Amidinourea 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:

26 April 2017 / 13 September 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

Version / remarks:

July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 16VL8189
- Expiration date of the lot/batch: 003 August 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (=< 30 deg C), dark, dry

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No

Analytical monitoring:

not required

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

Limit test: A weight of 499.3 – 501.1 mg of the test item was added directly into the test vessels 7 - 11 with a test volume of 500 mL to obtain a final concentration of 998.6 – 1002.2 mg/L. The pH of the test solution was measured before addition of inoculum. Full test: A Stock solution with 20 g/L test item was prepared (0, 2, 4, 7.2, 14.4 and 25.92 mL test item in stock solution). Five replicates of each concentration were prepared (80.0, 160.0, 288.0, 576.0 and 1037.0 mg/L). The pH of the test solutions were measured before addition of inoculum.

Reference item
Limit test: A weight of 24.8 mg of 3,5-dichlorophenol were dissolved in ultrapure water and filled up to 25 mL using a magnetic stirrer to get a stock solution with 1.0 g/L. 0.5, 5 and 15 mL of the stock solution were added to the reference vessels with a test volume of 500 mL in order to obtain final concentrations of 1, 10 and 30 mg/L.
Full test: A weight of 49.9 mg of 3,5-dichlorophenol were dissolved in deionised water and filled up to 50 mL using a magnetic stirrer to get a stock solution with 1.0 g/L. 0.5, 5 and 15 mL of the stock solution were added to the reference vessels in order to obtain final concentrations of 1, 10 and 30 mg/L.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge from the municipal wastewater treatment plant Staufener Bucht was used as test system. The treatment plant has a capacity of 140 000 inhabitant equivalents and clarifies predominantly domestic wastewater. Sampling date of activated sludge was 26 April 2017 (limit test) and 29 May 2017 (full test). The activated sludge was kept aerated until use at test temperature. Dry solid of the activated sludge was determined as 3.8 g/L (limit test) and 4.4 g/L (full test) by weight measurements before and after drying at 105°C (mean of triplicate measurements). The activated sludge was washed twice by settling the sludge, decanting the supernatant and re-suspending the sludge in chlorine-free tap water. Before using, the activated sludge was diluted to 3 g/L dry solids with chlorine-free tap water, in order to obtain a final concentration of 1.5 g/L dry solids in the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Remarks on exposure duration:

None

Post exposure observation period:

Not applicable

Hardness:

Not measured

Test temperature:

Limit test: 19.7 – 21.8°C.
Full test: 21.4 - 21.8°C

pH:

Limit test: 7.0 – 7.1. (adjusted)
Full test: 6.2- 7.1 (measured), 7.0 - 7.2 (adjusted)

Dissolved oxygen:

5.78 - 8.36 mg/L

Salinity:

Not applicable

Conductivity:

Not measured

Nominal and measured concentrations:

Limit test: 1000 mg/L
Full test: 80.0, 160.0, 288.0, 576.0 and 1037.0 mg/L

Details on test conditions:

Experimental set up
The different vessels werestarted separately with an interval of exactly 15 min in order to be able to measure the respiration rates one by one while making sure that the aeration time is 3 h in every vessel. At time "0", 16 ml synthetic sewage and the test item or reference compound were added to the test vessel and made up to 250 mL with chlorine-free tap water. After measurement and adjustment of pH, 250 mL of activated sludge (3 g/L dry solids) were added consequently obtaining a total volume of 500 ml with 1.5 g/L dry solids. For the controls the same procedure was made as with the test vessels, however without test or reference substance. All vessels were stirred and aerated throughout the contact time with room air to assure an oxygen saturation of 60-70% using a membrane pump. Before starting the test the oxygen saturation rate was measured.

