2-amino-6-methyl-1,2,4-triazolo[1,5-a]pyrimidin-5(1H)-one

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:

06 January 2015 to 04 February 2016

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:

yes

Remarks:

Additional evaluations were performed for this study although an instruction was not given to do them

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Nominal amounts of test item (50, 90 and 160 mg (five replicates of each)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at temperatures of between 20 °C and 21 °C, 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 100, 180 and 320 mg/L (five replicates of each).
The pH of the test item dispersions were measured after stirring using Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 to pH 8.0. As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation.
- Eluate: Water
- Controls: The control group was maintained under identical conditions but not exposed to the test item.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Not reported

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Severn Trent Water Plc sewage treatment plant at Loughborough,Leicestershire, UK
- Preparation of inoculum for exposure: 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 (10 litres) was fed synthetic sewage (500 mL).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

None

Test temperature:

21 to 22 °C

pH:

7.1 to 7.7 (definitive test)

Dissolved oxygen:

5.8 to 8.2 mg O2/L (definitive test)

Nominal and measured concentrations:

Nominal concentrations: 100, 180 and 320 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Volumetric flasks (500 mL)
- Aeration: Continuous aeration
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- Sludge concentration (weight of dry solids per volume): 3.0 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC)

OTHER TEST CONDITIONS
- Adjustment of pH: Adjusted to between pH 7.0 and 8.0
- Photoperiod: The test was conducted under normal laboratory lighting for the total exposure duration

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Dissolved oxygen was continuously recorded

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study: No statistically significant toxic effects were shown at the test concentration of 10 and 100 mg/L, however statistically significant toxic effects were shown at the test concentration of 1000 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None. No undissolved test item was visible in any vessel during the test period.
- Effect concentrations exceeding solubility of substance in test medium: No
- Blank controls oxygen uptake rate: 43.5 mg O2/L/h
- Coefficient of variation of oxygen uptake rate in control replicates: 2.8%

Results with reference substance (positive control):

The positive control EC50 was 7.8 mg/L and was within the the validation acceptance range for 3,5-dichlorophenol of 2 to 25 mg/L.

Reported statistics and error estimates:

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 for the control and all test concentrations 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).

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

-	Nominal Concentration (mg/L	Initial O2 Reading (mgO2/L)	MeasurementPeriod (minutes)	Final O2 Reading (mgO/L)	O2 Consumption Rates (mgO/L/hour)	% Inhibition
Control	R1	5.0	4	2.0	45.00	-
	R2	4.0	3	1.8	44.00	-
	R3	4.1	3	1.9	44.00	-
	R4	4.2	3	2.0	44.00	-
	R5	5.0	4	2.2	42.00	-
	R6	4.9	4	2.1	42.00	-
Test Item	100 (R1)	4.1	3	1.9	44.00	[1]
	100 (R2)	5.9	6	2.0	39.00	10
	100 (R3)	4.7	4	1.8	43.50	0
	100 (R4)	4.3	3	2.2	42.00	3
	100 (R5)	4.1	3	1.9	44.00	[1]
	180 (R1)	4.9	4	2.2	40.50	7
	180 (R2)	5.0	5	1.7	39.60	9
	180 (R3)	5.0	4	2.3	40.50	7
	180 (R4)	4.7	4	2.1	39.00	10
	180 (R5)	4.8	4	2.2	39.00	10
	320 (R1)	4.5	4	2.1	36.00	17
	320 (R2)	4.9	5	1.8	37.20	14
	320 (R3)	4.9	5	1.8	37.20	14
	320 (R4)	5.8	6	2.2	36.00	17
	320 (R5)	5.6	6	2.0	36.00	17
3,5-dichlorophenol	3.2	5.4	6	2.2	32.00	26
	10	6.7	10	3.6	18.60	57
	32	7.9	10	7.0	5.40	88

R1 - R6 = Replicates 1 to 6

[ Increase in rate as compared to control]

 

No statistically significantly toxic effects were shown at the test concentration of 100 mg/L. However, statistically significantly toxic effects (P < 0.05) were shown at the test concentrations of 180 and 320 mg/L. Based on these results the No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg/L.

Validity criteria fulfilled:

yes

Remarks:

The 3-h EC50 for 3,5-dichlorophenol was within the range 2 to 25 mg/L. The specific respiration rate of the blank controls is >= 20 mg O2/g dw/h. The coefficient of variation of oxygen uptake rate in control replicates is <30% at the end of the test.

Conclusions:

A study was performed to assess the effect of the test substance on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guideline 209. The No Observed Effect Concentration (NOEC) for the test substance after 3 hours exposure was 100 mg/L.

Executive summary:

A study was performed to assess the effect of the test substance on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guideline 209.

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 100, 180 and 320 mg/L (5 replicates) for a period of 3 hours at a measured temperature of between 21 and 22°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 item, 3,5-dichlorophenol. The reference item gave a 3-Hour EC50 value of 7.8 mg/L, 95% confidence limits 6.1 to 10 mg/L. 

No statistically significantly toxic effects were shown at the test concentration of 100 mg/L. However, statistically significantly toxic effects (p < 0.05) were shown at the test concentrations of 180 and 320 mg/L. Based on these results the No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg/L.

Description of key information

A study was performed to assess the effect of the test substance on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guideline 209. The No Observed Effect Concentration (NOEC) for the test substance after 3 hours exposure was 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

100 mg/L