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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011/06/03 to 2011/06/28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study performed under GLP conditions. The guidelines followed in this study are shown below:

- OECD Guidelines for the Testing of Chemicals, No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test, 2006.
- Commission Regulation (EC) No 761/2009 C.3: Algal Inhibition Test, 2009.
- JMAFF Test Guidelines, 2-7-7, Algae growth inhibition, 2005

No deviations occurred from these guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EC) No 761/2009 C.3: Algal Inhibition Test, 2009

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: JMAFF Test Guidelines, 2-7-7, Algae growth inhibition

Version / remarks:

2005

Deviations:

no

GLP compliance:

yes

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

Analytical monitoring:

yes

Details on sampling:

At the end of the test, a sample was taken from the control and from the test concentration of nominal 120 mg/L. The shape and size of the algal cells (Pseudokirchneriella subcapitata) were examined microscopically in these samples.

Test solutions

Vehicle:

no

Details on test solutions:

5 concentrations were tested with 1 control. The test design included three replicates per test concentration and six replicates of the control. The test medium of the highest nominal concentration of 120 mg/L was prepared by dissolving 120.09 mg of the test item completely in 1000 mL of test water using ultrasonic treatment for 5 minutes and intense stirring for 15 minutes at room temperature. The test medium of the highest test concentration was serially diluted with test water to prepare the test media of the different test concentrations. The test media were prepared just before the start of the test.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The algae (Pseudokirchneriella subcapitata) were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

Study design

Test type:

static

Water media type:

other: Reconstituted test water

Total exposure duration:

96 h

Test conditions

Hardness:

0.15 mmol/L (= 15 mg/L as CaCO3)

Test temperature:

21 °C

pH:

adjusted to 7.5

Nominal and measured concentrations:

Nominal concentration (mg/L): 0.38, 1.2, 3.8, 12, 38, 120, control group
Mean measured concentration (mg/L): 0.36, 1.2, 4.0, 13, 40 and 124

Details on test conditions:

An inoculum culture was set up three days before the start of the exposure. The algae (Pseudokirchneriella subcapitata) were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions. Potassium dichromate used as positive control, test performed in April 2011 to show that sensitivity of the test system was within the internal historical range. Reconstituted test water was prepared according to OECD test guideline 201, analytical grade salts were dissolved to obtain macro-nutrient and trace element concentrations.

50-mL Erlenmeyer flasks were used per replicate containing 15 mL of test solution. Each test flask was covered with a glass dish. The test flasks were labeled with the study number and all necessary additional information to ensure unique identification. During the test, the test solutions were continuously stirred by magnetic stirrers.

The test medium of the highest nominal concentration of 120 mg/L was prepared by dissolving 120.09 mg of the test item completely in 1000 mL of test water using ultrasonic treatment for 5 minutes and intense stirring for 15 minutes at room temperature. The test medium of the highest test concentration was serially diluted with test water to prepare the test media of the different test concentrations. The test media were prepared just before the start of the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 69 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

yield

Effect concentrations 2

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

40 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

yield

Effect concentrations 4

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 124 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 5

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

13 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

The test item had a significant inhibitory effect on the growth rate µ of the algae (Pseudokirchneriella subcapitata) after the test periods of 96 hours at the highest mean measured concentration of 40 mg/L. The 96-hour NOEC based on the growth rate µ was determined to be 13 mg/L, since up to and including this mean measured test concentration the growth rate of the algae (Pseudokirchneriella subcapitata) after 96 hours was not significantly lower than in the control.

The microscopic examination of the algal cells (Pseudokirchneriella subcapitata) at the end of the test showed no difference between the algae growing at the nominal test concentration of 120 mg/L and the algal cells in the control. The shape and size of the algal cells was obviously not affected by the test item up to at least this concentration.

Any other information on results incl. tables

Table 2

 

Summary of results  

Parameter	After 72 hours			After 96 hours
	AUC	Growth rate	Yield	AUC	Growth rate	Yield
EC50 [mg/L]  95% CI	97 74-124	>124 n.d	75 42-124	75 56-104	>124 n.d	69 52-92
EC20 [mg/L]  95% CI	35 18-49	90 76-104	8.6  2.3-16	33 16-47	97 86-105	36 20-49
EC10 [mg/L]  95% CI	21 7.7-33	45 29-58	2.8 0.32-6.9	22 7.9-34	61 46-72	26 11-37
NOEC [mg/L]	0.36	0.36	0.36	0.36	13	0.36
LOEC [mg/L]	1.2	1.2	1.2	1.2	40	1.2

95% CI: 95% confidence interval
n.d.: not determined

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The ErC50 is > 124 mg/L (growth rate) and NOEC 13 mg/L (growth rate).

Executive summary:

The influence of the Cyromazine on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 96-hour static test according to the OECD Guideline 201 (2006) and the Commission Regulation (EC) No 761/2009, C.3, as well as according to the JMAFF Test Guidelines, 2-7-7 (2005).

The nominal test item concentrations tested were 0.38, 1.2, 3.8, 12, 38 and 120 mg/L. Additionally, a control group was tested in parallel. The measured concentrations of Cyromazine technical in the test media of the test concentrations of 0.38 to 120 mg/L were between 95 and 105% of the nominal values at the start of the test and between 96 and 107% at the end of the test. Thus, the correct dosing of Cyromazine technical was confirmed and the test item was shown to be stable in the test media over the test period of 96 hours. The biological results were related to the mean measured concentrations of Cyromazine.

The mean measured concentrations (calculated as the arithmetic means of the concentrations measured at the start and end of the test) in the test concentrations of 0.38, 1.2, 3.8, 12, 38 and 120 mg/L were 0.36, 1.2, 4.0, 13, 40 and 124 mg/L, respectively.