.
Offspring: Produced by the parental animals in the course of this test.
When the parental animals are about seven days old, the first young Daphnia emerge from the brood pouch, thereafter a new batch appears every two to three days. These batches are called "broods" of the F1 (filial 1) generation.
Acclimatisation: The stock animals were bred under the same conditions as that used during the exposure period (holding water, temperature, background colour etc.), therefore additional acclimatisation to avoid stressing the parent animals prior to test was not necessary.
Animal health: Apparently healthy animals were used in this test with a known history (breeding method, pre-treatment).

Test type:

semi-static

Water media type:

other: Elendt M4 Medium

Limit test:

no

Total exposure duration:

21 d

Hardness:

Hardness of Water: Measured values: 295.5 mg/L (07 September 2021)
(as CaCO3) 252.1 mg/L (14 September 2021)
252.1 mg/L (21 September 2021)
249.2 mg/L (27 September 2021)
see Test conditions (measured in test vessels): water temperature, pH and dissolved oxygen concentration in "Any other information on results".

Test temperature:

Temperature: Measured in test vessels:
Control: 20.8 – 21.4°C
Highest test concentration: 20.8 – 21.4°C
Measured in the climate chamber: 20.5 – 21.7°C


see Test conditions (measured in test vessels): water temperature, pH and dissolved oxygen concentration in "Any other information on results".

pH:

pH: Control: 7.81 – 8.28
Highest test concentration: 7.91 – 8.26


see Test conditions (measured in test vessels): water temperature, pH and dissolved oxygen concentration in "Any other information on results"

Dissolved oxygen:

Dissolved Oxygen Concentration: Control: 7.46 – 8.32 mg/L
Highest test concentration: 7.64 – 8.40 mg/L


see Test conditions (measured in test vessels): water temperature, pH and dissolved oxygen concentration in "Any other information on results".

Nominal and measured concentrations:

see "Any other information on results"

Details on test conditions:

Test environment: Climate chamber
Test type: Semi-static test. The frequency of test solution renewal was three times per week (2d-2d-3d periods).
Temperature: 18 – 22 °C (the temperature should not vary by more than 2 °C within these limits in the test media as daily range)
Light: 16 h light : 8 h dark; intensity in the range of 1000-1500 lux (using cool white light)
pH: The pH should be within the range 6 - 9 (normally not vary by more
than 1.5 units in the test). The test was carried out without adjustment of pH.
Dissolved oxygen concentration: > 3 mg/L (the test vessels were not aerated)
Hardness of Water: > 140 mg/L (as CaCO3)
Duration of the Test: 21 days
Recording: Test conditions were measured with suitable instruments and documented in the raw data.

Note: Measured values are summarized in "Test Condition" and detailed in "Any other information on materials and methods".

Apparatus

Test vessels: Glass beakers, appropriate size for holding 50-100 mL test medium. Each test vessel was uniquely identified with study number, test group and replicate. Each test vessel was filled up with approx. 80 mL test medium.
Equipment: Normal laboratory equipment and the following were necessary:
• balance
• pH meter
• dissolved oxygen meter
• thermometer
• light-meter
• reagent set for determination of water hardness
• climate chamber
• microscope with calibrated slide

Preliminary Range-Finding Test

In order to select appropriate test concentrations for use in the definitive test, non-GLP preliminary range-finding test was conducted to determine the approximate toxicity of the test item.
The test item effect was investigated in a semi-static preliminary test in which the test solutions had been renewed periodically (three times per week) at the concentration levels of 0.1, 1, 10 and 100 mg/L. Concurrent control ran parallel.
The test solutions were prepared at each water renewal period as follows: a stock solution of (100 mg/L nominal loading) was first prepared by dissolving an appropriate amount of test item in Elendt M4 medium. Subsequent lower concentrations were prepared by appropriate serial dilution of this stock solution.
Five daphnids, divided into five replicates (1 parent animal per test vessel; held in approximately 40 mL test medium) were tested in each test group for a period of time that allowed the estimation of chronic toxicity (16 days in this case). The mortality among the parent animals and the total number of offspring produced by parents were observed during the test (results are presented below in "Any other information on materials and methods").

The preliminary range-finding test was not performed in compliance with the GLPRegulations and is excluded from the Statement of Compliance (Statement of the Study Director), but the raw data of this test will be archived under the study code of present study.

Description of the Test Procedure

Test Item Concentrations in the Definitive Test

Based on the results of the non-GLP preliminary range-finding test five test concentrations in a geometric series (with a spacing factor of 2.3) was used in the main test. Untreated control ran in parallel.
The following nominal concentrations were tested: 0.36, 0.82, 1.9, 4.3 and 10 mg/L.
Based on the results of analytical measurements the concentrations of test item has been maintained within ± 20 % of the nominal during the experiment, therefore the biological results and endpoints are based on the nominal concentrations.

