Methyl dihydrogen 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:

1 March 2018 to 19 July 2018

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

yes

Remarks:

The temperature in the Pre-test: 21.2 – 22.8°C The temperature in the main test: 20.3 – 22.7°C These deviations were stated as uncritical, as normal respiration activity of the control could be observed.

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

yes

Remarks:

The temperature in the Pre-test: 21.2 – 22.8°C The temperature in the main test: 20.3 – 22.7°C These deviations were stated as uncritical, as normal respiration activity of the control could be observed.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch 34448
Purity 67.9 - 71.2% Methyl dihydrogen phosphate
Expiry 30 March 2022

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

Due to the poor solubility of the test item, the test item was pipetted directly into the test vessels, using the density (1.55 g/cm3) given by the sponsor.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge from a biologic sewage treatment plant was used. The chosen plant treats mostly domestic sewage.

Source and Pre-Treatment
The sludge was taken from the activation basin of the sewage treatment plant in D-67480 Edenkoben. Upon arrival in the test facility, the sludge was filtrated, washed with tap water 3 times and re-suspended in tap water. The activated sludge was aerated until usage in the test and fed daily with 50 mL synthetic sewage feed /L.

The specification of the test system is given in the table below.

Preparations
On the day before the experiment, the inoculum was taken from its source, washed, aerated and the dry matter was determined. Volume was adapted to the desired content of dry matter. The nutrient solution was thawed and the sludge was fed with 50 mL nutrient solution / L sludge. On the day of the experiment, the dry matter of the inoculum was determined once more.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

After 3 hours, the content of the first vessel was shaken vigorously for 30 seconds, the narrow neck bottle was filled to the rim with an aliquot of the content of the first vessel, a magnetic stirrer was added, and the oxygen electrode was tightly fitted into the vessel. Care was taken that no air could reach the contents of the vessel. The oxygen concentration of the vessel was measured every 5–10 seconds over a period of 3-5 minutes. The interface of the oxygen meter was connected to a personal computer, feeding the measured values with their corresponding times in a table. The following vessels were measured likewise in 5 minutes intervals.

Test temperature:

20.3 - 22.7oC

pH:

Blank & Positive Control 7.8-8.0
Test 4.8-8.0
pH of 1000 mg/L 4.8-5.0
pH of 30 mg/L 7.1-7.2
pH of 100 mg/L 7.7
pH of 32 mg/L 7.9
pH of 10 mg/L 8.0-8.1

Nominal and measured concentrations:

Nominal test concentrations: 10, 32, 100, 320 and 1000 mg/L

Details on test conditions:

In the blank control vessels, 16 mL nutrient solution was mixed with 234 mL water. The positive control and the treatments were prepared by putting the appropriate amount of positive control solution respectively test item into the test vessel, adding 16 mL nutrient solution and water to give 250 mL. Then, in 5 minutes intervals, 250 mL inoculum was added and closed with sealed lids. After 3 hours, the content of the first vessel was shaken vigorously for 30 seconds, the narrow neck bottle was filled to the rim with an aliquot of the content of the first vessel, a magnetic stirrer was added, and the oxygen electrode was tightly fitted into the vessel. Care was taken that no air could reach the contents of the vessel. The oxygen concentration of the vessel was measured every 5–10 seconds over a period of 3-5 minutes. The interface of the oxygen meter was connected to a personal computer, feeding the measured values with their corresponding times in a table. The following vessels were measured likewise in 5 minutes intervals.

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

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

180 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

440 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Two experiments were performed. The first experiment is not relied upon here.

The coefficient of variation of oxygen uptake rate in control replicates was below 30% at the end of the test. The oxygen uptake rate of the blank controls was above 20 mg O2 per gram activated sludge in 1 hour.

The test item was tested using 5 concentrations ranging from 1000 to 10 mg/L nominal concentration. At the highest concentration 1000 mg/L 97.3 – 100.1 % inhibition was observed. In the concentration 320 mg/L the inhibition reached from 5.8 to 13.6 %. The NOEC can be stated as 100 mg/L, the EC10 as 180 mg/L (confidence interval 130 – 230 mg/L) and the EC50 was determined with 440 mg/L (confidence interval 350 – 590 mg/L). All replicates showed very good correlation. In both experiments inhibition at the concentration 1000 mg/L nearly complete inhibition was observed. At this concentration the pH was clearly lower than in the lower concentrated treatments. An influence of the low pH can not be excluded. However, at the lower concentration 320 mg/L also significant inhibition was observed and the pH at this concentration was in the range 7.1 - 7.2. The strong inhibition at the highest concentration is therefore undoubtedly mainly caused by the toxicity of the test substance. No inconsistencies in the dose-response estimation could be observed. Therefore, no further experiment was performed in order to discern between inhibition of nitrificators and inhibition of total population.

The result of the test can be considered valid.

Results with reference substance (positive control):

The EC50 of 3,5-Dichlorophenol in the second test was 9.9 mg/L which lies within the recommended limit of 2-25 mg/L.

Reported statistics and error estimates:

NOEC 100 mg/L
EC10 180 mg/L (confidence interval 130 – 230 mg/L)
EC50 440 mg/L (confidence interval 350 – 590 mg/L)

Validity criteria fulfilled:

yes

Conclusions:

NOEC 100 mg/L
EC10 180 mg/L (confidence interval 130 – 230 mg/L)
EC50 440 mg/L (confidence interval 350 – 590 mg/L)

Executive summary:

The effects seen at 320 mg/L were around 10% inhibition (average inhibition of 10.64%) and the pH was 7.1 -7.2 at the end of the test.

The effects seen at 1000 mg/L were close to 100% inhibition (average 98.44%) and the pH was 4.8 -5.0 at the end of the test.

In the first experiment, the effects seen at 1000 mg/L were close to 100% (average 100.9%) and the pH was 3.8 at the end of the test.

Given that the pH would have buffered somewhat over the 3 hour incubation period, it is possible that the pH at the start of the test was lower than that taken at the end of the test. Additionally, the pH differed by one whole unit (on a logarithmic scale) between two different runs at the same rate.

It cannot be excluded that free hydrogen ion concentration was the cause of the inhibition seen at 320 and 1000 mg/L due to the lack of pH data at the start of the test

Description of key information

The EC50 (3h) value was given as 440 mg/L and the NOEC (3 h) was reported as 100 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

440 mg/L

EC10 or NOEC for microorganisms:

100 mg/L