bis(diethylamino)-N,N-diethylmethaniminium chloride

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:

January 29, 2004

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

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Remarks:

OECD GLP

Analytical monitoring:

not specified

Vehicle:

no

Details on test solutions:

Water used for dilution of the sludge and for all toxicity testing was deionized water collected at Marblehead, Massachusetts with a hardness of 4 mg/L as CaCO3.

A stock solution of the test substance was prepared with a nominal concentration of 500 mg a.i./L (the highest stock concentration suggested by the OECD guideline) by bringing 0.7864 grams of test substance (based on correction for purity) to a total volume of 1,000 mL with deionized water in a 1,000 mL class A volumetric flask. The solution was mixed by multiple inversions of volumetric flask. Nominal concentrations [0 (control), 3.3, 10, 31, 93, and 280 mg a.i./L] were formulated by the addition of appropriate volumes of the stock solution to 500 mL of test solution without the use of a solvent.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge used for the test was obtained from the municipal wastewater treatment plant at Merrimac, Massachusetts. This treatment plant treats predominantly domestic waste. As collected, the suspended solids concentration of the sludge used in the definitive test was 6.5 g/L. The sludge was incubated at room temperature and aerated prior to use. It was adjusted to a suspended solids content of 4.3 g/L on the day of use for the definitive test. Because the sludge was not used the day it was collected, synthetic sewage, consisting of 16 g/L peptone, 11 g/L meat extract, 3 g/L urea, 0.7 g/L NaCl, 0.34 g/L CaCl2, 0.2 g/L MgSO4.7H2O, and 2.8 g/L K2HPO4 was combined with the sludge at the rate of approximately 50 mL/L. The pH of the sludge was adjusted to 7.0 with 1N sodium hydroxide at the time of use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

19.5 to 20.3 °C

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 3.3, 10, 31, 93, and 280 mg/L

Details on test conditions:

A range-finding test was not conducted. The definitive test was conducted under static conditions at 20 ± 2°C. At time 0 for each test vessel, 16 mL of synthetic sewage and an appropriate amount of the test substance stock solution (500 mg a.i./L) was brought up to 300 mL in dilution water in a 1.0 liter glass beaker. A 200 mL aliquot of microbial inoculum was added to this mixture within 5 minutes of its preparation. Test vessels were established at 15 minute intervals and vessels were aerated with 0.5 to 1.0 liters of air per minute for 3 hours. The first and last vessels established were controls.

After the 3 hour incubation period, a portion of the contents of each test vessel were transferred to a BOD bottle equipped with a magnetic stirrer and the dissolved oxygen concentration in this sealed bottle was monitored for 10 minutes. Test vessels were maintained under ambient laboratory lighting during the test. Dissolved oxygen was measured with a YSI Model 57 meter.

A respiration inhibition test was performed with the reference toxicant, 3,5-dichlorophenol, using the same batch of sludge used for this definitive test. The test was performed with three nominal concentrations of 3,5-dichlorophenol (5.0, 12, and 30 mg/L) under test conditions described above.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 280 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The control respiration rates were within 15% of each other and the temperature during the test was 19.5 to 20.3°C.

The test substance did not inhibit respiration of the activated sludge in the tested range of concentrations. The 3-hour EC50 for activated sludge exposed to the test substance was greater than 280 mg a.i./L, the highest tested concentration. Insoluble material was not observed at any tested concentration during the test.

Results with reference substance (positive control):

The EC50 determined during a reference toxicant test with this batch of activated sludge and 3,5-dichlorophenol was 6.9 mg/L, which is within the acceptable range of 5 to 30 mg/L.

Reported statistics and error estimates:

The respiration rate was calculated as the mg oxygen/liter/hour consumed between 6.5 (or the initial dissolved oxygen concentration, if less than 6.5 mg/L) and 2.5 mg/L, or over a 10-minute period if the respiration rate was low. The percent inhibition, which was used to calculate EC50 values, was calculated as (1 minus [2 times the respiration rate of the test substance concentration] divided by the sum of the 2 control respiration rates) times 100.

The 3-hour EC50 value for the definitive activated sludge respiration inhibition test could not be calculated for the test substance by standard statistical techniques because there was less than 50% inhibition at all tested concentrations. The 3-hour EC50 value for the reference substance was calculated by the probit method.

Activated sludge respiration data from the test with the test substance

Nominal Concentration of Test Substance (mg a.i./L)	R	Dissolved Oxygen Concentration (mg/L)		Time to 6.5 mg/L Oxygen (minutes)	Time to 2.5 mg/L Oxygen (minutes)	Respiration Rate (mg/L/hr)	Inhibition (%)
		Initial	Final
0 (control)	1	6.7	0.1	0.5	3.0	80	--
	2	7.8	0.1	1.0	4.5	69	--
3.3	1	6.8	0.1	0.5	3.5	80	0
	2	5.9	0.1	--	2.5	102	0
10	1	7.3	0.1	0.5	4.0	69	7
	2	7.3	0.1	1.0	4.0	80	0
31	1	7.4	0.1	1.0	4.5	69	7
	2	7.1	0.1	0.5	4.5	60	20
93	1	7.8	0.1	1.5	5.5	60	20
	2	6.8	0.1	0.5	4.0	69	7
280	1	8.0	0.7	2.0	7.5	44	41
	2	7.3	0.1	1.0	5.5	53	29

R = Replicates

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study the 3-hour EC50 for active sludge exposed to the test material based on the inhibition of total respiration was > 280 mg/L (active ingredient), the highest concentration tested.

Executive summary:

The effect of the test material to microorganisms was investigated in accordance with the standardised guidelines, OECD 209, under GLP conditions. 

The test was performed at 20 ± 2 °C under static conditions with a control and five concentrations of test material (3.3, 10, 31, 93, and 280 mg/L). Nominal concentrations of the active ingredient were used for all calculations.

The total suspended solids content of the activated sludge was 6.5 g/L on the day of collection and 4.3 g/L on the day of the respiration inhibition test.  The EC50 determined during a reference toxicant test with this batch of activated sludge and 3,5-dichlorophenol was 6.9 mg/L, which is within the acceptable range of 5 to 30 mg/L.

The test material did not inhibit respiration of the activated sludge in the tested range of concentrations.

Under the conditions of this study the 3-hour EC50 for active sludge exposed to the test material based on the inhibition of total respiration was > 280 mg/L (active ingredient), the highest concentration tested. 

Description of key information

Boeri, Wyskiel, Ward (2003)

Under the conditions of this study the 3-hour EC50 for active sludge exposed to the test material based on the inhibition of total respiration was > 280 mg/L (active ingredient), the highest concentration tested. 

Key value for chemical safety assessment

EC50 for microorganisms:

280 mg/L

Additional information

Boeri, Wyskiel, Ward (2003)

The effect of the test material to microorganisms was investigated in accordance with the standardised guidelines, OECD 209, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test was performed at 20 ± 2 °C under static conditions with a control and five concentrations of test material (3.3, 10, 31, 93, and 280 mg/L).  Nominal concentrations of the active ingredient were used for all calculations.

The total suspended solids content of the activated sludge was 6.5 g/L on the day of collection and 4.3 g/L on the day of the respiration inhibition test.  The EC50 determined during a reference toxicant test with this batch of activated sludge and 3,5-dichlorophenol was 6.9 mg/L, which is within the acceptable range of 5 to 30 mg/L.

The test material did not inhibit respiration of the activated sludge in the tested range of concentrations.

Under the conditions of this study the 3-hour EC50 for active sludge exposed to the test material based on the inhibition of total respiration was > 280 mg/L (active ingredient), the highest concentration tested.