Preparation of Test Solutions

The test solutions used in the test were prepared by mechanical dispersion without using any solubilising agent, as the substance is well water soluble.
For each water renewal period, a stock solution of 50 mg/L nominal concentration was first prepared by dissolving an amount of 0.025 g test item in 500 mL dilution water. Test solutions of chosen test concentrations were prepared by appropriate dilution of this stock solution. (see Table 2.). After the formulation procedure the test animals were immediately introduced into the test solutions, at each water renewal period.

Performance of the Test

Ten Daphnia in control and in each test concentration were divided into ten replicates [1 parent animal/test container (the animals were held individually)], each of them in approx. 80 mL test medium. Volume of test medium was identical in each vessel.
For test results to be satisfactory, treatments and addition of test organisms were randomly assigned to the test vessels, furthermore test vessels were randomly located within the test area.
This reproduction test was carried out in a semi-static renewal system. The frequency of test solution renewal was three times per week (2d-2d-3d periods; see detailed experimental schedule).
When the medium was renewed a second series of test vessels were prepared and the parent animals transferred to them by a pipette of suitable diameter. The volume of test medium transferred with the Daphnia was minimized.
The Daphnia were fed daily with concentrated suspension of living algal cells of Raphidocelis subcapitata. Concentrated algal suspension was fed in order to minimise the volume of algal culture medium transferred to the test vessels. The amount of food supplied was based on the amount of organic carbon (C) provided to each parent animal. A total of 0.1-0.2 mg C/Daphnia/day was fed. Determination of the total organic carbon (TOC) content of algal suspension used for feeding is performed preferably annual. Recent TOC measurement was performed on 02 February 2021. Measured values are detailed in "Any other information on materials and methods", the correlation between algal cell number and TOC concentration is shown in "illustration".
The environmental conditions were measured as described below.

Test Parameters

Offspring: The living offspring produced by each parent animals were removed and counted daily from the appearance of the first brood. The presence of any aborted eggs and dead, aborted offspring were recorded.
Mortality: The mortality (immobility) among the parent animal was recorded daily.
Measurement of Environmental Parameters: The water temperature, dissolved oxygen concentration and pH values were measured at each water renewal in fresh and old media, in the control and in the highest test item concentration.
The ambient temperature was monitored continuously using a min-max thermometer within the climate chamber.
Measurement of water hardness was performed at least once a week and in each batch of reconstituted water prepared for the experiment.
The light intensity was measured and recorded at the start of the test within the climate chamber.
Growth Measurement and Other Parameters: The length of the parent animals (i.e. body length excluding the anal spine) was measured at the end of the test.
Other parameters including time to production of first brood (and subsequent broods), number of broods per animal, number of aborted broods, presence of male neonates or ephippia were assessed during the test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate. Tested at least twice a year. Date of the last study (Study number: 392-202-6327) with reference item Potassium dichromate 14– 15 September 2021. result of study, the 24h EC50 was determined to be 0.92 mg/L.

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Validity of the Test

 The mortality of parent animals (female Daphnia) did not exceed 20 % in the control at the end of the test (no mortality was observed). Furthermore, the same validity criterion (mortality ≤ 20%), was fulfilled for accidental and inadvertent parental mortality for each of the test concentrations.
 The mean number of living offspring produced per parent animal survived at the end of the test was 81.1 (≥ 60) with variation coefficient of 12.57 % (≤ 25 %) in the control.
All validity criteria were met, therefore the study was considered to be valid.

Test Conditions

Temperature: Measured in test vessels:
Control: 20.8 – 21.4°C
Highest test concentration: 20.8 – 21.4°C
Measured in the climate chamber: 20.5 – 21.7°C

pH: Control: 7.81 – 8.28
Highest test concentration: 7.91 – 8.26

Dissolved Oxygen Concentration: Control: 7.46 – 8.32 mg/L
Highest test concentration: 7.64 – 8.40 mg/L

Note: Measured values of Water Temperature, pH and Dissolved Oxygen Concentration are detailed in "Any other information on results".

Light Intensity: 1068 lux measured at the start of the test.
Hardness of Water: Measured values: 295.5 mg/L (07 September 2021)
(as CaCO3) 252.1 mg/L (14 September 2021)
252.1 mg/L (21 September 2021)
249.2 mg/L (27 September 2021)

Analytical Measurements

Nominal concentrations of 0.36, 0.82, 1.9, 4.3 and 10 mg/L plus a control were investigated in the definitive test and analysed on one renewal occasion weekly during the test when freshly prepared and at renewal.
The mean of the measured concentrations of the test solutions were in the range of 95 – 115 % of the nominal during the test.
All measured values remained within the range of ± 20 % of the nominal, therefore the biological results are based on the nominal test item concentrations.
Test item was not detected in the control samples.

