N-cyclopropyl-1,3,5-triazine-2,4,6-triamine

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1979/08/06 to 1979/09/24

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Study was not performed under GLP conditions. The guidelines followed in this study are not stated. No deviations occurred in the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Report date:

1979

Materials and methods

Test guideline

Qualifier:

no guideline followed

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-cyclopropyl-1,3,5-triazine-2,4,6-triamine

EC Number:

266-257-8

EC Name:

N-cyclopropyl-1,3,5-triazine-2,4,6-triamine

Cas Number:

66215-27-8

Molecular formula:

C6H10N6

IUPAC Name:

N2-cyclopropyl-1,3,5-triazine-2,4,6-triamine

Specific details on test material used for the study:

The test material is cyromazine.

Sampling and analysis

Details on sampling:

One ml of the suspension in each flask (during aeration) was removed and diluted to 10 ml with sterile tap water. A uniform suspension of solids was maintained by vortexing. Plate counts from the serial dilutions of each sludge flask were tabulated in numbers of colony forming units per (CFU) mL of sludge suspension. Protozoa were examined by placing one 25 µL drop of sludge suspension from each flask on a glass slide for examination by phase contrast microscopy (100X). Motile protozoa greater than 50 µm in length were counted.

Test solutions

Details on test solutions:

Seven hundred milliliters of activated sludge suspension (suspended solids adjusted to 2.5 g/L), 43 mL of synthetic sewage stock and 1357 mL of aged tap water were added to each chamber. Aeration of 500 ml/min was begun and continued for 23 hours to complete the acclimation period.

Test organisms

Test organisms (species):

activated sludge

Study design

Test type:

static

Limit test:

no

Total exposure duration:

5 d

Test conditions

Test temperature:

21 ± 2°C

Dissolved oxygen:

>3.0 ppm

Nominal and measured concentrations:

Nominal concentration (mg/L): 0.1, 1.0, 10.0, 50.0, 100.0

Details on test conditions:

Test chambers had a total volume of 2800 mL (2100 mL operating volume) and were fed compressed air at a rate of 500 mL/min. Purged air exited via a volatiles and CO2 trap. The daily feed volume was 1400 mL with 700 mL of settled sludge remaining in the system from the previous cycle.

Before initiating the study, the suspended solids were adjusted to 1.3 g/L and a 23-hour acclimatization cycle was run the test material was mixed with non-labelled test material and DMSO to produce solution A, which had a specific activity of 0.519 µCi/mg An aliquot (0 1 mL) of solution A was added to 9.9 mL of DMSO to give solution B.

In establishing the individual test chambers 700 mL of fresh activated sludge suspension (adjusted to 2.5 g/L), 43 mL of synthetic sewage stock and 1375 ml of aged tap water was added to each vessel. Aeration was begun and maintained throughout the 23-hour acclimatization period. At the end of each 23-hour cycle, but prior to discontinuing aeration, 210 mL of suspensions were removed to determine microbial counts, protozoa counts, suspended solids, % settled solids, effluent turbidity and to obtain solids and supernatant samples for radio chemical analysis.

Aeration was then discontinued for 30 minutes allowing solids to settle, during which time the dissolved oxygen, pH and temperature of each flask was measured. Finally 1200 mL of the supernatant fluid was withdrawn and all flasks were recharged with 1400 mL of diluted synthetic sewage stock plus the daily dose of chemical Therefore during the 5 day test the concentration started at 0.1 mg/kg and increased daily to 100 mg/kg on day 5.

Reference substance (positive control):

yes

Remarks:

positive (HgCl2) control negative (DMSO) control

Results and discussion

Effect concentrations

Key result

Dose descriptor:

EC50

Effect conc.:

> 100 other: mg/kg

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth inhibition

Details on results:

The temperature of test chambers remained at 21 ± 2°C in all flasks and the dissolved oxygen content (D O ) was greater than 3 0 ppm for the control and cyromazine treated flasks throughout the five day study. Each cycle lasted 23-hours compared to the normal test time of 3 hours The suspended solids, which were, initially 1,300mg/L increased to 1800 mg/L in the cyromazine treatment and 1700 mg/L in the HgCl2 and DMSO controls. However, the flocculated solids were higher in the positive control compared to the cyromazine treatment and the DMSO control.

The turbidity of the 10 ppm and above HGCl2 treated flasks also increased significantly compared to the cyromazine treatment and the negative (DMSO control). The pH of the cyromazine treated flasks and the DMSO control dropped from 7 0 to ~5 4, whereas the pH of the HgCl2 treated flasks increased from 7.0 to 7.9

Higher microbial counts (bacteria, yeasts and actinomycetes) were recorded in the HgCl2 treated flasks but no protozoa were observed in these flasks (essential for eating pathogenic microbes in sewage). Radioactive recovery studies demonstrated that 94% of the recovered radioactive cyromazine was in the supernatant and 1 3 % was in the solids. Less than 0 1% of the radioactivity observed was in the 14C volatile or CO2 trap. No radioactive melamine was recovered

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this activated sludge model, the test material at increasing concentration up to 100 mg/kg had minimal effect on the system therefore, the EC50 is > 100 mg/kg test material (the highest rate tested).

Executive summary:

The effect of cyromazine on the operating parameters and microbial populations of an activated sludge system was investigated under controlled laboratory conditions. The effect of radiolabelled CGA 72662 on the test system was compared to a negative (DMSO) and positive (HgCl₂) control. The study was not performed under GLP conditions. The guidelines followed in this study are not stated. No deviations occurred in the study. 

 

The suspended solids, which were, initially 1300 mg/L increased to 1800 mg/L in the CGA 72662 treatment and 1700 mg/L in the HgCl₂ and DMSO controls. However, the flocculated solids were higher in the positive control compared to the cyromazine treatment and the DMSO control. The turbidity of the 10 ppm and above HGCl₂ treated flasks also increased significantly compared to the cyromazine treatment and the negative (DMSO control).

 

The pH of the cyromazine treated flasks and the DMSO control dropped from 7.0 to ~5.4, whereas the pH of the HgCl₂ treated flasks increased from 7.0 to 7.9 Higher microbial counts (bacteria, yeasts and actinomycetes) were recorded in the HgCl₂ treated flasks but no protozoa were observed in these flasks (essential for eating pathogenic microbes in sewage). Radioactive recovery studies demonstrated that 94% of the recovered radioactive cyromazine was in the supernatant and 1.3 % was in the solids. Less than 0.1% of the radioactivity observed was in the ¹⁴C volatile or CO₂ trap No radioactive melamine was recovered.

 

In this activated sludge model cyromazine at increasing concentration up to 100 mg/kg had minimal effect on the system therefore, the EC₅₀ is > 100 mg/kg cyromazine (the highest rate tested)