Bis(2-chloroethyl) ether

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06-nov-2016 to 24-feb-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TEST MATERIAL
- Names (as cited): 2,2`- Dichlorodiethyl ether
- Purity: 99.76%

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch n°20150706
- Expiration date of the lot/batch: 01 June 2016
- Purity test date: 28 August 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled Room Temperature (15-25°C, below 70 RH%) Protected from light

Analytical monitoring:

yes

Details on sampling:

- Concentrations: single concentration (100 mg/L nominal)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Vehicle: algal growth medium (OECD Medium)
- Concentration of vehicle in the final test medium: 100 mg/L (nominal), 79.44 mg/L (measured)

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: 61.81 SAG (synonyms: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY
- Method of cultivation: Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.
- Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22.8 - 23.1°C

pH:

7.45 - 8.91

Nominal and measured concentrations:

Nominal concentration: 100 mg/L
Measured concentration (mean 0-72h): 79.44 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type: open (air-permeable stoppers)
- Fill volume: 100 mL
- Aeration: through diffusion
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 74.3E+04
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 8145 - 8249 lux (equivalent to ~110 - 111 µE/m2/s); cool white fluorescent tubes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: limit test

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 79.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

79.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 79.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities observed
- Unusual cell shape: no
- Any stimulation of growth found in any treatment: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
Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions. The date of the last study (Study Code: 15/428-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 27-30 October 2015.

The 72h ErC 50 : 0.84 mg/L, (95 % confidence limits: 0.77 – 0.92 mg/L)
The 72h EbC 50 : 0.59 mg/L, (95 % confidence limits: 0.54 – 0.65 mg/L)
The 72h EyC 50 : 0.55 mg/L, (95 % confidence limits: 0.50 – 0.60 mg/L)

These values are within the range of laboratory ring test data (see ISO Guideline No. 8692)

RANGE FINDING TEST:

The concentration levels used and results (72 h) of the preliminary range-finding test are summarised in the following table:

Nominal concentration (mg/L)	unteated control	0.1	1	10	100
Average cell number at 72h (10^4 cell/mL)	75.0	73.5	74	72	71.5

FINAL TEST:

-Validity:

The cell density in the control culture increased by the factor of 74.33 within three days.

The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 13.70 %.

The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.47 %.

All validity criteria were met, therefore the study can be considered as valid.

- Cell counts:

The cell number in each flask was determined at the 24^th, 48^th, 72^ndhours. The results are shown in the following table:

Concentration (mg/L)		Number of cells (x 10^4 cell/mL)
Nominal	Measured (geom. mean)		0 h	24 h	48 h	72 h
Control	-		1	4	22	76
			1	5	23	75
			1	4	18	72
			1	4	23	73
			1	4	21	75
			1	4	20	75
		Mean	1.00	4.17	21.17	74.33
		SD	0.0	0.4	1.9	1.5
100	79.44		1	4	22	72
			1	5	22	76
			1	4	21	70
			1	4	18	75
			1	4	18	76
			1	5	18	74
		Mean	1.00	4.33	19.83	73.83
		SD	0.0	0.5	2.0	2.4

- Morphological deviations of algal cells:

There were no morphological deviations of the algal cells. All cells observed to be normal both in the control and in the applied concentrations.

- Average specific growth rate:

The results of the statistical evaluation (based on 2 Sample t-Test; a=0.05) show that the 0-72 h average specific growth rate was not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 79.44 mg/L (measured). Growth rate dat are detailed in the following table:

Concentration (mg/l)			Growth rate (µ) and inhibition of µ (%)
Nominal		Measured (geom. mean)	0-24h		0-78h		0-72h
			µ	%	µ	%	µ	%
Control		-	0.0593	-	0.0653	-	0.0598	-
100		79.44	0.0609	-2.6	0.0621	2.2	0.0597	0.2

- Areas under the growth curves:

The results of the statistical evaluation (based on 2 Sample t-Test;a=0.05) show that the 0-72 h areas were not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 79.44 mg/L (measured). AUC data are detailed in the following table:

Concentration (mg/l)			Area under the growth curve (A) and inhibition of A (%)
Nominal		Measured (geom. mean)	0-24h		0-78h		0-72h
			A	%	A	%	A	%
Control		-	38.0	-	318.0	-	1440.0	-
100		79.44	40.0	-5.3	306.0	3.8	1406.0	2.4

- Yield:

The results of the statistical evaluation (based on 2 Sample t-Test;a=0.05) show that the 0-72 h yield was not statistically significantly different from the untreated control value; accordingly the No Observed Effect Concentration (NOEC) was determined as 79.44 mg/L (measured). Yield data are detailed in the following table:

Concentration (mg/l)			Yield (Y) and inhibition of Y (%)
Nominal		Measured (geom. mean)	A			%
Control		-	73.3			-
100		79.44	72.8			0.7

Validity criteria fulfilled:

yes

Conclusions:

The 72h LC50 of Bis(2-chloro)ethyl ether on Pseudokirchneriella subcapitata was determined to be > 79.44 mg/L. As no effects were observed on the algal growth rate, the NOAEC was 79.44 mg/L.

