N-(2-nitrophenyl)phosphoric triamide

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-12-09 - 2006-03-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

50 mg test item were dissolved in 100 ml water. To accelerate the process it was ultrasonic-treated for 3 minutes. At the start of the test the formulation, 16 ml synthetic sewage feed and 200 ml inoculum were mixed in the test vessel and filled up to 500 ml with water. During the contact time the vessels were continuously aerated.

Test organisms (species):

activated sludge

Details on inoculum:

The inoculum was derived from a treatment plant (Gemeinschaftsklarwerk Bitterfeld-Wolfen) which processes predominantly domestic sewage. A fresh sample of activated sludge was washed with tap water and the sample was adjusted for a level of 4 g/l suspended solids. The activated sludge was aerated until use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20.6°C

pH:

7.7 - 7.9

Nominal and measured concentrations:

100 mg/l nominal

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Contact time	Test point	Inhibition(%)
3 hours	Test item, 100 mg/1	0
	Reference substance, 4 mg/1	12
	Reference substance, 12 mg/1	53
	Reference substance, 36 mg/1	84

Validity criteria fulfilled:

yes

Conclusions:

After a contact of 3 hours no inhibition of the respiration rate by the test item at a concentration of 100 mg/1 was measured. For this reason in the present study the test item seems to be non-toxic in the respiration test at the concentration tested. Thus, the EC50 of the test item is greater than 100 mg/1.

Executive summary:

The inhibitory effect of N-(2-Nitrophenyl)phosphoric triamide on bacteria was tested by measuring the respiration rate of activated sludge. The study was performed according to the EU guideline No. C. 11. The bacteria of activated sludge from a sewage treatment plant were exposed to the test item at a concentration of 100 mg/1. The test item solution, activated sludge and synthetic sewage feed were mixed in a test vessel and the content of the vessel was aerated during the incubation period. After a contact time of 3 hours the respiration rate was measured by an oxygen electrode. No inhibition of the respiration activity of the bacteria was measured after the 3 hours contact time. The result demonstrates that the median effective concentration (EC50) is greater than the test concentration of 100 mg/1.

Reference 2

Endpoint:

activated sludge nitrification inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-06-16 - 2014-09-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 9509 (Toxicity test for assessing the inhibition of nitrification of activated sludge microorganisms)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

A stock solution of the test item with a concentration of 1.0018 g/L was prepared.

Test organisms (species):

activated sludge

Details on inoculum:

Specification
Activated sludge from a biologic sewage treatment plant was used. The chosen plant treats mostly household sewage.
Source and Pre-Treatment
Source
The sludge was collected from the activation basin of the ESN (Stadtentsorgung Neu-stadt) sewage treatment plant, Im Altenschemel, 67435 NW-Lachen-Speyerdorf.
Date of collection: 17. July 2014
Pre-Treatment
Upon arrival in the test facility, the sludge was filtered, washed with tap water and re-suspended in tap water. The activated sludge was aerated until use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

4 h

Nominal and measured concentrations:

100 mg/l

Reference substance (positive control):

yes

Remarks:

N-Allylthiourea in a concentration of 11.6 mg/L was used as positive control for inhibition of nitrification. At this concentration, complete inhibition of nitrification was reached.

Key result

Duration:

4 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Results with reference substance (positive control):

N-Allylthiourea in a concentration of 11.6 mg/L was used as positive control for inhibition of nitrification. At this concentration, complete inhibition of nitrification was reached.

Inhibition of Nitrification and Oxygen Concentration

Number	Content	Inhibition in %	O2concentration in mg/L
1	Control without urea	0 %	8.2
2	Control with urea	--	8.2
3	Positive Control without urea	100 %	8.2
4	Positive Control with urea	--	8.4
5	Test Item with urea 3.2mg/L	--	7.9
6	Test Item with urea 1 mg/L	--	8.3
7	Test Item with urea 0.32 mg/L	--	8.0
8	Test Item with urea 0.1 mg/L	--	8.3
9	Test Item with urea 0.032 mg/L	--	8.0
10	Test Item with urea 0.01mg/L	--	8.3
11	Test Item without urea 100 mg/L	5.03 %	8.0

Validity criteria fulfilled:

yes

Conclusions:

The test item was tested using six concentrations ranging from 0.01 to 3.2 mg/L test item and with urea and one concentration containing 100 mg/L without urea. At the highest test concentration 100 mg/L, no significant inhibition of nitrification could be observed (5 %).