Measured data and the percentage of the nominal concentrations are detailed in "Any other information on results".

A detailed description of the chemical analysis (method, determination, results) is seen in the sampling and analysis section.

Biological Results

All biological results are based on the nominal test concentrations.

Reproduction

The mean number of offspring produced per surviving parent animal was 81.1 in the control group.
The mean number of offspring produced per surviving parent animal was 69.9, 72.3, 82.3, 100.4 and 98.2 in the concentrations of 0.36, 0.82, 1.9, 4.3 and 10 mg/L respectively.
Based on the results of the statistical evaluation, no statistically significant reduction in reproduction was observed in any of the test concentrations compared to the control.
Accordingly, the 21-day NOEC for reproduction was determined to be 10 mg/L and the LOEC as higher than 10 mg/L.
The 21-day EC50, EC20 and EC10 values for reproduction were determined to be higher than 10 mg/L.
In conclusion, the test item had no effect on reproduction of Daphnia magna up to at least the highest tested concentration of 10 mg/L (i.e. the maximum required test concentration for chronic effects testing). Accordingly, the test item had no effect on reproduction of Daphnia magna up to at least the limit concentration of 10 mg/L.

The offspring produced during the test are detailed in "Any other information on results" and summarised in "Any other information on results".
Results of the statistical evaluation for reproduction are detailed in "illustration".
Graphical presentations of reproduction are shown in "illustration".

Mortality of Parent Animals

No mortality was observed in the control group during the test. No statistically significant mortality (up to 20 %) was noticed in any of the test concentrations compared to the control.
Accordingly, the 21-day NOEC for mortality was determined to be 10 mg/L and the LOEC as higher than 10 mg/L.
The mortalities observed during the test (and the day on which mortality occurred) are presented in Any other information on results".
Results of the statistical evaluation for mortality are detailed in "illustration".

Body Length of Parent Animals

Based on the results of the statistical evaluation, parent body length values in the two lowest test concentrations (0.36 and 0.82 mg/L) was found to be statistically significantly lower compared to the control. As no statistically significant reduction of body length was observed in the concentrations from above (1.9, 4.3 and 10 mg/L), no concentrationresponse pattern was determined and that lower body length values were considered to be due to natural variability rather than a test substance related effect. Accordingly, the test item had no effect on the growth of Daphnia magna up to at least the limit concentration of 10 mg/L.

Body lengths of parent animals measured at the end of the test are presented in "Any other information on results".
Results of the statistical evaluation for body lengths are detailed in "illustration".

Other Parameters

No aborted broods, difference in size of broods, presence of male neonates or ephippia were noted in the control or in any of the test concentrations during the test period.

No statistically significant different increase in time to production of first brood was observed in any of the test concentrations compared to the control.

The time to production of first brood per parent animal is detailed in "Any other information on results".
Results of the statistical evaluation for time to production of first brood are detailed in "illustration".

Results with reference substance (positive control):

For the evaluation of the reliability of the applied test system and the experimental conditions, Potassium dichromate is tested in a Daphnia magna acute toxicity test at least twice a year.
The date of the last study (Study number: 392-202-6327) with reference item Potassium dichromate was: 14 – 15 September 2021.
As result of that study, the 24h EC50 was determined to be 0.92 mg/L.

Name and data of Reference Item
Name: Potassium dichromate (K2Cr2O7)
Batch No.: K50498364
CAS No.: 7778-50-9
Description: Orange crystalline
Expiry Date: 31 July 2023
Storage: Room temperature
Supplier: MERCK KGaA

Reported statistics and error estimates:

The statistical analysis was performed by ToxRat Professional (Version 3.3.0) software. Statistical procedures are detailed in "illustration".

Number of offspring (NOEC/LOEC, ECx values):
To determine NOEC/LOEC values for reproduction, Multiple Sequentially-rejective Welsht-test after Bonferroni-Holm (α = 0.05, one-sided smaller) was used. Pretesting on normal distribution was performed by Shapiro-Wilk´s test and on variance homogeneity by Levene’s test.
To determine the ECx values for reproduction no statistical analysis was necessary as no toxic effects were observed.

Mortality:
Mortality results was evaluated using Fisher’s Exact Binominal test with Bonferroni Correction (α = 0.05, one-sided greater).

Body length of parent animals:
Comparison of body length of parents was performed using Dunnett`s Multiple t-test Procedure (α = 0.05, one-sided smaller). Pretesting on normal distribution was performed by Shapiro-Wilk’s test and on variance homogeneity by Levene’s test.

