Dimolybdenum trizinc nonaoxide

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

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

GLP compliance:

yes

Analytical monitoring:

not required

Details on sampling:

Analytical monitoring not a requirement of the test guideline

Vehicle:

no

Details on test solutions:

The test item was dispersed directly in water.

Nominal amounts of test item (5.0, 16, 50, 160 and 500 mg (five replicates of each)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and sealed with nescofilm prior to being subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing.

Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 32, 100, 320 and 1000 mg/L (five replicates of each).

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained for the definitive test from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. On the day of collection the activated sewage sludge (14 liters) was fed synthetic sewage (700 mL). The pH of the sample on the day of the test was 7.3 measured using a Hach HQ40d Flexi handheld meter. 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 pre-weighed GF/A filter paper* using a Buchner funnel which was then rinsed three times with 10 mL of deionized 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 3.0 g/L prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

Standard as per test guideline

Hardness:

Not specified

Test temperature:

20 to 21°C

pH:

pH at the start and end of test in test vessels ranged from 7 to 7.9.

Dissolved oxygen:

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L.

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Range-finder (nominal): 0, 10, 100 and 1000 mg/L
Definitive (nominal): 0, 10, 32, 100, 320 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL conical flask
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 500 mL (synthetic sewage, water and inoculum)
- Aeration: Yes
- No. of vessels per concentration (replicates): 5 replicates of each
- No. of vessels per control (replicates): 5
- No. of vessels per vehicle control (replicates): 6
- No. of vessels per abiotic control (replicates): NA
- Sludge concentration (weight of dry solids per volume): 3 g/L prior to use
- Nutrients provided for bacteria: Synthetic sewage meeting the OECD guideline requirements
- Nitrification inhibitor used (delete if not applicable): none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the range-finding and definitive tests was deionized reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC).
- Particulate matter: None specified

OTHER TEST CONDITIONS
- Adjustment of pH: Yes adjusted as appropriate in a small number of replicates at varying concentrations at the start of study to get to the guideline specified pH values.
- Photoperiod: Continuous light
- Light intensity: Not specified
- Details on termination of incubation: 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 Yellow Springs 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 7 mg O2/L and 2 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.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 3 hour calculation of percentage inhibition compared with controls.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Standard as per OECD guidelines to enable determination of an appropriate EC50 and NOEC
- Justification for using fewer concentrations than requested by guideline: NA
- Range finding study
- Test concentrations: 0, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes, results of the range-finder were used to determine the concentrations for the main phase of the study.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

29 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.:

360 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

No statistically significant toxic effects were shown at the test concentrations of 10 and 32 mg/L; however statistically significant toxic effects (P < 0.05) were shown at the test concentrations of 100, 320 and 1000 mg/L. Based on these results the NOEC after 3 hours exposure was 32 mg/L.

In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 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.

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L.

The specific respiration rate of the controls was 29.78 mg oxygen per gram dry weight of sludge per hour and the coefficient of variation of oxygen uptake in the control vessels was 7.2%.

Results with reference substance (positive control):

- Results with reference substance valid?
- Relevant effect levels: 3 hour EC50 7 mg/L. The EC50 (3-Hour contact time) for 3,5-dichlorophenol should lie within the range 2 to 25 mg/L for total respiration.

Reported statistics and error estimates:

The percentage inhibition values were plotted against concentration, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values for the reference item and the EC10, EC20 and EC50 values for the test item determined from the equation for the fitted line. The EC80 value for the test item was determined by inspection of the inhibition of respiration rate data.

The 95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).
In order to determine the NOEC, one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data after 3 hours to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Inhibition of respiration rates:

	Test substance		3,5-DCP
	ECx mg/L (3 hours)	95% confidence limits (mg/L)	ECx mg/L (3 hours)	95% confidence limits (mg/L)
EC10	29	-	1.4	-
EC20	64	-	2.1	-
EC50	360	-	7	5.3 – 9.2
EC80	> 1000	-	2	-
NOEC	32	-	-	-

Oxygen consumption rates and percentage inhibition values after 3 hours contact time in the definitive test

Nominal conc. (mg/L)		Initial O2 reading (mgO2/L)	Measurement period (minutes)	Final O2 reading (mgO2/L)	O2 Consumption rates (mgO2/L)	Inhibition (%)
Control	R1	3.6	2	2	48	-
	R2	3.4	2	2	42	-
	R3	3.8	2	2.3	45	-
	R4	2.3	1	1.5	48	-
	R5	3.9	3	1.9	40	-
	R6	3.6	2	2.1	45	-
Test substance	10 R1	3.6	2	2.2	42	6
	10 R2	4.0	2	2.5	45	[1]
	10 R3	4.3	3	2.1	44	1
	10 R4	4.5	4	1.7	42	6
	10 R5	4.6	2	3.1	45	[1]
	32 R1	4.1	4	1.5	39	13
	32 R2	4.0	3	2	40	10
	32 R3	3.8	3	1.8	40	10
	32 R4	4.4	4	1.9	37.5	16
	32 R5	3.3	2	1.9	42	6
	100 R1	4.7	5	2	32.4	27
	100 R2	5.4	7	2	29.14	35
	100 R3	4.3	4	1.9	36	19
	100 R4	4.6	6	1.6	30	33
	100 R5	4.4	4	2	36	19
	320 R1	5.8	9	1.8	26.67	40
	320 R2	4.9	6	1.9	30	33
	320 R3	6.1	10	2.4	22.2	50
	320 R4	6.3	10	3.4	17.4	61
	320 R5	5.9	9	3	19.33	57
	1000 R1	6.5	10	3.6	17.4	61
	1000 R2	6.6	10	4.1	15	66
	1000 R3	6.4	10	4.1	13.8	69
	1000 R4	5.8	10	2.9	17.4	61
	1000 R5	6.7	10	4.5	13.2	70
3,5-DCP	3.2	4.5	5	1.9	31.2	30
	10	6.2	9	3.5	18	60
	32	7.3	10	6.3	6	87

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of 360 mg/L. The NOEC after 3 hours exposure was 32 mg/L.

Description of key information

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of 360 mg/L. The NOEC after 3 hours exposure was 32 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

360 mg/L

EC10 or NOEC for microorganisms:

32 mg/L