Ethylene dinitrate

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-07-27 to 2010-07-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethylene dinitrate

EC Number:

211-063-0

EC Name:

Ethylene dinitrate

Cas Number:

628-96-6

Molecular formula:

C2H4N2O6

IUPAC Name:

ethane-1,2-diyl dinitrate

Specific details on test material used for the study:

Test substance was produced on site in a micro-reactor and used on the same day it was produced. Purity was reported as >99% using HPLC; batch number was EGDN-LN-20100727. Stability of the test solutions was checked at the start and end of the study.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The measured concentrations at t = 0 h were in a range of 96 - 128 % of the nominal concentrations. The measured concentrations at t = 3 h were in a range of 85 - 105 % of the nominal concentrations and 77 - 101 % of the start concentrations. Therefore the determination of the results was based on the mean of measured concentrations.

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

activated sludge, domestic

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test conditions

Test temperature:

20 ± 2 °C

Details on test conditions:

16 mL nutrient solution was mixed with 225 mL water in the first control vessel, then 259 mL inoculum were added. The bottle was closed immediately and stirred on a mag-netic stirrer without aeration. Five minutes later, the second control vessel was prepared likewise.
Again five minutes later, the first positive control treatment was prepared by mixing 16 mL nutrient solution with the appropriate amount of the stock solution and adding water to give 241 mL. Then 259 mL inoculum was added. The following positive control, test item and control treatments were prepared likewise in five minute intervals.
The content of the test item in the test vessels was measured at the start after one minute stirring and at the end of the test before oxygen measurement.
After 2 hours and 59 minutes the test vessels were shaken for one minute to guarantee a sufficient oxygen concentration in the test solution. Then the narrow neck bottle was filled to the rim with an aliquot of the content of the first vessel, a magnetic stirrer was added and the oxygen electrode was tightly fit into the vessel. Care was taken that no air could reach the contents of the vessel. The oxygen concentration of the vessel was measured every 5 - 10 seconds over a period of approx. 3 - 5 minutes. The interface of the oxygen meter was connected to a personal computer, feeding the measured values with their corresponding times in a table.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (CAS 591-35-5) was used as positive control. A stock solution in deionised water containing 500 mg/L (nominal) was freshly prepared for each experiment.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 5.6 - < 23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95 % Conf. interval

Effect concentrations 2

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 160 - < 530 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95 % Conf. interval

Results with reference substance (positive control):

3,5-Dichlorophenol (CAS 591-35-5) was used as positive control. A stock solution in deionised water containing 500 mg/L (nominal) was freshly prepared for each experiment.

Any other information on results incl. tables

Nominal Conc. in mg/L	Measured Conc. in mg/L		% Recovery	% of Nominal Concentration		Mean of Measured Conc. in mg/L
			after 3 h
	0 h	3 h		0 h	3 h
1	1.3	1.0	80	128	102	1.2
3.2	3.3	2.7	82	104	85	3.0
10	11.3	8.7	77	113	87	10.0
32	32.3	29.8	92	101	93	31.0
100	107.0	105.0	98	107	105	106.0
320	317.5	313.7	99	99	98	315.6
1000	968.8	975.5	101	97	98	972.1
1000	958.1	956.1	100	96	96	957.1

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The inhibitory effect of ethylene glycol dinitrate on the respiration rate of activated sludge was determined in a study according to OECD guideline 209.
The following results were determined
3h NOEC = 10 mg/L (95% confid. interval: not determinable)
3h EC10 = 13 mg/L (95% confid. interval: 5.6 - 23 mg/L)
3h EC20 = 37 mg/L (95% confid. interval: 20 - 62 mg/L)
3h EC50 = 260 mg/L (95% confid. interval: 160 - 530 mg/L)

Executive summary:

One valid experiment was performed.

The main study was performed using seven concentrations, ranging from 1000 to 1.0 mg/L (nominal concentration).

Due to the volatility of the test item, the study was performed in a closed system without aeration and with analytical determination.

 

The dry matter of the activated sludge was determined as 2.9 g suspended solids/L, giving a concentration of 1.5 g suspended solids/L in the test. 3,5-Dichlorophenole was used as positive control. Four concentrations were tested; an EC50 of 7.7 mg/L (95% confidence interval: 2.8 – 12 mg/L) was determined, which lies within the range of 5 – 30 mg/L.

 

The following results for the test item Ethylene Glycol Dinitrate were determined:

Biological Results Test Item

Parameter	Value	95% Confidence Interval
NOEC	10 mg/L	n. d.
3h-EC10	13 mg/L	5.6 – 23 mg/L
3h-EC20	37 mg/L	20 – 62 mg/L
3h-EC50	260 mg/L	160 – 530 mg/L