Time to production of first brood:
Comparison of time to production of first brood was performed using Multiple Sequentiallyrejective Welsh-t-test after Bonferroni-Holm (α = 0.05, one-sided greater). Pretesting on normal distribution was performed by Shapiro-Wilk’s test and on variance homogeneity by Levene’s test.

Concentrations measured at the renewal period of September 08-10, 2021 

Nominal concentration mg/L	Start		End
	Concentration, mean (mg/L)	% of the nominal	Concentration, mean (mg/L)	% of the nominal
Control	not detected	-	not detected	-
0.36	0.384	107	0.341	95
0.82	0.865	105	0.820	100
1.9	1.89	99	1.91	101
4.3	4.30	100	4.34	101
10	10.1	101	10.5	105

 

Concentrations measured at the renewal period of September 15-17, 2021 

Nominal concentration mg/L	Start		End
	Concentration, mean (mg/L)	% of the nominal	Concentration, mean (mg/L)	% of the nominal
Control	not detected	-	not detected	-
0.36	0.388	108	0.379	105
0.82	0.896	109	0.873	106
1.9	1.94	102	2.02	106
4.3	4.53	105	4.62	107
10	10.3	103	11.0	110

 

Concentrations measured at the renewal period of September 20-22, 2021 

Nominal concentration mg/L	Start		End
	Concentration, mean (mg/L)	% of the nominal	Concentration, mean (mg/L)	% of the nominal
Control	not detected	-	not detected	-
0.36	0.351	97	0.383	106
0.82	0.829	101	0.884	108
1.9	2.03	107	2.05	108
4.3	4.79	111	4.87	113
10	11.1	111	11.5	115

 

Concentrations measured at the renewal period of September 27-29, 2021 

Nominal concentration mg/L	Start		End
	Concentration, mean (mg/L)	% of the nominal	Concentration, mean (mg/L)	% of the nominal
Control	not detected	-	not detected	-
0.36	0.396	110	0.378	105
0.82	0.864	105	0.861	105
1.9	1.97	104	2.01	106
4.3	4.65	108	4.60	107
10	10.7	107	11.1	111

 

Test conditions (measured in test vessels): water temperature, pH and dissolved oxygen concentration

Measurement occasion		Control	pH	10 mg/L	Dissolved oxygen concentration [mg/L]        Control              10 mg/L		Temperature [°C]        Control              10 mg/L
Day 0	fresh	8.28		8.26	8.21	8.23	21.2	21.3
	old	–		–	–	–	–	–
Day 2	fresh	8.01		8.03	7.46	7.73	21.4	21.4
	old	7.96		8.20	7.88	8.21	21.3	21.4
Day 5	fresh	7.89		8.04	8.07	8.04	21.3	21.2
	old	8.06		8.06	7.84	7.87	21.3	21.3
Day 7	fresh	8.23		8.22	7.94	8.05	21.3	21.3
	old	8.12		8.06	7.72	7.64	21.2	21.3
Day 9	fresh	7.92		7.91	7.71	8.08	21.2	21.3
	old	7.99		8.05	7.78	7.81	21.2	21.2
Day 12	fresh	7.88		8.03	8.26	8.33	21.2	21.3
	old	8.03		8.06	8.20	8.21	21.2	21.2
Day 14	fresh	8.16		8.14	8.14	8.40	21.2	21.2
	old	8.11		8.12	8.32	8.14	21.3	21.2
Day 16	fresh	8.10		8.12	8.25	8.19	21.2	21.1
	old	8.06		8.10	8.01	8.14	21.1	21.1
Day 19	fresh	7.96		8.02	8.17	8.31	20.9	20.8
	old	8.01		8.05	8.27	8.23	21.0	20.9
Day 21	fresh	–		–	–	–	–	–
	old	7.81		8.14	8.09	8.33	20.8	20.8
	min	7.81		7.91	7.46	7.64	20.8	20.8
	max	8.28		8.26	8.32	8.40	21.4	21.4

The full record of offspring production during the test in the Control 

Test group: Control
Time (day)					Replicate
	1	2	3	4	5	6	7	8	9	10
1.	0	0	0	0	0	0	0	0	0	0
2.	0	0	0	0	0	0	0	0	0	0
3.	0	0	0	0	0	0	0	0	0	0
4.	0	0	0	0	0	0	0	0	0	0
5.	0	0	0	0	0	0	0	0	0	0
6.	0	0	0	0	0	0	0	0	0	0
7.	9	9	9	0	4	0	7	12	0	13
8.	0	0	0	0	0	0	0	0	0	0
9.	0	0	0	13	0	9	0	0	11	0
10.	5	0	0	0	0	0	0	0	0	0
11.	0	0	0	0	0	0	0	0	0	0
12.	11	14	9	27	15	17	13	11	17	13
13.	20	13	17	0	17	0	18	15	16	0
14.	0	0	0	0	0	0	0	0	0	0
15.	0	0	0	25	0	18	0	0	18	0
16.	6	7	8	0	9	0	5	8	0	9
17.	0	0	0	0	0	0	0	0	0	0
18.	20	0	0	18	0	15	0	0	15	0
19.	0	24	21	0	19	0	18	24	0	22
20.	0	0	0	0	0	0	0	0	0	0
21.	19	15	12	11	17	0	23	13	13	15
Total / replicate	90	82	76	94	81	59	84	83	90	72
Mean					81.1
SD					10.19
CV					12.57

