2-{3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazol-3-ium-5-yl}ethyl hydrogen phosphate

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:

16 Mar 2017

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))

Version / remarks:

adopted July 2010

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, Medicines & Healthcare products Regulatory Agency (27 Jun 2016)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Individually weighed quantities of the test item were directly added to the appropriate test vessels.
- Differential loading: Yes
- Controls: Untreated test medium (dechlorinated tap water) + inoculum

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: The inoculum was collected on 15 Mar 2017 from the sludge return line at Burley Menston Sewage Treatment Works (West Yorkshire, U.K) with a predominantly domestic catchment, ensuring a sample relatively free of exogenous material.
- Preparation and Maintenance: The sludge was transported to the test facility in a closed container with adequate headspace to prevent the sample becoming anoxic. On arrival, the sludge was aerated with compressed air and the suspended solids concentration was determined gravimetrically following homogenisation and adjusted to 3 g/L (± 0.3 g/L) using dechlorinated tap water. The sludge was maintained at 20 ± 2 °C with aeration and fed with synthetic sewage concentrate at a rate of 50 mL/L.
- Inoculum: The concentration was 3 g/L after adjustment with dechlorinated tap water. The pH was 5.98 and was adjusted to 7.23 using 0.5 mL 2M NaOH to within the acceptable range of 7.5 ± 0.5.
- Initial biomass concentration: 3 g/L after adjustment with dechlorinated tap water

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C (incubation period)
20 °C (during measurement with Strathtox instrumentation)

pH:

7.26 - 7.39 (control)
7.00 - 7.45 (treatments)

Nominal and measured concentrations:

1, 10, 100, and 1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass conical flasks containing a total volume of 250 mL.
- Aeration: Yes, air was bubbled through the test system for ca. 3 h. The rate of aeration was sufficient to keep the test samples adequately mixed.
- No. of vessels per concentration (replicates): 2 aliquots of each test vessel were used as individual replicates for respiration assessments and 3 at the highest level concentration (1000 mg/L).
- No. of vessels per control (replicates): 6
- Sludge concentration (weight of dry solids per volume): 3 g/L after adjustment with dechlorinated water
- Weight of dry solids per volume of reaction mixture per unit of time: 1.5 g/L suspended solids in test vessels
- Nutrients provided for bacteria: 125 mL synthetic sewage concentrate per test vessel (Prepared with premade concentrate from Strathkelvin, using 1 packet per 250 mL of water)
- Nitrification inhibitor used: N-allylthiourea (ATU)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the sludge was 5.98 and was adjusted to 7.23 using 0.5 mL 2M NaOH, in order to bring it into the acceptable range of 7.5 ± 0.5.
- Details on termination of incubation: After 3 h incubation, 20 mL of each test preparation was transferred to a sample tube containing a PTFE stirrer. N-allylthiourea was added at least 10 min prior to oxygen consumption measurements, where necessary. The dissolved oxygen electrode was sealed in the neck of the flask, ensuring air was completely excluded and the flask contents were constantly stirred during measurements.

EFFECT PARAMETERS MEASURED:
Oxygen consumption was measured over a period of 10 min after 3 h incubation of the test vessels.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: Range-finder/limit test according to guideline
- Test concentrations: 1, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: A range-finder/limit test was undertaken to determine the appropriate conentration levels for a definitive test. The range-finder/limit test showed no inhibition of total, nitrifcation or heterotrophic respiration. As there were sufficient control replicates at the start and end of each exposure period and the highest concentration (1000 mg/L) was conducted in triplicate (according to OECD guideline 209), it was not necessary to carry out any further testing.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (3,5-DCP)

Duration:

3 h

Dose descriptor:

other: NOAEC

Remarks:

No Observed Adverse Effect Concentration

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other:

Remarks:

for total, heterotrophic and nitrification respiration

Details on results:

- Blank controls oxygen uptake rate: The blank control respiration was ≥ 20 mg/g/h.
- Coefficient of variation of oxygen uptake rate in control replicates: The coefficient of variation of the blank control respiration rates were ≤ 30%.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50 (total respiration) = 3.8 mg/L, EC50 (nitrification respiration) = 1.4 mg/L

RANGE-FINDER/LIMIT TEST

Total and heterotrophic nitrification respiration rates were similar across the controls and all treatment levels. Nitrification respiration showed negative inhibition in some levels. No significant inhibition was observed for any type of respiration up to and including 1000 mg/L.

As there was < 50% inhibition observed in the test, the EC50 for total, heterotrophic and nitrification respiration could not be determined and is classed as > 1000 mg/L, which is the highest concentration level used in this test.

As there were slight enhancement effects (observed as negative inhibition), the endpoint is reported as No Observed Adverse Effect Concentration (NOAEC).

VALIDITY CRITERIA

The study satisfied the validity criteria of OECD guideline 209 (Table 1).

 

Table 1: Validity criteria OECD 209.

Criterion from the guideline	Study results	Validity criterion fulfilled
The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.	The blank control respiration rate was ≥ 20 mg/g/h.	Yes
The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.	The coefficient of variation of the blank control respiration rates were ≤ 30%.	Yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

NOAEC (3 h) > 1000 mg/L (nominal, OECD 209)

Additional information

There is one study available in which the toxicity of 2-{3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazol-3-ium-5-yl}ethyl hydrogen phosphate (CAS 10023-48-0) to microorganisms of activated sludge was investigated according to OECD guideline 209 and GLP.

In a range-finder/limit test, domestic activated sludge at a nominal suspended solids concentration of 1.5 g/L was exposed to 1, 10, 100 and 1000 mg/L test item under constant aeration for 3 h. Test vessels with a reference substance (3,5-DCP) and nitrification inhibitor (ATU) were run in parallel. After 3 h incubation, dissolved oxygen consumption (respiration rate) was measured during 10 min with an electrode.

After 3 h, no inhibition of total, nitrification or heterotrophic respiration was found and the determination of an EC50 was not possible as the observed inhibition was < 50%. Since slight enhancement effects were observed (negative inhibition), the endpoint for this test was expressed as No Observed Adverse Effect Concentration. The reported NOAEC (3 h) is 1000 mg/L.