Bis(2-(2-methoxyethoxy)ethyl) ether

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2010-01-13 to 2010-02-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to OECD guideline and GLP.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-methoxyethyl) ether

EC Number:

203-924-4

EC Name:

Bis(2-methoxyethyl) ether

Cas Number:

111-96-6

IUPAC Name:

1-methoxy-2-(2-methoxyethoxy)ethane

Constituent 2

Reference substance name:

Diglyme

IUPAC Name:

Diglyme

Details on test material:

- Name of test material (as cited in study report): Diethylenglykoldimethylether
- Molecular formula: C6H14O3
- Molecular weight: 134.18 g/mol
- Physical state: Liquid, colourless
- Analytical purity: 99.9 % (Bis(2-methoxyethyl)ether)
- Storage condition of test material: Room temperature, protected from moisture and light.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: In the first week analyses of all concentration levels of the testitem and the control was carried out for a 48 hour and 72 hour exposure interval. Thereafter, all concentrations and the control were analytically verified in the fresh media (0 h) and old media (48 or 72 h) via GC-MS at least once every 7days
- Sampling method: For the fresh media samples of the freshly prepared test solutions were taken and analysed. For the old media samples without test organisms were taken and stored in a closed 4 mL vial without headspace under test conditions until analysis (after 48 or 72 h). The method was validated according to SANCO 3029/99 rev.4 (11/07/00).
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until start of analysis, if necessary.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1000 mg/L was freshly prepared with dilution water in a measuring flask by directly pipetting of 1.06 mL test item/L.
- Eluate: Dilution water
- Differential loading: 10.0 - 32.0 - 100 - 320 - 1000 mg/L
- Controls: 10 replicates of dilution water without test item.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Clone 5
- Justification for species other than prescribed by test guideline: Daphnia magna STRAUS is recommended in the guideline
- Source: Own breeding (Origin: Institut fuer Wasser- Boden- und Lufthygiene)
- Age of parental stock (mean and range, SD): > 14 days
- Feeding during test
- Food type: Mix of Pseudokirchneriella subcapitata and Desmodesmus subspicatus
- Amount: 0.2 mg C/daphnia per day
- Frequency: daily


ACCLIMATION
- Acclimation period: 2 h in dilution water
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: During acclimation the daphnids were not fed
- Feeding frequency: None
- Health during acclimation (any mortality observed): Healthy

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: The parent animals were removed from the culture medium and the juveniles collected over a sieve and flushed into fresh medium

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not observed

Test conditions

Hardness:

Total Hardness [mg/L] as CaCO3
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Jan. 13th Jan. 15th Jan. 20th Jan. 22nd Jan. 27th Jan. 29th
1000 178 172 167 182 174 178
Control 180 170 164 189 180 175

Test temperature:

Temperature [°C]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Jan. 13th Jan. 15th Jan. 20th Jan. 22nd Jan. 27th Jan. 29th
1000 21.0 21.2 19.8 20.6 20.3 20.8
Control 20.0 21.5 19.1 20.6 19.3 21.0

pH:

pH-Values
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Jan. 13th Jan. 15th Jan. 20th Jan. 22nd Jan. 27th Jan. 29th
1000 7.38 7.42 7.86 7.39 7.50 7.17
Control 7.92 7.47 7.93 7.14 7.87 7.18

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Jan. 13th Jan. 15th Jan. 20th Jan. 22nd Jan. 27th Jan. 29th
1000 8.42 7.35 8.41 7.17 8.66 4.48
Control 8.36 6.57 8.49 7.29 9.17 6.07

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to Any other information on materials and methods

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: 100 mL glass beaker, filled with 50 mL test solution
- Aeration: No
- Renewal rate of test solution (frequency): 3 x per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.



OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark per day
- Light intensity: Max. 20 µE*m-2*s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Adult mortality: daily
- Number of juveniles: daily
- Stillborn juveniles and aborted eggs: daily
- Appearance of first brood
- Intrinsic rate of natural increase: test end
- Growth (total length and dry weight): test end


RANGE-FINDING STUDY

Immobilization Rates of the GLP Acute Immobilization Test with
Diethylenglykoldimethylether
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Test Item
Concentration IMMOBILIZATION [%]
24 h 48 h
Replicates Replicates
[mg/L] 1. 2. 3. 4. MV 1. 2. 3. 4. MV
2000 100 100 100 100 100 100 100 100 100 100
1000 0 0 0 0 0 80 80 100 20 70
500 0 0 0 0 0 0 0 0 0 0
250 0 0 0 0 0 0 0 0 0 0
125 0 0 0 0 0 0 0 0 0 0
Control 0 0 0 0 0 0 0 0 0 0
NOEC (48 h) 500 mg/L
LOEC (48 h) 1000 mg/L
EC50 (48 h)
with 95 % confidence limits (Cl) 943 mg/L (Cl: 899 - 989 mg/L)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality of parent animals:
The test item induced biologically significant adult mortality of 50 % at the concentration level of 1000 mg/L. At the tested concentration levels of 10.0 to 320 mg/L and the control no biologically significant adult mortality was observed within the exposure phase of 21 days. At the control all parental daphnids survived till the end of the test after 21 days. At the concentration level 10.0 mg/L an adult mortality of 10 % and at the concentration levels 32.0 to 320 mg/L an adult mortality of 20 % was observed, which is not biologically significant.
The EC50-value for the adult mortality was estimated directly from the observation data to be ≥ 1000 mg/L, because effects > 50 % did not occur within the tested concentration range.

- No. of offspring produced per day per female:
The first brood was released till day 9 by all parent animals of the control and the concentration levels of 10.0 to 320 mg/L. Four to five broods were observed at the concentration levels 10.0 to 320 mg/L during the course of the study. At the concentration level 1000 mg/L one parental daphnid released its first brood biologically significantly delayed on day 18. Release of the first brood after day 9 is regarded to be too late and biologically significant. As a result of that only two broods were observed at the daphnid concerned.
The average number of juveniles per parent alive at the end of the test in the control group was 116 after 21 days. A statistically significant reduction of the reproductive output in comparison to the control was determined at the nominal test concentration of 1000 mg/L (45 %). The reduction of the reproductive output in the range of 5 to 20 % in comparison to the control group at the other concentration levels of 10.0 to 320 mg/L was not statistically or biologically significant (One Way Analysis of Variance, DUNNETT’S method, p = 0.05).


- Body length and weight of parent animals:
The mean values of the body length of the surviving parental daphnids at the test groups in the range of 4.65 to 5.09 mm per daphnid were comparable to the control group with 5.23 mm per daphnid. The total body length of the surviving daphnids of the treatment groups were compared to the control by KRUSKAL-WALLIS One Way Analysis of Variance on Ranks (p = 0.05), because equal variance test failed.
The mean dry body weight was determined to be in the range of 0.54 to 0.75 mg per daphnid at the test groups and 0.77 mg per daphnid at the control, respectively.

- Type and number of morphological abnormalities: None
- Type and number of behavioural abnormalities: None
- Number of males and females (parental): No males occurred

- Time to first brood release or time to hatch: The first brood was released till day 9 by all parent animals of the control and the concentration levels of 10.0 to 320 mg/L. Four to five broods were observed at the concentration levels 10.0 to 320 mg/L during the course of the study.
At the concentration level 1000 mg/L one of four parental daphnids released its first brood biologically significantly delayed on day 18. Release of the first brood after day 9 is regarded to be too late and biologically significant. As a result of that only two broods were observed at the daphnid concerned

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.20 (CI 1.08 - 1.34)

Reported statistics and error estimates:

