Diquat dibromide

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:

22 Mar 2009 to 23 Mar 2009

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

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

For the purpose of the test, the test material was dissolved directly in water. The following test concentrations were assigned to the test: 1.0, 3.2, 10, 32, 100, 320, and 1000 mg test material/L. An amount of test material (2000 mg) was dissolved in water and the volume adjusted to 1 L to give a 2000 mg/L stock solution from which serial dilutions were made to give 200, 20 and 2.0 mg/L stock solutions. An aliquot (250 mL) of the 2.0 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (200 mL) and water, to a final volume of 500 mL, to give the required concentration of 1.0 mg/L. Similarly, aliquots (80 and 250 mL) of the 20 mg/L stock solution, aliquots (80 and 250 mL) of the 200 mg/L stock solution and aliquots (80 and 250 mL) of the 2000 mg/L stock solution were used to prepare the test concentrations of 3.2, 10, 32, 100, 320 and 1000 mg test material/L respectively. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.
The control group was maintained under identical conditions but not exposed to the test material.

Test organisms (species):

activated sludge

Details on inoculum:

- Activated sewage sludge micro-organisms: A mixed population of activated sewage sludge micro-organisms was obtained on 23 March 2009 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

A synthetic sewage of the following composition, was added to each test vessel and dissolved in 1L of water with the aid of ultrasonication. to act as a respiratory substrate:
- Peptone: 16 g
- Meat extract: 11 g
- Urea: 3 g
- NaCl: 0.7 g
- CaCl2.2H2O: 0.4 g
- MgSO4.7H2O: 0.2 g
- K2HPO4: 2.8 g

- Preparation of inoculum: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and was used on the day of collection. The pH of the sample was 7.6. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a preweighed filter paper using a Buchner funnel which was then rinsed 3 times with 10 mL of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.9 g/L prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

21 ± 1 °C

pH:

7.6 - 8.2

Nominal and measured concentrations:

- Nominal concentrations: 0 (negative control) 1.0, 3.2, 10, 32, 100, 320 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL conical flask
- No. of vessels per concentration: 1
- No. of vessels per control: 2
- No. of vessels per positive control/concentration: 1

PREPARATION OF THE TEST SYSTEM
At time "0" 16 mL of synthetic sewage was diluted to 300 mL with water and 200 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 – 1 L/min. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added. Finally a second control was prepared.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the definitive test was laboratory tap water dechlorinated by passage through an activated carbon filter and partly softened giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

EFFECT PARAMETERS MEASURED
As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 6.5 mg O2/L and 2.5 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period. Observations were made on the test preparations throughout the test period. Observations of the test material vessels at 0 hours were made prior to addition of activated sewage sludge and synthetic sewage.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

An overview of the results is provided in Table 1 and Table 2 in 'Any other information on results incl. tables'.

- Observation: Observations made at 0 hours prior to the addition of activated sewage sludge and synthetic sewage showed that the test concentrations of 1.0, 3.2 and 10 mg/L contained clear colourless solutions. The test concentrations of 32 and 100 mg/L contained very pale brown solutions and the test concentrations of 320 and 1000 mg/L contained pale brown solutions. Observations made after 30 minutes and 3 hours contact time showed that all test concentrations contained dark brown dispersions with no undissolved test material visible.

- Inhibition of Respiration Rate: The initial dissolved oxygen concentrations in the 2 replicates control groups are 6.7 and 5.3 mg O2/L. Their final (after 3 hours contact) dissolved oxygen concentrations were 2.9 and 2.8 mg O2/L. Variation in respiration rates of controls 1 and 2 after 3 hours contact was ± 0%. The test item showed a dose-related inhibiting effects on the total respiration rate of activated sludge microorganisms. After 3 hours contact, in comparison to the inoculum controls, the respiration rates of the activated sludge were slightly inhibited at test concentrations of 1.0 and 3.2 and 10 mg/L with 2%, 5% and 5% inhibition respectively. The inhibition was 10%, 17%, 29% and 40% for test item concentrations of 32, 100, 320 and 1000 mg/L, respectively. In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O2/L and 2.5 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace. It was not possible to obtain EC50 or EC80 values for the test material as no concentration tested resulted in greater than 50% inhibition. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Results with reference substance (positive control):

- Inhibition of Respiration Rate: In comparison to the controls the total respiration rate of the activated sludge was inhibited by 26.0% at the lowest nominal concentration of 3.2 mg/L. At the nominal concentrations of 10 and 32 mg reference item/L, the respiration rate was inhibited by 71% and 86%, respectively. The 3-h EC50 for the reference substance was determined to be 6.6 mg/L. The 3-h NOEC was determined to be 1.7 mg/L.