The full record of offspring production during the test in concentration of 0.36 mg/L 

Test group: 0.36 mg/L nominal concentration
Time (day)					Replicate
	1	2	3	4	5	6	7	8	9	10
1.	0	0	0	0	0	0	0	0	0	0
2.	0	0	0	0	0	0	0	0	0	0
3.	0	0	0	0	0	0	0	0	0	0
4.	0	0	0	0	0	0	0	0	0	0
5.	0	0	0	0	0	0	0	0	0	0
6.	0	0	0	0	0	0	0	0	0	0
7.	12	10	0	7	10	9	0	9	10	0
8.	0	0	0	0	0	0	5	1	0	0
9.	0	0	10	0	0	0	0	0	0	0
10.	0	0	0	0	0	0	0	0	0	0
11.	0	0	0	0	0	0	0	0	0	0
12.	13	11	15	11	14	13	12	8	11	12
13.	12	14	0	17	19	16	17	10	8	15
14.	0	0	0	0	0	0	0	0	0	0
15.	0	0	17	0	0	0	0	0	0	0
16.	8	8	0	9	11	5	7	10	8	8
17.	0	0	0	0	0	0	0	0	0	0
18.	5	–	13	0	0	0	0	0	0	0
19.	20	–	0	21	19	21	17	22	20	24
20.	0	–	1	0	0	0	0	0	0	0
21.	11	–	12	15	11	0	0	0	0	18
Total / replicate	81	–	68	80	84	64	58	60	57	77
Mean					69.9
SD					10.72
CV					15.33

-: no data (due to parent mortality); mortality of parent animal was considered to be inadvertent mortality (i.e. non substance related mortality with not known cause), therefore this replicate was excluded from data analysis

 

The full record of offspring production during the test in concentration of 0.82 mg/L 

Test group: 0.82 mg/L nominal concentration
Time (day)					Replicate
	1	2	3	4	5	6	7	8	9	10
1.	0	0	0	0	0	0	0	0	0	0
2.	0	0	0	0	0	0	0	0	0	0
3.	0	0	0	0	0	0	0	0	0	0
4.	0	0	0	0	0	0	0	0	0	0
5.	0	0	0	0	0	0	0	0	0	0
6.	0	0	0	0	0	0	0	0	0	0
7.	0	6	7	8	10	0	0	5	0	0
8.	0	0	0	0	0	0	0	0	11	0
9.	10	0	0	0	0	9	10	0	0	14
10.	0	0	0	0	0	0	0	0	0	0
11.	0	0	0	0	0	0	0	0	0	0
12.	17	12	9	14	11	15	18	11	18	22
13.	0	–	–	15	15	0	0	14	0	0
14.	0	–	–	0	0	0	0	0	16	0
15.	19	–	–	0	0	22	20	0	0	16
16.	0	–	–	6	9	0	0	10	0	0
17.	0	–	–	0	0	0	0	0	0	18
18.	12	–	–	0	0	15	15	0	0	0
19.	0	–	–	23	22	0	0	18	0	0
20.	0	–	–	0	0	11	10	0	14	16
21.	0	–	–	21	18	0	0	0	0	0
Total / replicate	58	–	–	87	85	72	73	58	59	86
Mean					72.3
SD					12.83
CV					17.75

-: no data (due to parent mortality); mortality of parent animal was considered to be inadvertent mortality (i.e. non substance related mortality with not known cause), therefore this replicate was excluded from data analysis

The full record of offspring production during the test in concentration of 1.9 mg/L 