The NOEC and the LOEC for the reproductive output as the most sensitive effect were determined directly from the observation data. Significant deviations were determined in comparison to control using statistical standard procedures as Normality Test, Equal Variance Test and One Way Analysis of Variance (ANOVA).
Prior to running a One Way Analysis of Variance a Normality Test and an Equal Variance Test were performed. P-values for both Normality and Equal Variance Test were 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) was a=0.05.
For the determination of significant deviations for the reproduction rates One Way Analysis of Variance, DUNNETT’S method (p = 0.05) was used. Statistical evaluation of the intrinsic rates of natural increase was carried out using KRUSKAL-WALLIS, One Way Analysis of Variance, DUNN’S method (p = 0.05), because normality test failed. Statistical evaluation of the total body length was carried out using KRUSKAL-WALLIS, One Way Analysis of Variance (p = 0.05), because equal variance test failed. The coefficients of variation around the mean number of living offspring produced per parent in the control and the test groups were evaluated.

Any other information on results incl. tables

Number of Juveniles in the Control and Test Groups after 21 Days

Nominal Test Item Conc. [mg/L]												No. of Parents prod. Juv.	Mean No. of Juveniles per Parent producing Juveniles			Comparison versus Control
	Number of Juveniles in Replicate No.										Total No.				CV
																Red.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD		[%]	[%]
1000	80	--	3	--	79	--	--	91	66	--	319	5	64 ±	35	55*	45	yes
320	128	112	104	103	83	101	--	93	--	107	831	8	104 ±	13	13	10	no
100	--	70	99	94	71	115	77	105	--	113	744	8	93 ±	18	20	20	no
32.0	120	--	86	114	132	--	118	91	104	114	879	8	110 ±	15	14	5	no
10.0	98	99	118	90	80	--	104	100	99	95	883	9	98 ±	10	10	15	no
Control	153	95	97	123	140	124	124	103	97	104	1160	10	116 ±	20	17	—	—

First Appearance of Living Juveniles in the Individual Groups

Nominal Test Item Concentration [mg/L]	Day of First Appearance of Living Juveniles										First
	in Replicate No.										Appearance
	1	2	3	4	5	6	7	8	9	10	Mean Day
1000	8	--	18*	--	7	--	--	8	8	--	9.8
320	8	8	8	8	8	8	--	8	--	8	8.0
100	--	8	8	8	8	8	8	8	--	8	8.0
32.0	8	--	8	8	8	--	8	8	8	8	8.0
10.0	8	9	8	8	8	--	8	8	8	8	8.1
Control	8	8	8	8	8	8	8	8	8	8	8.0

Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure

                 (n = 10)         

Nominal Test Item Concentration [mg/L]	Adult Mortality [%]
	7 days	14 days	21 days
1000	0	40*	50*
320	0	20	20
100	0	20	20
32.0	0	0	20
10.0	0	0	10
Control	0	0	0

Total Body Length of the Parental Daphnids

Nominal Test Item Concentration [mg/L]	Total Length of the Parent Animals [mm]										MV	N	Dry Weight [mg]
	Replicate No.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
1000	4.75	--	3.75	--	4.75	--	--	5.25	4.75	--	4.65	5	3.5	0.70
320	5.50	5.25	5.00	5.00	5.00	5.00	--	5.00	--	5.00	5.09	8	6.0	0.75
100	--	4.50	5.25	4.75	4.75	5.00	5.00	5.25	--	5.25	4.97	8	4.9	0.61
32.0	5.25	5.00	--	5.00	4.75	--	5.50	4.75	5.00	5.00	5.03	8	5.0	0.63
10.0	5.00	5.00	5.25	4.75	5.00	--	5.25	5.00	5.00	5.00	5.03	9	4.9	0.54
Control	5.50	5.50	5.00	5.50	5.50	5.00	5.25	5.00	4.75	5.25	5.23	10	7.7	0.77

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The Lowest Observed Effect Concentration (LOECReproduction) and the No Observed Effect Concentration (NOECReproduction) after 21 days based on the reduction of the reproductive output and the intrinsic rates of natural increase as the most sensitive effects in this study were determined directly from the observed reduction of the reproductive output. The EC50-values were estimated directly from the observation data to be > 1000 mg/L for the reduction of the reproductive output and ≥ 1000 mg/L for the adult mortality, because effects > 50 % did not occur within the tested concentration range.

