Bis(2-chloroethyl) ether

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 28-jan-2016 to 17-mar-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TEST MATERIAL
- Name (as cited): 2,2’- Dichlorodiethyl ether
- Purity: 99.76%

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 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:

no

Details on sampling:

- Concentrations: 1, 10, 100, 1000 mg/L

Vehicle:

no

Details on test solutions:

Just before the start of the experiment a concentrated stock solution (3200 mg/L) was prepared by dissolving test item in deionised water. The test solutions used in the test were freshly prepared by dilution of the stock solution by mechanical dispersion at the beginning of the experiment, in the testing laboratory. The pH of the solutions was checked but not adjusted, because they were in the range of 7–8.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Laboratory culture: prior to use the sludge was fed daily with 50 mL synthetic sewage per litre and kept aerated at 20 ± 2°C until use
- Name and location where inoculum was collected: domestic sewage (Veszprém, Hungary)
- sampling date: two days before the start of the experiment
- Initial biomass concentration: 3 g per litre (on dry weight basis)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.8 - 21.7°C

Nominal and measured concentrations:

Nominal concentrations tested: 10, 100 and 1000 mg/L

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

IC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

- Results with reference substance valid: yes

REFERENCE TEST SUBSTANCE:

The following nominal concentrations of the reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as in the case of the test item: 1.0, 3.2, 10.0 and 32.0 mg/L.

In comparison to the controls the inhibition of the respiration rate of the activated sludge was 74.64 % at the highest nominal concentration of 32.0 mg/L.

At the nominal concentrations of 1.0, 3.2 and 10.0 mg/L 10.96 %, 17.19% and 35.28 % inhibition of the respiration rate was calculated respectively. The results are summarised in the table below.

The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 13.64 mg/L with 95% confidence limits of 10.36 – 17.97 mg/L.

TEST ITEM:

In comparison to the inoculum controls the inhibition of the respiration rate in the case of the activated sludge was between -2.07 % and 35.92 % in the examined range of 10 – 1000 mg/L of 2,2’- Dichlorodiethyl ether test item.

The average specific respiration rate (RS) of the untreated (Blank) controls (B1-B5) was 22.48 mg O2/L/h/g solid.

The variation coefficient of oxygen uptake rate in above untreated replicates was 3.46 %.

The influence on the respiration rate of activated sludge is presented in the table below.

ID	Conc. of test item in test mix. (mg/L)	Total O2 consumption rate (mg O2/L/h)	Specific respiration rate (mg/O2/L/h/g solid)	Inhibition (%)
Abiotic
A1	1000	-0.21	-	-
A2	1000	-1.08	-	-
A3	1000	-0.54	-	-
A4	1000	-0.83	-	-
A5	1000	-0.29	-	-
Blank
B1	0	33.11	22.07	-
B2	0	33.68	22.45	-
B3	0	34.80	23.20	-
B4	0	34.88	23.25	-
B5	0	32.12	21.42	-
Ref. item
REF1	1.00	29.44	19.62	10.96
REF2	3.20	27.33	18.22	17.19
REF3	10.00	21.23	14.16	35.28
REF4	32.00	7.96	5.31	74.64
Test item
TI1	10	33.83	22.55	-2.07
TI2	100	28.79	19.20	12.86
TI3	1000	21.02	14.01	35.92

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation), the 3-hour EC50 of 2,2’-Dichlorodiethyl ether was determined to be > 1000 mg/L.

Executive summary:

In this study, the effect of 2,2’-Dichlorodiethyl ether on the total respiration rate of activated sludge microorganisms was investigated according to OECD Guideline No. 209 under GLP.

The purpose of the 3-hour toxicity test was to evaluate the influence of the test item on the activity of activated sludge by measuring the total respiration rate under defined conditions, and to calculate the inhibition effect, based on the measured data. The test method and the sensitivity of the activated sludge inoculum was checked, using 3,5-Dichlorophenol (3,5-DCP). The IC50 result of 3,5-DCP was compared to the result of international ring test, organized in 2004.

Under the conditions of this Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) the observed endpoint for the effect of 2,2’-Dichlorodiethyl ether was the following: 3-hour IC50 > 1000 mg/L. Validity criteria were fulfilled.

Based on the results of this study, the test item 2,2’-Dichlorodiethyl ether had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at the highest concentration tested. Therefore, further testing for nitrification inhibition is not necessary.