Test group: 1.9 mg/L nominal concentration
Time (day)					Replicate
	1	2	3	4	5	6	7	8	9	10
1.	0	0	0	0	0	0	0	0	0	0
2.	0	0	0	0	0	0	0	0	0	0
3.	0	0	0	0	0	0	0	0	0	0
4.	0	0	0	0	0	0	0	0	0	0
5.	0	0	0	0	0	0	0	0	0	0
6.	0	0	0	0	0	0	0	0	0	0
7.	9	5	0	0	0	4	0	0	0	0
8.	0	0	3	2	0	0	8	5	8	0
9.	0	0	0	0	0	0	1	6	0	11
10.	0	0	0	0	0	0	0	0	0	0
11.	0	0	0	0	0	0	0	0	0	0
12.	13	15	5	4	0	13	12	16	11	19
13.	23	29	0	10	0	26	21	0	23	0
14.	0	0	7	0	12	0	0	0	0	0
15.	0	4	0	0	0	0	0	26	0	19
16.	17	9	0	11	0	12	12	0	14	0
17.	0	0	16	0	10	0	0	0	0	22
18.	26	22	0	0	0	19	0	23	0	0
19.	0	0	0	17	0	27	3	0	32	2
20.	0	0	12	0	15	0	0	0	0	22
21.	27	25	0	0	0	17	26	–	9	0
Total / replicate	115	109	43	44	37	118	83	–	97	95
Mean					82.3
SD					32.59
CV					39.59

-: no data (due to parent mortality); mortality of parent animal was considered to be inadvertent mortality (i.e. non substance related mortality with not known cause), therefore this replicate was excluded from data analysis

 

The full record of offspring production during the test in concentration of 4.3 mg/L 

Test group: 4.3 mg/L nominal concentration
Time (day)					Replicate
	1	2	3	4	5	6	7	8	9	10
1.	0	0	0	0	0	0	0	0	0	0
2.	0	0	0	0	0	0	0	0	0	0
3.	0	0	0	0	0	0	0	0	0	0
4.	0	0	0	0	0	0	0	0	0	0
5.	0	0	0	0	0	0	0	0	0	0
6.	0	0	0	0	0	0	0	0	0	0
7.	0	0	0	0	0	0	0	0	0	0
8.	10	7	9	6	7	8	8	0	6	9
9.	0	0	0	0	0	0	0	13	0	0
10.	0	0	0	0	0	0	0	0	0	0
11.	0	0	0	0	0	0	0	0	0	0
12.	17	16	17	14	12	17	14	3	12	20
13.	21	23	22	26	22	21	0	19	–	–
14.	0	0	0	0	0	0	21	0	–	–
15.	0	0	0	0	0	0	0	24	–	–
16.	10	8	15	9	14	16	0	0	–	–
17.	0	0	0	0	0	0	13	0	–	–
18.	0	0	0	0	1	0	0	25	–	–
19.	39	24	33	39	38	25	27	0	–	–
20.	0	0	0	0	0	0	0	0	–	–
21.	23	8	12	0	22	5	0	20	–	–
Total / replicate	120	86	108	94	116	92	83	104	–	–
Mean					100.4
SD					13.73
CV					13.68

-no data (due to parent mortality); mortality of parent animal was considered to be inadvertent mortality (i.e. non substance related mortality with not known cause), therefore this replicate was excluded from data analysis

The full record of offspring production during the test in concentration of 10 mg/L 

Test group: 10 mg/L nominal concentration
Time (day)					Replicate
	1	2	3	4	5	6	7	8	9	10
1.	0	0	0	0	0	0	0	0	0	0
2.	0	0	0	0	0	0	0	0	0	0
3.	0	0	0	0	0	0	0	0	0	0
4.	0	0	0	0	0	0	0	0	0	0
5.	0	0	0	0	0	0	0	0	0	0
6.	0	0	0	0	0	0	0	0	0	0
7.	0	0	0	0	0	0	0	0	0	8
8.	5	4	9	0	0	0	0	0	0	0
9.	0	0	0	15	14	12	12	13	12	0
10.	0	0	0	0	0	0	2	0	0	0
11.	0	0	0	0	0	0	0	0	0	0
12.	12	16	14	27	22	26	22	25	24	18
13.	26	24	8	0	0	0	0	0	0	18
14.	0	0	13	0	0	0	0	0	0	3
15.	0	0	0	27	23	25	28	32	24	14
16.	12	21	9	0	0	0	0	0	0	0
17.	0	0	0	0	22	5	0	3	9	0
18.	0	4	0	20	0	10	15	19	15	16
19.	27	20	3	0	0	0	0	0	0	0
20.	0	0	0	13	20	20	18	26	21	0
21.	15	26	0	0	0	0	0	0	0	16
Total / replicate	97	115	56	102	101	98	97	118	105	93
Mean					98.2
SD					16.84
CV					17.15

 

The total number of offspring produced per parent animal

Replicates			Concentration [mg/L]
	Control	0.36	0.82	1.9	4.3	10
1	90	81	58	115	120	97
2	82	–	–	109	86	115
3	76	68	–	43	108	56
4	94	80	87	44	94	102
5	81	84	85	37	116	101
6	59	64	72	118	92	98
7	84	58	73	83	83	97
8	83	60	58	–	104	118
9	90	57	59	97	–	105
10	72	77	86	95	–	93
Mean	81.1	69.9	72.3	82.3	100.4	98.2
SD	10.19	10.72	12.83	32.59	13.73	16.84
CV	12.57	15.33	17.75	39.59	13.68	17.15
% Reduction		13.8	10.9	-1.5	-23.8	-21.1