After 3 h the aeration of the first vessel was shut off and the respiration rate was measured in a BOD-bottle with an oxygen electrode while mixing with a magnetic stirrer.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

145.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other:

Remarks:

95%-CL: 1.175 – 15679.727 mg/L

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

< 80 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Details on results:

Limit Test
A limit test with the concentration of 1000 mg/L was performed. The pH in the test vessels was between 6.2 and was adjusted to 7.0 – 7.1.
An inhibition of the total respiration of activated sludge between 62 – 75% was observed in the test vessels with 1000 mg/L test item concentration. In accordance with the sponsor a full test with five concentrations was prepared afterwards.
The respiration rate of six blanks were within 31.2 – 54.7 mg/(L*h) that is equivalent to 20.8 – 36.5 mg/(L*h) per one gram activated sludge. The coefficient of variation (CV) was 19% and therefore less than 30%. The pH in the blank vessels was 7.3.
The temperature range within the test duration was between 19.7 – 21.8°C.

Test item
The inhibiting effect of the test item on the total respiration of activated sludge was observed with five concentrations (80.0, 160.0, 288.0, 576.0 and 1037.0 mg/L).
EC50, EC10, LOEC and NOEC are given by ToxRat Professional Version 3.2.1, © ToxRat Solutions GmbH
The results for the inhibition of the total respiration of activated sludge are therefore:
EC50 = 145.4 mg/L (95%-CL: 1.175 – 15679.727 mg/L)
EC10 = 0.603 mg/L (95%-CL: 0.011 – 31.807 mg/L)
LOEC <= 80.0 mg/L
NOEC < 80.0 mg/L

Blanks
The respiration rate of six blanks were within 19.2 – 32.0 mg/(g sludge*h). The coefficient of variation (CV) was 21.3% and therefore less than 30%. The pH in the blank vessels was between 7.3 – 7.4.
The temperature range within the test duration was between 21.4 and 21.8°C.

Criteria met for the validity of the study
The coefficient of variation (CV) of the blank respiration rates was less than 30%
The blank controls oxygen uptake rate was higher than 20 mg oxygen per one gram of
activated sludge per hour The EC50 of the reference item was within the acceptable range of 2 to 25 mg/l

Results with reference substance (positive control):

In the limit test the EC50 of 3,5-dichlorphenol was calculated as 4.10 mg/l and was therefore within the acceptable range of 2 to 25 mg/l.
In the full test the EC50 of 3,5-dichlorphenol was calculated as 3.047 mg/l (Appendix III) and was therefore within the acceptable range of 2 to 25 mg/l. The pH in the reference vessels was 7.4.

Validity criteria fulfilled:

yes

Conclusions:

Limit test with test concentration 1000 mg/L:
An inhibition between 62 and 75% was observed in the test vessels with 1000 mg/L test concentration (five replicates).

Full test with five concentrations (80.0, 160.0, 288.0, 576.0 and 1037.0 mg/L):
EC50 = 145.4 mg/L (95%-CL: 1.175 – 15679.727 mg/L)
LOEC <= 80.0 mg/L
NOEC < 80.0 mg/L
(ToxRat Professional Version 3.2.1, © ToxRat Solutions GmbH)

Executive summary:

An OECD 209 Activated sludge inhibition test (2010) was conducted with the test item. This was to assess the inhibition effect of different concentrations of test item (80.0, 160.0, 288.0, 576.0 and 1037.0 mg/L) to micro-organisms of activated sludge by measuring the respiration rate after adding a standard amount of synthetic sewage feed. The test met all guideline validity criteria.  

The results for the inhibition of the total respiration of activated sludge were:

EC50 = 145.4 mg/L (95%-CL: 1.175 – 15679.727 mg/L)

EC10 = 0.603 mg/L (95%-CL: 0.011 – 31.807 mg/L)

LOEC <= 80.0 mg/L

NOEC < 80.0 mg/L

Description of key information

An OECD 209 Activated sludge inhibition test (2010) was conducted with the test item. This was to assess the inhibition effect of different concentrations of test item (80.0, 160.0, 288.0, 576.0 and 1037.0 mg/L) to micro-organisms of activated sludge by measuring the respiration rate after adding a standard amount of synthetic sewage feed. The test met all guideline validity criteria.  

The results for the inhibition of the total respiration of activated sludge were:

EC50 = 145.4 mg/L (95%-CL: 1.175 – 15679.727 mg/L)

EC10 = 0.603 mg/L (95%-CL: 0.011 – 31.807 mg/L)

LOEC <= 80.0 mg/L

NOEC < 80.0 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:

145.4 mg/L