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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD UK

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

TEST ITEM
The test item was dissolved directly in water. Nominal amounts of test item (450, 800 and 1400 mg) were dissolved separately in water with the aid of ultrasonication for approximately 10 minutes and the volumes adjusted to 1 liter to give 450, 800 and 1400 mg/L stock solutions. An aliquot (200 mL) of the 450 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 180 mg/L. Similarly, aliquots (200 mL) of the 800 and 1400 mg/L stock solutions were used to prepare the test concentrations of 320 and 560 mg/L. There were five replicates for all test concentrations. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions. The pH of the test item stock solutions was measured and adjusted to between pH 7.0 and 8.0 using a Hach HQ40d Flexi handheld meter. The control group was maintained under identical conditions but not exposed to the test item.

REFERENCE SUBSTANCE
A reference item, 3,5-dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 15 minutes. The pH of this stock solution was measured to be pH 4.8 and was adjusted to pH 7.2 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to a final volume of 500 mL to give the required concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 18 January 2016 for the range-finding test and on 01 February 2016 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

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

21°C

pH:

7.1 - 7.6

Dissolved oxygen:

3.6 - 8.0 mg/L

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 180, 320 and 560 mg/L (based on range-finding test).

Details on test conditions:

TEST WATER
The test water used for the range-finding and definitive tests was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).

SYNTHETIC SEWAGE
A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:16 g Peptone; 11 g Meat extract; 3 g Urea; 0.7 g NaCl; 0.4 g CaCl2·2H2O; 0.2 g MgSO4·7H2O; 2.8 g K2HPO4 dissolved in a final volume of 1 liter of water with the aid of ultrasonication. The pH of the synthetic sewage stock used to feed the activated sewage sludge was pH 7.2 and the pH of the synthetic sewage stock used for the definitive test was pH 7.3. The pH values were measured using a Hach HQ40d Flexi handheld meter.

PREPARATION OF INOCULUM
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 (9.9 liters) was fed synthetic sewage (495 mL). The pH of the sample on the day of the test was 7.5 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 3 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 one 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.

PREPARATION OF THE TEST SYSTEM
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 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 0.5 to 1.0 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added. Two additional control vessels were then prepared prior to the test item vessels being prepared as described under 'Details on test solutions'. Finally two further control vessels were prepared. The test was conducted under normal laboratory lighting in a temperature controlled room at a measured temperature of 21 ºC.

PARAMETERS MEASURED
- Respiration rates: 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.
- Appearence: The appearance of the mixtures in the vessels before the addition of activated sludge were documented. The observation of the mixtures were continued during the exposure period and changes in the appearance were recorded.
- pH measurements: The pH of the test preparations were measured at the test start (i.e. after the addition of activated sludge) and at the end of the incubation period.
- Oxygen Concentration: The oxygen concentration was checked at least once during the incubation period in all aerated test media.

EFFECT PARAMETERS MEASURED
The percentage inhibition of respiration was calculated based on comparison with blank controls.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

180 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

At a test concentration of 180 mg/L no statistically significantly inhibition of respiration was observed. At test concentrations of 320 mg/L and 560 mg/L the percentages inhibition were 10-15% and 15-18%, respectively. Only a 3-h NOEC is reported with a value of 180 mg/L.

Results with reference substance (positive control):

3-h EC50: 6.5 mg/L (95% C.L. 5.0 - 8.4 mg/L).

Reported statistics and error estimates:

The percentage inhibition values were plotted against concentration for the reference item only, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values determined from the equation for the fitted line. 95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949). 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 in order to determine the NOEC value. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Validity criteria fulfilled:

yes

Conclusions:

The 3-h NOEC is 180 mg/L in activated sewage sludge.

Executive summary:

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)". Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 180, 320 and 560 mg/L (5 replicates) for a period of 3 hours at a measured temperature of 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 item, 3,5-dichlorophenol. The No Observed Effect Concentration (NOEC) for the test item after 3 hours exposure was 180 mg/L. The reference item gave a 3-Hour EC50 value of 6.5 mg/L, 95% confidence limits 5.0 to 8.4 mg/L.

No statistically significantly toxic effects were shown at the test concentration of 180 mg/L, however statistically significantly toxic effects (P < 0.05) were shown at the test concentrations of 320 and 560 mg/L. Based on these results the No Observed Effect Concentration (NOEC) after 3 hours exposure was 180 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.

Description of key information

The 3-h NOEC value is 180 mg/L as observed in a respiration inhibition study with activated sludge of a predominantly domestic sewage.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

180 mg/L

Additional information

The toxicity of the substance to microorganisms was investigated in an activated sludge respiration inhibition study according to OECD guideline No 209 and in compliance with GLP criteria. In this study activated sludge from a predominantly domestic sewage was exposed to nominal test substance concentrations of 0, 180, 320 and 560 mg/L for 3 hours. Test concentrations were not analytically verified. The effects on microorganisms was determined by measuring total respiration rates and comparing these to an unexposed control. Inhibition of respiration rates was observed at 320 mg/L (0 -15%) and 560 mg/L (15 -18%). The 3-h NOEC is determined at 180 mg/L.