-: no data (due to parent mortality); mortality of parent animal was considered to be inadvertent mortality (i.e. non substance related mortality with not known cause), therefore this replicate was excluded from data analysis

 

The mortality of parent animals and the day on which mortality occurred 

Replicates			Concentration [mg/L]
	Control	0.36	0.82	1.9	4.3	10
1	0	0	0	0	0	0
2	0	1 (day 17)	1 (day 12)	0	0	0
3	0	0	1 (day 12)	0	0	0
4	0	0	0	0	0	0
5	0	0	0	0	0	0
6	0	0	0	0	0	0
7	0	0	0	0	0	0
8	0	0	0	1 (day 20)	0	0
9	0	0	0	0	1 (day 12)	0
10	0	0	0	0	1 (day 12)	0
Total mortality	0	1	2	1	2	0
% mortality	0.0	10.0	20.0	10.0	20.0	0.0

The body length [mm] of parent animals measured at the end of the test 

Replicates			Concentration [mg/L]
	Control	0.36	0.82	1.9	4.3	10
1	4.0	3.9	4.0	4.0	4.0	4.0
2	4.0	–	–	4.1	3.9	4.0
3	4.0	4.0	–	3.9	4.0	4.0
4	4.1	3.8	3.9	3.9	4.0	4.1
5	4.0	4.0	3.8	3.9	4.0	4.0
6	4.0	4.0	4.0	4.0	3.9	4.0
7	4.1	4.0	3.9	4.0	3.9	4.1
8	4.0	4.0	4.0	–	4.1	4.0
9	4.0	3.8	3.8	4.0	–	4.0
10	4.0	4.0	3.9	4.0	–	4.1
Mean	4.02	3.94	3.91	3.98	3.98	4.03
SD	0.04	0.09	0.08	0.07	0.07	0.05
CV	1.05	2.24	2.13	1.68	1.78	1.20
% Reduction		1.9	2.7	1.1	1.1	-0.2

-: no data (due to parent mortality); mortality of parent animal was considered to be inadvertent mortality (i.e. non substance related mortality with not known cause), therefore this replicate was excluded from data analysis

 

Time to production of first brood per parent animal [day]

Replicates			Concentration [mg/L]
	Control	0.36	0.82	1.9	4.3	10
1	7	7	9	7	8	8
2	7	7	7	7	8	8
3	7	9	7	8	8	8
4	9	7	7	8	8	9
5	7	7	7	14	8	9
6	9	7	9	7	8	9
7	7	8	9	8	8	9
8	7	7	7	8	9	9
9	9	7	8	8	8	9
10	7	12	9	9	8	7
Mean	7.6	7.8	7.9	8.4	8.1	8.5
SD	0.97	1.62	0.99	2.07	0.32	0.71
CV	12.71	20.76	12.59	24.59	3.90	8.32

Note: The statistical software use “Age of First Reproduction” (see "illustration") instead of “Time to production of first brood” (which is indicated in OECD TG 211 [2]) and set the age of parents arbitrarily as half day at the start of exposure. This causes a half day difference in mean values of time to production of first brood (Table 12 above) and age of first reproduction (Table indicated in "illustration"). However, this difference has no influence on comparisons of treatment groups with control and on the output of the study.

 

 

Validity criteria fulfilled:

yes

Conclusions:

In this 21-day chronic toxicity test with Daphnia magna, the obtained results showed that the test item had no effect on Daphnia magna up to at least the highest tested concentration of 10 mg/L (i.e. the maximum required test concentration for chronic effects testing). Accordingly, the test item 1-ethoxy-2-(2-methoxyethoxy)ethane had no chronic effects on Daphnia magna up to at least the limit concentration of 10 mg/L.

All validity criteria were met, therefore the study was considered to be valid.