All effect values given are based on the nominal concentrations of Diethylenglykoldimethylether.

Endpoints assessed

Diethylenglykoldimethylether
Nominal Test Item Concentration
[mg/L]
NOECReproduction 320
LOECReproduction 1000
EC50, Reproduction > 1000
EC50, Adult Mortality ≥ 1000

Executive summary:

The Daphnia magna Reproduction Test (Semi-Static, 21 d) of the test item Diethylenglykoldimethylether(batch No. DEG4071791) was conducted according to OECD 211 (2008) from 2010-01-13 to 2010-02-04 with the definitive exposure phase between 2010-01-13 to 2010-02-03 at Dr.U.Noack-Laboratorien, Käthe-Paulus-str. 1, D-31157Sarstedt, Germany .

Test system was Daphnia magna STRAUS (Clone 5). 10 test organisms, individually held, were used per concentration level and control. At test start they were 2 to 24 h old. The study was carried out undersemi-static conditions with a three times per week renewal of the test solutions. Aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects or parameters such as time of production of intrinsic rate of natural increase, first brood, adult mortality, occurrence of aborted eggs and stillborn juveniles and body length of the parental daphnids.

Nominal concentrations of Diethylenglykoldimethylether were selected as follows: 10.0 - 32.0 - 100 - 320 - 1000 mg/L.

The tested concentrations levels of the test item Diethylenglykoldimethylether and the control were analytically verified via GC-MS of samples on days 0, 2, 7, 14 (fresh media, 0 h) and on days 2, 9, 16 (old media, 48 h) and 5 (old media, 72 h). Details of the analytical method are presented in part 11. The analytical results are presented in part 4.2.2. The recovery rates in the fresh (0 h) and old media (48 or 72 h) were determined to be in the range ± 20 % of the nominal values at the biologically relevant test concentrations of 320 mg/L (NOEC) and 1000 mg/L (LOEC). Therefore, all effect values given are based on the nominal test item concentrations.

·   The average number of juveniles per parent alive at the end of the test in the control group was 116 after 21 days. A statistically significant reduction of the reproductive output in comparison to the control was determined at the nominal test concentration 1000 mg/L (45 %). The reduction of the reproductive output in the range of 5 to 20 % in comparison to the control group at the other concentration levels of 10.0 to 320 mg/L was not statistically or biologically significant (One Way Analysis of Variance, Dunnett’s method, p = 0.05). The NOEC was assessed to be 320 mg/L (the highest test concentration with no significant reduction of the reproductive output). The EC₅₀-value for the reduction of the reproductive output was estimated directly from the observation data to be > 1000 mg/L, because effects > 50 % did not occur within the tested concentration range. Details are presented in Table 1, Table 4, Figure 1 and Figure 2.

·   The coefficient of variation of the number of living offspring produced per parent was 17 % at the control. At the test groups the coefficient of variation came to a biologically significant maximum of 55 % at the concentration level 1000 mg/L. Coefficients of variation > 25 % are regarded to be biologically significant (Table 1 and Table 4).

·   The Lowest Observed Effect Concentration (LOEC_Reproduction) and the No Observed Effect Concentration (NOEC_Reproduction) after 21 days based on the reduction of the reproductive output and the intrinsic rates of natural increase as the most sensitive effects in this study are summarized in Table 1.

The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The mean IR of the surviving daphnids of the treatment groups were compared to the control by Kruskal-Wallis One Way Analysis of Variance on Ranks, Dunn’smethod (p = 0.05), because normality test failed. There was a statistically significant difference at the tested concentration level 1000 mg/L when compared to the control. At the other concentration levels 10.0 to 320 mg/L no statistically significant difference in comparison to the control was determined.

