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

Long-term toxicity to fish

Administrative data

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983/07/27 to 1983/08/28

Reliability:

1 (reliable without restriction)

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

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

Test material form:

solid: particulate/powder

Remarks:

Colorless powder

Specific details on test material used for the study:

The test material is cyromazine.

Sampling and analysis

Analytical monitoring:

yes

Remarks:

Analytical determinations of the test substance concentrations were made at test start and 7, 14, 21 and 28 days

Details on sampling:

Fifty healthy fertile eggs were randomly assigned to each eggcup and 24 and 48 hours after introduction any dead, or fungally infected eggs were removed and the number off eggs per cup was standardised to 32. From the third day of exposure the fish were fed a mixture of newly emerged brine shrimp and brine shrimp puree. Test chambers were cleaned daily and any dead fish were noted and removed. The quality of the water was monitored throughout the 32-day exposure period. At the end of the test all surviving fish were weighed and measured. Samples were taken on Days 0, 7, 14, 21, and 28 of this study.

Test solutions

Details on test solutions:

A proportional diluter was used to deliver five test concentrations and a control. Stock solutions of cyromazine were prepared with distilled water in a 12-gallon glass carboy. The stock solution was stirred continuously and delivered to the diluter with a COLE-Parmer Masterflex Pump. The diluter was calibrated to deliver 100%, 51%, 27%, 15% and 6% cyromazine and dilution water for the controls. Test solution was delivered every 14.9 minutes.

Test organisms

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ANIMALS
- Source: External Non-Lab source
- Age at study initiation: Early life stage

50 healthy fertile eggs used in study, fertilized flathead minnows Pimephales promelas, <48 hours old, were used for the early life stage test.

Study design

Test type:

flow-through

Limit test:

no

Total exposure duration:

32 d

Test conditions

Hardness:

34 - 48 mg CaCO3/L

Test temperature:

24.2 - 26.0°C

pH:

6.7 - 6.8

Dissolved oxygen:

4.2 - 7.5 mg/L

Conductivity:

95-107 µmhos/cm2

Nominal and measured concentrations:

Nominal concentrations (mg/L): 17, 43, 77, 147 and 290

Details on test conditions:

2 replicate exposure chambers were used for each treatment and control
Exposure chambers were made out of glass, 19.7 cm wide 40 cm long drain hole 4.4 cm from bottom on one end
Total volume: 3.5 L
3 - 4 cm in from the drain dole, a 400-micron mesh nitrex screen was sealed to glass with silicone cement. Each chamber contained a eggcup with a 1 pint glass jar. The bottom of the jar was replaced with a 600-micron nitrex net, this was suspended from a motor driven rocker arm to ensure test organism was submerged in test solution and to allow test solution to flow in and out of egg chamber.

Fresh test solution (200 - 230ml) was automatically delivered to each test chamber every 14.9 minutes giving daily volume exchanges of between 6.4 and 7.2 times

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

> 36 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

adult mortality

Effect concentrations 2

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

> 22 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: Geometric mean value based on length and mass (>14 mg/l and <36 mg/L)

Details on results:

Egg mortality was low in all  chambers,  with  hatching commencing on day 2 and being complete by day 5 At the termination of the experiment it was discovered that uneven numbers of eggs had been left in the chambers (as a result of clumping of eggs in the first 48 hours) however, this did not affect the integrity of the results or their statistical evaluation. Scoliosis (curvature of the spine) occurred in the 147 and 290 mg/L cyromazine treatments, although not all fish were affected. However, in the highest of these two treatments even those fish that were not scoliotic, were not normal in appearance. The Dunnett's test detected statistically significant differences in mean length between the control and 36, 73 and 264 mg/L cyromazine treatments were noted. However when the mean mass of the fish were analysed with the same test all cyromazine of 36 mg/L and above were statistically different from the control treatments.

Reported statistics and error estimates:

The effects of test material on the early life stages of the fathead minnow Pimephales promelas were statistically analysed using the Chi-square approach No significant difference between the control and any of the test material treatments was apparent for the number of fertile eggs at 48-hours, the percentage of eggs that produced live fish at the end of the test and the number of hatched fry which subsequently produced life fish at the end of the test. However, in the 73 mg/L treatment statistically significant differences (95% confidence level) in the percentage of eggs that produced live fry and the number of eggs that produced live, healthy fry. At 127mg and 264 mg/L a significant difference was observed to the control was also apparent for the percentage of eggs, which produced normal fish at the end of the test, and for the highest of these treatments, also for the percentage of hatched fry that subsequently produced healthy fish at the end of the test.

Any other information on results incl. tables

Physical parameters monitored: Water quality throughout the duration of the study remained within acceptable levels with an average temperature (across all test chambers) of 24 2-26 0°C, and pH between 6 7 and 6 8 Dissolved oxygen ranged from 4 2-7 5mg/L Total water hardness was 34-48 mg CaCO₃/L and the conductivity was 95-107 µmhos/cm².

 

Analytical findings: The measured concentrations were 67 5 to 107 8% of nominal values with the initial measured concentrations being 79 3 to 93 5% of nominal values All results are presented in terms of mean measured values.

 

Mortality and symptoms of toxicity:

 

Table 1

 

Effect of test material, in a flow-through system, on the early life stages of the fathead minnow Pimephales promelas

 

Treatment concentration (mg/L)		% Healthy fertile eggs at 48- hours	% Eggs producing live fry ^a	% Eggs producing live normal fry ^a	% Eggs producing live fish at the end ^a%	% Eggs producing live normal fish at the end ^a%	% Hatched producing live fish at end ^a	% Hatched producing live normal fish at the end ^a
Nominal	Mean measured
Control	Control	83	100	100	97	97	97	97
17	14	88	100	98	89	89	89	89
43	36	88	99	99	99	99	100	100
77	73	80	90 *	90*	87	87	96	96
147	127	78	93	93	86	83*	93	89
290	264	87	95	95	89	69*	93	72*

^a            After egg reduction at 48 hours

*            Significantly different from the control based on a Chi-square analysis of 2x2 contingency tables

 

 

 

Table 2

Mass and length of the surviving  fish  in  each  test  chamber  after  exposure  to test material under flow-through conditions

Test chambers/ concentrations			Mean mass (g)	Mean length (mm)
Nominal Conc	Mean measured conc	Replicate
Control	-	1	0 0925	21 85
		2	0 1154	23 07
17	14	1	0 0836	22 08
		2	0 1035	22 20
43	36	1	0 0887	21 43
		2	0 0830	20 92
77	73	1	0 0822	20 74
		2	0 1122	22 29
147	127	1	0 0823	21 37
		2	0 1061	22 35
290	264	1	0 0535	17 67
		2	0 0474	16 13

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

not specified

Conclusions:

The 32-day NOEC of cyromazine under flow-through conditions in a fish early life stage test with the fathead minnow (Pimephales promelas) was determined to be 22 mg/L based on length and weight.

Executive summary:

The 32-day NOEC of cyromazine under flow-through conditions in a fish early life stage test with the fathead minnow (Pimephales promelas). The test was conducted according to ERT Bioassay Protocol D-SC from July 27, 1983 until August 28, 1983. The final test concentrations were based on the 96-hour LC₅₀ value of 715 mg/L based on measured concentrations. This was determined in a static system on 13-day-old fish prior to the initiation of the flow-through study. 

The 32-day NOEC of cyromazine under flow-through conditions in a fish early life stage test with the fathead minnow (Pimephales promelas) was determined to be >22 mg/L based on length and weight of fish.