Carbazamidine sulphate

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see Remarks

Remarks:

This study was performed according with OECD Guideline No. 209 and in accordance with GLP principles. Deficiencies: the pH was not adjusted in the highest test concentration (1000 mg/L). Nonetheless, the test is deemed acceptable as the EC50 exceeded 1000 mg/L.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Guanidinium phosphate (1:1)

EC Number:

226-551-9

EC Name:

Guanidinium phosphate (1:1)

Cas Number:

5423-22-3

Molecular formula:

CH5N3.H3O4P

IUPAC Name:

Guanidium phosphate (1:1)

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 10 g/L was prepared by adding 5.0 g test substance to 500 mL of tap water purified by reverse osmosis. Vigorous mixing (vortex) was used to dissolve the test substance in the test medium. Volumes of the stock solution were then added to the test media to prepare the desired test concentration. Magnetic stirring was applied during the distribution of the stock solution into the test vessels to ensure homogeneity.
- Controls: blank control, nitrification control, positive (reference substance) control and abiotic control
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Method of cultivation: The sludge was coarsely sieved, washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The batch of sludge was used one day after collection; therefore 50 mL of synthetic sewage feed was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Hardness:

Not reported

Test temperature:

19.2 - 20.8 °C

pH:

Before addition of sludge: 6.2 - 7.8
End of exposure period: 6.3 - 7.9

Nominal and measured concentrations:

- Nominal: blank control, 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass, 1 litre test bottles
- Aeration: Clean, oil-free air. The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension
- No. of vessels per concentration (replicates): 3 for the highest test concentration; 1 for the lowest test concentrations
- No. of vessels per control (replicates)
Blank control: 2
Nitrification control: 1
Abiotic control: 1
Positive control (reference substance): 1
- Biomass loading rate (suspended solids): 1.5 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis
- Culture medium different from test medium: Yes
- Intervals of water quality measurement: pH at the begining and the end of the exposure period. Temperature at the end of the exposure period.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Oxygen consumption (respiration rate). After the 3-hour contact time the oxygen consumption was recorded for a period of 15 to 17 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. This procedure was repeated for all test/reference substance concentrations and controls.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- The combined limit/range-finding test showed 19%, 22% and an average of 38% inhibition of the respiration rate at 10, 100 and 1000 mg/L, respectively. The EC50 was above the highest concentration rate tested (EC50>1000 mg/L). Since Guanidine phosphate (1:1) decreased the respiration rate of waste water (activated sludge) bacteria at the highest concentration tested, a NOEC could not be determined.

- There was no oxygen uptake from abiotic processes. The results at 1000 mg/L with a nitrification inhibitor showed 39% heterotrophic inhibition of the respiration rate.

- Acceptability of the test:
1. The mean control oxygen uptake rate was 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour. The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test.
2. The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (9.4 mg/L)

Results with reference substance (positive control):

3,5-dichlorophenol: EC50 was 9.4 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Guanidine phosphate (1:1) significantly inhibited the respiration rate at a concentration of 1000 mg/L. The NOEC could not be determined and the EC50 exceeded 1000 mg/L.

Executive summary:

In a 3 h activated sludge respiration inhibition study conducted according to OECD guideline 209, microorganisms in activated sludge were exposed to Guanidine phosphate (1:1) at the following nominal concentrations: 0 (blank control), 10, 100 and 1000 mg/L. The test substance significantly inhibited the respiration rate at a concentration of 1000 mg/L. The NOEC could not be determined and the EC50 was >1000 mg/L. The study is considered to be reliable with restrictions since the pH was not adjusted in the highest test concentration (1000 mg/L). Nonetheless, the test is deemed acceptable as the EC50 exceeded 1000 mg/L.