Table 1. Oxygen Consumption Rates and Percentage Inhibition Values in the Definitive Test after 3 Hours Contact Time

Nominal Concentration (mg/L)		Initial O2 Reading (mg O2/L)	Measurement Period (minutes)	Final O2 Reading (mg O2/L)	O2 Consumption Rates (mg O2/L/min)	% Inhibition
Control	Replicate1	6.7	9	2.9	0.42	-
	Replicate2	5.3	6	2.8	0.42	-
Test Material	1.0	6.9	10	2.8	0.41	2
	3.2	6.5	9	2.9	0.40	5
	10	6.8	10	2.8	0.40	5
	32	7.0	10	3.2	0.38	10
	100	7.2	10	3.7	0.35	17
	320	7.5	10	4.5	0.30	29
	1000	7.7	10	5.2	0.25	40
3,5-dichlorophenol	3.2	6.9	10	3.8	0.31	26
	10	6.9	10	5.7	0.12	71
	32	8.5	10	7.9	0.06	86

 

Table 2. Results of effect concentrations.

	The test substance		3,5-dichlorophenol
	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC20	340	-	2.1	-
EC50	>1000	-	6.6	5.1 - 8.5
EC80	>1000	-	21	-
NOEC	200	-	1.7	-

 

Validity criteria fulfilled:

yes

Conclusions:

The 3-hour EC50 value of the test substance was determined to be > 1000 mg test material/L and the NOEC was 200 mg test material/L.

Executive summary:

A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The study was in accordance with OECD TG 209 and in compliance with GLP criteria.  Activated sewage sludge was exposed to an aqueous solution of the test material at concentrations of 1.0, 3.2, 10, 32, 100, 320 and 1000 mg test material/L for a period of 3 hours at a temperature of approximately 21 °C with the addition of a synthetic sewage as a respiratory substrate.The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol. The initial dissolved oxygen concentrations in the 2 replicates control groups are 6.7 and 5.3 mg O2/L. Their final (after 3 hours contact) dissolved oxygen concentrations were 2.9 and 2.8 mg O2/L. Variation in respiration rates of controls 1 and 2 after 3 hours contact time was ± 0%. After 3 hours contact, in comparison to the controls, the inhibition of the reference material on the total respiration rate of the activated sludge was 26.0% at the lowest nominal concentration of 3.2 mg/L. At the nominal concentrations of 10 and 32 mg reference item/L, the respiration rate was inhibited by 71% and 86%, respectively. The test item showed a dose-related inhibiting effects on the total respiration rate of activated sludge microorganisms. After 3 hours contact, in comparison to the inoculum controls, the respiration rates of the activated sludge were slightly inhibited at test concentrations of 1.0 and 3.2 and 10 mg/L with 2%, 5% and 5% inhibition respectively. The inhibition was 10%, 17%, 29% and 40% for test item concentrations of 32, 100, 320 and 1000 mg/L, respectively. In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O2/L and 2.5 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace. It was not possible to obtain EC50 or EC80 values for the test material as no concentration tested resulted in greater than 50% inhibition. Based on the findings, the 3-hour EC50 value was determined to be > 1000 mg/L for test material, equivalent to >411 mg pure test substance/L. The NOEC was 200 mg test material/L, corresponding to 82.2 mg pure test substance/L.

Description of key information

3-h NOEC = 82.2 mg test substance/L, Activated sludge, inhibition of total respiration, OECD TG 209, Clarke 2009

3-h EC50 >411 mg test substance/L, Activated sludge, inhibition of total respiration, OECD TG 209, Clarke 2009

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

82.2 mg/L

Additional information

A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The study was in accordance with OECD TG 209 and in compliance with GLP criteria.  Activated sewage sludge was exposed to an aqueous solution of the test material at concentrations of 1.0, 3.2, 10, 32, 100, 320 and 1000 mg test material/L for a period of 3 hours at a temperature of approximately 21 °C with the addition of a synthetic sewage as a respiratory substrate.The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol. The initial dissolved oxygen concentrations in the 2 replicates control groups are 6.7 and 5.3 mg O2/L. Their final (after 3 hours contact) dissolved oxygen concentrations were 2.9 and 2.8 mg O2/L. Variation in respiration rates of controls 1 and 2 after 3 hours contact time was ± 0%. After 3 hours contact, in comparison to the controls, the inhibition of the reference material on the total respiration rate of the activated sludge was 26.0% at the lowest nominal concentration of 3.2 mg/L. At the nominal concentrations of 10 and 32 mg reference item/L, the respiration rate was inhibited by 71% and 86%, respectively. The test item showed a dose-related inhibiting effects on the total respiration rate of activated sludge microorganisms. After 3 hours contact, in comparison to the inoculum controls, the respiration rates of the activated sludge were slightly inhibited at test concentrations of 1.0 and 3.2 and 10 mg/L with 2%, 5% and 5% inhibition respectively. The inhibition was 10%, 17%, 29% and 40% for test item concentrations of 32, 100, 320 and 1000 mg/L, respectively. In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O2/L and 2.5 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace. It was not possible to obtain EC50 or EC80 values for the test material as no concentration tested resulted in greater than 50% inhibition. Based on the findings, the 3-hour EC50 value was determined to be > 1000 mg/L for test material, equivalent to >411 mg pure test substance/L. The NOEC was 200 mg test material/L, corresponding to 82.2 mg pure test substance/L.