Description of key information

In a study conducted according to OECD Guideline No. 209 under GLP, the effect of Bis(2-chloroethyl) ether on the total respiration rate of activated sludge microorganisms was investigated. The test method and the sensitivity of the activated sludge inoculum were checked, using 3,5-Dichlorophenol (3,5-DCP). The EC50 result of 3,5-DCP was compared to the result of international ring test, organized in 2004. Under the conditions of this Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation), no bacterial inhibition following 3h exposure to 1000 mg/L of test substance was observed. Bis(2-chloroethyl) ether had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge and is considered non-toxic towards micro-organisms.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

Five studies were available to evaluate the toxicity of Bis(2-chloroethyl) ether on aquatic microorganisms.

Bis(2-chloroethyl) ether toxicity towards domestic activated sludge was tested using the test method desribed in OECD Guideline No. 209 under GLP. The purpose of this 3-hour toxicity test was to evaluate the influence of the test item on the activity of activated sludge by measuring the total respiration rate under defined conditions, and to calculate the inhibition effect, based on the measured data. The test method and the sensitivity of the activated sludge inoculum was checked, using 3,5-Dichlorophenol (3,5-DCP). The EC50 result of 3,5-DCP was in agreement with the result of an international ring test organized in 2004. Under the conditions of this Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation), Bis(2-chloroethyl) ether showed low toxicity towards the bacterial inoculum. The 3-hour IC50 was determined to be > 1000 mg/L. This result was obtained based on a guideline study conducted under GLP and validity criteria were fulfilled. Accordingly, it has been assigned a reliability score of 1. Considering additional available studies were not conducted using standard methods and that the results of these studies confirm the low toxicity of Bis(2-chlorethyl) ether towards micro-organisms, the results of this guideline study were selected as key information.

In a published study by Cho et al. (Environ. Technol. Lett., 1989, 10:875-86), acute bacterial toxicity responses to Bis(2-chloroethyl) ether was determined using a modified Standard method spread plate count technique. The growth inhibition activity of the chemical was tested on three bacterial inocula origination from the three waste stabilization ponds of a chemical and plastics company. The three ponds are arranged in series, so the primary waste stabilization pond received high strength process wastes plus a small portion of domestic wastewater, the secondary waste stabilization pond received the primary pond effluent, a small amount of domestic sewage, and additional low strength process wastewaters, and finally the tertiary waste stabilization pond containing influent from the secondary waste stabilization pond. The three bacterial inocula showed IC50 ranging from 1640 to 3100 mg/L of Bis(2-chloroethyl) ether. The IC10 ranged from 380 to 900 mg/L. However, statistical analyses (not detailed in the publication) did not reveal any significant differences (P < 0.05) between the microbial responses of the three pond influents. The data were pooled to estimated IC50 and IC10 were 2160 and 600 mg/L, respectively. Based on the results of this study, Bis(2-chloroethyl) ether did not exhibit significant toxicity towards the selected microbial communities. This non guideline study is based on an accpetable methodology. In addition, raw data on Bis(2-chloroethyl) ether are provided and the publication has been subject to peer-review. Accordingly, is is judge acceptable for assessment and has been assigned a reliability score of 2

In Johnson & Young (JWPCF, 1983, 55(12):1441 -9), the results of a batch bioassay technique using serum bottles to determine the inhibitory effect of Bis(2-chloroethyl) ether are reported. Inhibition of anaerobic cultures was monitored and the conditions affecting recovery from this inhibition were examined. Bacterial activity was monitored from methane and carbon dioxide production as measured by GC-TCD. Under the conditions of this test, Bis(2-chloroethyl) ether was found to induce very limited inhibition of gas production by anaerobic bacteria. IC50 after 5h and after 285h were both > 100 mg/L. This study did not follow any recommended guideline and detailed results on Bis(2-chloroethyl) ether are not available in the original publication. However, the study is based on an accpetable methodology, which is described in sufficient details. In addition, it has been subject to peer-review. Accordingly, is is judge acceptable for assessment and has been assigned a reliability score of 2.

The study reported by Nirmalakhandan et al. (1994) relates to assays done on a surrogate microorganism: Polytox, a proprietary blend of 12 streams of aerobic bacteria. Polytox has been recommended by EPA as an inexpensive culture for use in effluent toxicity evaluations (page c-3, EPA/4-87-005). The objective of the study was to analyze joint effects of two or more chemicals on microorganisms. To this end, a dataset on the single effect of 50 chemicals (chosen from the list of "chemicals of concern to the US Air Force", which includes Bis(2-chloroethyl) ether) was aquired. Considering the scope of this study, no detailed information on each chemicals is provided in the original publication. Based on the Polytox assay, the IC50 of Bis(2-chloroethyl) ether was determined to be 1600 mg/L. In a sister study (Prakash et al., 1996) using the very same method, the IC50 was determined at 1298 mg/L.

Conclusion:

The results from five independent studies confirm that the toxicity of Bis(2-chloroethyl) ether towards heterpotrophic bacteria is very low. None of the studies found IC50 values < 100 mg/L.