Executive summary:

One valid experiment was performed.

The result of a non-GLP pre-test was used to define the test conditions and the treatments to be tested in the experiment under GLP conditions.

The test for inhibition of nitrification was performed as a limit test at the concentration 100 mg/L.

The test item is a urease inhibitor. Therefore, for testing whether formation of ammonium from urea in presence of the test item was affected, additional replicates (control, positive-control and test replicates) were tested. In these additional replicates, the nutrient medium was supplemented with 2.15 g/L urea (CH₄N₂O), corresponding to 1 g/L nitrogen from urea (CH₄N₂O-N). The positive control with urea was not necessary for evaluation of the results.

In the main study, the test item was tested using six concentrations ranging from 0.01 to 3.2 mg/L test item in nutrient medium supplemented with urea and one concentration containing 100 mg/L in nutrient medium without urea. Urease inhibition was estimated by the content of total inorganic nitrogen (NH₄-N, NO₂-N and NO₃-N) at the end of the test in the test replicates with urea, compared with the control replicate with urea.

The duration of the test was four hours. Activated sludge was used as the inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 3.14 g suspended solids/L, giving a concentration of 1.57 g suspended solids/L in the test.

N-Allylthiourea in a concentration of 11.6 mg/L was used as positive control for inhibition of nitrification. At this concentration, complete inhibition of nitrification was reached.

 

The following results were detected for the test item 2-NPT:

 

Inhibition of nitrification:

4h EC50 > 100 mg/L

 

Urease inhibition:

4h EC50 = 10 µg/L

Description of key information

EU C.11 guideline study, GLP: EC50: >100 mg/l

ISO 9509 guideline study, GLP: EC50: > 100 mg/l

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L

Additional information

EU C.11:

The inhibitory effect of N-(2-Nitrophenyl)phosphoric triamide on bacteria was tested by measuring the respiration rate of activated sludge. The study was performed according to the EU guideline No. C. 11. The bacteria of activated sludge from a sewage treatment plant were exposed to the test item at a concentration of 100 mg/1. After a contact time of 3 hours the respiration rate was measured by an oxygen electrode. No inhibition of the respiration activity of the bacteria was measured after the 3 hours contact time. The result demonstrates that the median effective concentration (EC50) is greater than the test concentration of 100 mg/1.

ISO 9509:

The test for inhibition of nitrification was performed as a limit test at the concentration 100 mg/L.

The test item is a urease inhibitor. Therefore, for testing whether formation of ammonium from urea in presence of the test item was affected, additional replicates (control, positive-control and test replicates) were tested. In these additional replicates, the nutrient medium was supplemented with 2.15 g/L urea (CH₄N₂O), corresponding to 1 g/L nitrogen from urea (CH₄N₂O-N). The positive control with urea was not necessary for evaluation of the results.

In the main study, the test item was tested using six concentrations ranging from 0.01 to 3.2 mg/L test item in nutrient medium supplemented with urea and one concentration containing 100 mg/L in nutrient medium without urea. Urease inhibition was estimated by the content of total inorganic nitrogen (NH₄-N, NO₂-N and NO₃-N) at the end of the test in the test replicates with urea, compared with the control replicate with urea.

The duration of the test was four hours. Activated sludge was used as the inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 3.14 g suspended solids/L, giving a concentration of 1.57 g suspended solids/L in the test.

N-Allylthiourea in a concentration of 11.6 mg/L was used as positive control for inhibition of nitrification. At this concentration, complete inhibition of nitrification was reached.

 

The following results were detected for the test item 2-NPT:

 

Inhibition of nitrification:

4h EC50 > 100 mg/L

 

Urease inhibition:

4h EC50 = 10 µg/L