The biological endpoints are based on the nominal concentrations and are listed below:

21-day NOEC (for reproduction): 10 mg/L
21-day LOEC (for reproduction): > 10 mg/L

21-day EC10 (for reproduction): > 10 mg/L
21-day EC20 (for reproduction): > 10 mg/L
21-day EC50 (for reproduction): > 10 mg/L


21-day NOEC (for mortality): 10 mg/L
21-day LOEC (for mortality): > 10 mg/L

Executive summary:

Test design:
Test item:	1-ethoxy-2-(2-methoxyethoxy)ethane (Lot No.: 09511)
Test species:	Daphnia magna (Straus)
Dilution water:	Elendt M4 medium; prepared in the laboratory of TOXI-COOP ZRT.
Concentrations:	Nominal concentrations of 0.36, 0.82, 1.9, 4.3 and 10 mg/L were investigated in the main study. Concurrent control ran in parallel.
Test design:	Young female Daphnia (the parent animals) aged less than 24 hours at the start of the test were exposed to aqueous test media containing the test item at a range of concentration for 21 days in a semi-static test. Fully characterized (content, physico-chemical parameters) Elendt M4 Medium was used as dilution water. Ten Daphnia were investigated in each test group. The test animals were held individually (ten replicates/treatment). Each test vessel contained approximately 80 mL test medium. The Daphnia were fed daily, with concentrated algal cell suspension. The living offspring produced by each parent animal were removed and counted daily from the appearance of the first brood. The mortality of parent animals was recorded daily. Body length measurements of parents were performed at the end of the test.
Analytics:	For determination of the test item concentrations, samples were taken from each test concentration and from the control when freshly prepared and at renewal on one occasion weekly during the test. Concentrations were determined by GC-MS method.
Endpoints:	21-day EC10, EC20, EC50, NOEC and LOEC for reproduction; 21-day NOEC and LOEC for mortality.
Statistics:	The statistical analysis was performed by ToxRat Professional (Version 3.3.0) software.

To determine NOEC/LOEC values for reproduction Multiple Sequentiallyrejective Welsh-t-test after Bonferroni-Holm was used. To determine the EC_x no statistical procedure was necessary. 

Mortality results was evaluated using Fisher’s Exact Binominal test with Bonferroni Correction. 

Comparison of body length of parents was performed using Dunnett`s Multiple ttest Procedure.

Comparison of time to production of first brood was performed using Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm.

 

Results:                     

             

Validity: All validity criteria were met and therefore the study was considered to be valid (see section 8.1)

             

Analytical results: Based on the results of analytical measurements the concentrations of test item has been maintained within ± 20 % of the nominal during the experiment. 

             

Biological results: Biological results and endpoints are based on the nominal concentrations.

                                 Reproduction:

No statistically significant reduction in reproduction was observed in any of the test concentrations compared to the control. 

The 21-day NOEC for reproduction was determined to be 10 mg/L and the LOEC as > 10 mg/L. 

The 21-day EC₅₀, EC₂₀ and EC₁₀ values for reproduction were determined to be > 10 mg/L.

Mortality of parents:

No statistically significant mortality was noticed in any of the test concentrations compared to the control.

The 21-day NOEC for mortality was determined to be 10 mg/L and the LOEC as > 10 mg/L. 

Body length of parents:

No concentration-response pattern was observed in reduction of parent body length compared to the control.

Other parameters:

No aborted broods, difference in size of broods, presence of male neonates or ephippia were noted in the control or in any of the test concentrations during the test period.

No statistically significant different increase in time to production of first brood was observed in any of the test concentrations compared to the control.

 

 In conclusion: In this 21-day chronic toxicity test with Daphnia magna, the obtained results showed that the test item had no effect on Daphnia magna up to at least the highest tested concentration of 10 mg/L (i.e. the maximum required test concentration for chronic effects testing). Accordingly, the test item 1-ethoxy-2-(2-methoxyethoxy)ethane had no chronic effects on Daphnia magna up to at least the limit concentration of 10 mg/L.

The biological endpoints are based on the nominal concentrations and are listed below:

 

 

21-day NOEC (for reproduction):	10 mg/L
21-day LOEC (for reproduction):	> 10 mg/L
21-day EC10 (for reproduction):	> 10 mg/L
21-day EC20 (for reproduction):	> 10 mg/L
21-day EC50 (for reproduction):	> 10 mg/L
21-day NOEC (for mortality):	10 mg/L
21-day LOEC (for mortality):	> 10 mg/L

 

Description of key information

In this 21-day chronic toxicity test with Daphnia magna, the obtained results showed that the test item had no effect on Daphnia magna up to at least the highest tested concentration of 10 mg/L (i.e. the maximum required test concentration for chronic effects testing). Accordingly, the test item 1-ethoxy-2-(2-methoxyethoxy)ethane had no chronic effects on Daphnia magna up to at least the limit concentration of 10 mg/L.

The biological endpoints are based on the nominal concentrations and are listed below:

 

 

21-day NOEC (for reproduction):	10 mg/L
21-day LOEC (for reproduction):	> 10 mg/L
21-day EC10 (for reproduction):	> 10 mg/L
21-day EC20 (for reproduction):	> 10 mg/L
21-day EC50 (for reproduction):	> 10 mg/L
21-day NOEC (for mortality):	10 mg/L
21-day LOEC (for mortality):	> 10 mg/L

 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

10 mg/L

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

> 10 mg/L