Executive summary:

The purpose of the study was to determine the effect of the Test Item 2,2`-Dichlorodiethyl ether on the growth of the unicellular green algal species Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum). The study was conducted according to OECD Guideline No. 201 under GLP.

In a preliminary study, exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to various concentrations of the Test Item over several generations under defined conditions. The algal growth in relation to a control culture was determined over a fixed period of 72 hours. The method of application and the test species Pseudokirchneriella subcapitata was recommended by the test guidelines.

Because no toxic response was observed during the preliminary concentration range-finding test, a limit test was carried out using a single test concentration (100 mg/L, nominal) and a control group. The test design included eight replicates at the nominal concentration of 100 mg/L Test Item and eight replicates for the untreated controls.

The Test Item concentration was analytically determined at 24 intervals during the experiment. As the measured concentration deviated more than 20 per cent from the nominal at 48h and 72h measurements, biological results are based on the measured geometric mean concentration which is 79.44 mg/L. Not effect on algal growth was obserbed at this concentration.

Accordingly, the LC50 (based on growth rate) towards the test organism was determined to be > 79.44 and the NOAEC was 79.44 mg/L. This data has been assigned a reliability score of 1 (reliable without restrictions). Accordingle, Bis(

Description of key information

In a study performed according to OECD TG 201 under GLP, the effect of Bis(2-chloroethyl) ether on the growth of the unicellular green algal species Pseudokirchneriella subcapitata was determined. The algal growth in relation to a control culture was determined over a fixed period of 72 hours at 100 mg/L of test substance. Measured test substance concentration over the exposure period (geometric mean) was determined to be 79.44 mg/L. At this concentration, no effect was observed on algal growth and no EC50 could be determined. The substance is not classified as toxic towards algae according to GHS-UN criteria.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

79.44 mg/L

Additional information

Two study reports include results related to the evaluation of the acute toxicity of the substance on aquatic algae.

The purpose of the first study (key study) was to determine the effect of Bis(2-chloroethyl) ether on the growth of the unicellular green algal species Pseudokirchneriella subcapitata (formerly known asSelenastrum capricornutum). The study was conducted according to OECD Guideline No. 201 under GLP. In a preliminary test, exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to various concentrations of the Test Item over several generations under defined conditions. The algal growth in relation to a control culture was determined over a fixed period of 72 hours. The method of application and the test species Pseudokirchneriella subcapitata was recommended by the test guidelines. As no toxic response was observed during the preliminary concentration range-finding test, a limit test was carried out using only one test concentration (100 mg/L, nominal) and one control group. The test design included eight replicates at the nominal concentration of 100 mg/L Test Item and eight replicates for the untreated controls. The Test Item concentration was analytically determined at 24 intervals during the experiment. As the measured concentration deviated more than 20 per cent from the nominal at 48h and 72h measurements, biological results are based on the measured geometric mean concentration which is 79.44 mg/L. Not effect on algal growth was obserbed at this concentration. Accordingly, the LC50 (based on growth rate) towards the test organism was determined to be > 79.44 and the NOAEC was 79.44 mg/L. This data has been assigned a reliability score of 1 (reliable without restrictions).

The second study was conducted on behalf of the Japanese Ministry of Environment (MOE). The methodology used was not detailed but it can reasonnably be assumed that a standard method was used. The study summary does not report any details regarding the experimental conditions (including the test species used). Accordingly, a reliability factor of 4 was assigned to this study. The EC50 based on growth rate was determined to be 390 mg/L. The NOEC (growth rate) was 72 mg/L.

Conclusion: Both studies support the fact that the substance has low toxicity towards aquatic algae. While the study conducted on behalf of the Japanese Ministry of Environment was assigned a low reliability score (due to a lack of information provided in the study report), the NOEC (72 mg/L) obtained from this study is very similiar to the key NOEC (79.44 mg/L) estimate. Based on an overall assessment of existing data, the substance does not met the GHS-UN classification criteria.