·   The first brood was released till day 9 by all parent animals of the control and the concentration levels of 10.0 to 320 mg/L. Four to five broods were observed at the concentration levels 10.0 to 320 mg/L and control during the course of the study.
At the concentration level 1000 mg/L one parental daphnid released its first brood biologically significantly delayed on day 18. Release of the first brood after day 9 is regarded to be too late and biologically significant. As a result of that only two broods were observed at the daphnid concerned (Table 8).

·   No stillborn juvenilesand aborted eggs were produced by the control group. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was biologically not significant at the tested concentration levels (Table 7).

Table 1:        Test Item Related Effects on Reproduction (NOEC, LOEC, EC₅₀)

                      (based on the nominal test item concentrations)

Effects	Diethylenglykoldimethylether Nominal Test Item Concentration [mg/L]
	Control	10.0	32.0	100	320	1000
Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)	116 ± 20	98 ± 10	110 ± 15	93 ± 18	104 ± 13	64 ± 35^1)
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent [%]	17	10	14	20	13	55^2)
Mean Intrinsic Rates of Natural Increase	0.48	0.46	0.48	0.46	0.48	0.39^3)
NOECReproduction	320 mg/L
LOECReproduction	1000 mg/L
EC50, Reproduction	> 1000 mg/L

A summary of test item related effects concerning the adult mortality and growth (dry weight, body length) is presented in Table 2.

·   The test item induced biologically significant adult mortality of 50 % at the concentration level of 1000 mg/L. At the tested concentration levels of 10.0 to 320 mg/L and the control no biologically significant adult mortality was observed within the exposure phase of 21 days. At the control all parental daphnids survived till the end of the test after 21 days. At the concentration level 10.0 mg/L an adult mortality of 10 % and at the concentration levels 32.0 to 320 mg/L an adult mortality of 20 % was observed, which is not biologically significant. The EC₅₀-value for the adult mortality was estimated directly from the observation data to be ≥ 1000 mg/L, because effects > 50 % did not occur within the tested concentration range of 10.0 to 1000 mg/L. Details are presented in Table 2, Table 9 and Figure 4.

·   The mean values of the body length of the surviving parental daphnids at the test groups in the range of 4.65 to 5.09 mm per daphnid were comparable to the control group with 5.23 mm per daphnid. The total body length of the surviving daphnids of the treatment groups were compared to the control by Kruskal-Wallis One Way Analysis of Variance on Ranks (p = 0.05), because equal variance test failed.
The mean dry body weight was determined to be in the range of 0.54 to 0.75 mg per daphnid at the test groups and 0.77 mg per daphnid at the control, respectively (Table 2 and Table 10).

Table 2:    Test Item Related Effects on Adult Mortality and Growth

Effects	Diethylenglykoldimethylether Nominal Test Item Concentration [mg/L]
	Control	10.0	32.0	100	320	1000
Adult Mortality after 21 Days [%]	0	10	20	20	20	50^1)
Parent Animals: Mean Body Length [mm]	5.23	5.03	5.03	4.97	5.09	4.65
Parent Animals: Mean Dry Weight [mg]	0.77	0.54	0.63	0.61	0.75	0.70

¹⁾    = Adult mortality > 20 % biologically significant

 

·       No males and ephippia (winter eggs) were observed at the control or test groups.

 

·   Water quality parameters as pH-value, dissolved oxygen, water hardness and temperature were determined to be within the acceptable limits.

·          In order to prove the validity of the test system and test conditions at the test facility, an acute immobilization test according to DIN 38412 L 11 is carried out with potassium dichromateas reference item once per month.
The EC₅₀ of the reference item at 1.20 mg/L after 24 h is within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS P 9/2 (05/1996) 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.