Bis(2-butoxyethyl) ether

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-04-30 to 2012-05-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP and no defiencies

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-butoxyethyl) ether

EC Number:

204-001-9

EC Name:

Bis(2-butoxyethyl) ether

Cas Number:

112-73-2

Molecular formula:

C12H26O3

IUPAC Name:

1-[2-(2-butoxyethoxy)ethoxy]butane

Details on test material:

- Name of test material (as cited in study report): Diethyleneglycoldibutylether
- Physical state: Colourless liquid
- Water solubility: 2.8 g/L (20°C)
- Density: 0.883 g/cm3
- pH: 6.5 - 7.5 (20°C, 100 g/L in isopropanol : water 4 : 1)
- Stability under test conditions: Not specified
- Storage condition of test material: Room temperature, protected from light, in original container

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The concentrations of the test item were analytically determined from all test concentrations and the control after 0 hours (test start) and 72 hours (test end). Samples at test start were taken from an additional replicate without algae. At end of the test samples were taken directly from the pooled test replicates.
All original samples were stored at 6 ± 2 °C until sample preparation, if necessary. Prepared samples were stored at room temperature until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

APPLICATION
- Method: Stock solution 100 mg/L, freshly prepared with dilution water.
Dispersion treatment Agitation
- Eluate: Dilution water
- Differential loading: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Dilution water: 0.24 mmol Ca+mg/L.

Test temperature:

min.: 22.0 max.: 23.0, mean value: 22.5

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
100 7.78 7.84
31.6 7.82 8.00
10.0 7.80 8.20
3.16 7.78 8.22
1.00 7.82 7.89
Control 7.80 7.84

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

See window "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 103 cells/mL, Actual: mean 4910 cells/mL
- Control end cells density: Mean 1713179 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6



GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1, mean value: 68.3



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS
- Range finding study:Yes
Results of the Range Finding Test (0-72 h)
Nominal Test Item Concentration
[mg/L] Specific Growth Rate Inhibition Yield Inhibition
[%] [%]
2800 100 100
1400 100 100
700 100 100
350 100 100
100 82 99
10.0 4 20
1.00 0 1

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

41.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 39.5-43.6 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

16.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 14.4-17.8 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
EC50 Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.540 0.686 ± 0.470
95 % confidence interval 0.528 - 0.551
Yield Inhibition
EyC50 0.324 0.319 ± 0.140
95 % confidence interval 0.304 - 0.347

Reported statistics and error estimates:

NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s Test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

Any other information on results incl. tables

 Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours	48 hours	72 hours
100		1		4910		9713	9151	7902
		2		4910		11317	8762	7959
		3		4910		11534	9357	7262
		Mean		4910		10855	9090	7708
31.6		1		4910		17156	48881	208842
		2		4910		18636	53664	218449
		3		4910		20304	66906	242658
		Mean		4910		18699	56484	223316
10.0		1		4910		35628	237073	1272260
		2		4910		34109	209472	1409082
		3		4910		32589	229579	1576030
		Mean		4910		34109	225375	1419124
3.16		1		4910		29207	244276	1687803
		2		4910		27213	245806	1810001
		3		4910		27230	272501	1897402
		Mean		4910		27883	254194	1798402
1.00		1		4910		31150	260755	1719013
		2		4910		29684	239605	1845408
		3		4910		32734	244960	1750493
		Mean		4910		31189	248440	1771638
Control		1		4910		37456	274159	1705743
		2		4910		32883	247052	1561610
		3		4910		34714	239646	1877634
		4		4910		37233	324542	1812709
		5		4910		30930	250200	1693557
		6		4910		32352	241839	1627821
		Mean		4910		34261	262906	1713179

Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to

control values are marked (+), not significant differences are marked (-).

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		0.159	91.9		2992	99.8
	2		0.161	91.8		3049	99.8
	3		0.130	93.3		2352	99.9
	Mean	(+)	0.150	92.3	(+)	2798	99.8
31.6	1		1.25	35.9		203932	88.1
	2		1.27	35.2		213539	87.5
	3		1.30	33.4		237748	86.1
	Mean	(+)	1.27	34.8	(+)	218406	87.2
10.0	1		1.85	5.05		1267350	25.8
	2		1.89	3.31		1404172	17.8
	3		1.92	1.39		1571120	8.03
	Mean	(+)^1	1.89	3.25	(+)	1414214	17.2
3.16	1		1.95	0.22		1682893	1.49
	2		1.97	-0.97		1805091	-5.67
	3		1.99	-1.78		1892492	-10.8
	Mean	(-)	1.97	-0.84	(-)	1793492	-4.99
1.00	1		1.95	-0.09		1714103	-0.34
	2		1.98	-1.3		1840498	-7.74
	3		1.96	-0.4		1745583	-2.18
	Mean	(-)	1.96	-0.60	(-)	1766728	-3.42
Control	1		1.95			1700833
	2		1.92			1556700
	3		1.98			1872724
	4		1.97			1807799
	5		1.95			1688647
	6		1.94			1622911
	Mean		1.95			1708269

Section-by-Section and Average Specific Growth Rates of the Control Group

(0 – 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	2.03	1.99	1.83	1.95	0.108	5.53	5.86
	2	1.90	2.02	1.84	1.92	0.0879	4.58
	3	1.956	1.93	2.06	1.98	0.0673	3.39
	4	2.03	2.17	1.72	1.97	0.228	11.6
	5	1.84	2.09	1.91	1.95	0.129	6.61
	6	1.89	2.01	1.91	1.94	0.0675	3.49
				Mean	1.95
				SD ±	0.02
				CV [%]	1.16

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study Diethyleneglycoldibutylether was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-values for inhibition of specific growth rate and inhibition of yield after 72 hours were 10.0 mg/L and 3.16 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 hours were 31.6 mg/L and 10.0 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 hours were 41.3 (39.5 – 43.6) and 16.4 (14.6 – 18.8) mg/L, respectively.
All effect levels are given based on nominal concentrations of Diethyleneglycoldibutylether.

Executive summary:

The toxicity of Diethyleneglycoldibutylether (batch number: DEG4136740) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from 2012-04-30 to 2012-05-03. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 4910 cells/mL. Based on a preliminary test 5 concentration levels were tested in a geometrical series with a nominal dilution factor of : 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of Diethyleneglycoldibutyletherwere analytically verified by GC-MS at test start and test end of all test concentrations and the control. The measured concentrations of Diethyleneglycoldibutyletherat test start were in the range of 102 to 115 % and after 72 hours in the range of 89 to 112 % of the nominal values. Therefore, all effect values given are based on the nominal test item concentrations of the test item Diethyleneglycoldibutylether.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Diethyleneglycoldibutylether
(0-72 hours)

               based on nominal test item concentrations [mg/L]

                               

	Growth Rate Inhibition
NOEC	10.0
LOEC	31.6
ErC10	16.1 (14.4 – 17.8)
ErC20	22.8 (21.4 – 23.9)
ErC50	41.3 (39.5 – 43.6)
	Inhibition of Yield
NOEC	3.16
LOEC	10.0
EyC10	8.48 (7.25 – 9.83)
EyC20	10.6 (9.37 – 12.0)
EyC50	16.4 (14.6 – 18.8)