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

Sediment toxicity

Administrative data

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004/01/22 to 2004/04/01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Proposal for a BBA-Guideline, 1995: "Effects of plant protection products on the development of sediment-dwelling larvae of Chironomus riparius in a water-sediment system"

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 219 (Sediment-Water Chironomid Toxicity Test Using Spiked Water)

Version / remarks:

OECD Guidelines for Testing of Chemicals, Proposal for a new Guideline 219, Draft Document February 2001: "Sediment-Water Chironomid Toxicity Test Using Spiked Water"

Deviations:

no

GLP compliance:

yes

Remarks:

with the exception of the preliminary range finder tests

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

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

From each application solution two samples were taken after dosing the test system on Day 0. Water, pore water and sediment samples from the test systems were taken on Day 0 (approximately one hour after the test item application), on Day 7 and at test termination on Day 26. The test systems of the middle and the highest test concentration (nominal 25 µg/L and 100 µg/L) and the control were sampled (control on Days 0 and 26 only). On Day 26 analytical samples were taken out of one test beaker from the biological test. The analytical samples from Day 0 and 7 were taken from test beakers prepared exclusively for analytical measurements in parallel to the biological test.

Test substrate

Vehicle:

no

Details on sediment and application:

An artificial sediment (5% sphagnum peat, 20% kaolin clay, 75% sand and 0 2% CaCO3) was adjusted with KCl to give a final pH of 7.1 and was added to the bottom of the beaker to a depth of approximately 3 cm corresponding to about 675 g wet weight with 46% water content (or approximately 462 g dry sediment). Without disturbing the sediment 1600 mL of test water (M7 medium, initial pH 7.9 ± 0 3) was carefully added to give a water column of 12cm depth. The vessels were prepared seven days before addition of the test animals, and were acclimatized during this period to the test conditions.

Test organisms

Test organisms (species):

Chironomus riparius

Details on test organisms:

At the date when the test animals were placed into the test beakers, the larvae (Chironomus riparius) were 2-3 days old (first-instar larvae).

Five days before adding the larvae (Chironomus riparius) into the test beakers some fresh egg masses were taken from the test organism culture and deposited into small vessels in test water with a small amount of food.

Study design

Study type:

laboratory study

Test type:

static

Water media type:

other: Reconstituted water ("M7-medium") with an initial pH of 7.9

Type of sediment:

artificial sediment

Limit test:

no

Exposure duration

Duration:

26 d

Exposure phase:

total exposure duration

Remarks:

impact on full maturation of the larvae to adult midges

Test conditions

Hardness:

3.4 mmol/L

Test temperature:

Water temperature: 19.6 - 21.1 °C during the experiment

pH:

pH= 7.7 - 8.3

Dissolved oxygen:

6.0 mg/L (= 65% oxygen saturation value at the measured minimal water temperature of 19.6 °C)

Ammonia:

0.38 mg/L

Nominal and measured concentrations:

nominal concentrations: 6.3, 12.5, 25, 50 and 100 µg/L

Details on test conditions:

Four test vessels each of which contained 20 larvae (Chironomus riparius) at the first larval stage (2-3 days old) were established for each test treatment and control. The test vessels were glass beakers (3 L, approximately 13 cm in diameter), which were covered with watch-glasses. Additionally each test beaker was covered with a mosquito net to prevent the escape of emerged adult test animals. An artificial sediment was adjusted with KCl to give a final pH of 7.1 and was added to the bottom of the beaker to a depth of approximately 3 cm corresponding to about 675 g wet weight with 46% water content (or approximately 462 g dry sediment). Without disturbing the sediment 1600 mL of test water (M7 medium, initial pH 7.9 ± 0.3) was carefully added to give a water column of 12 cm depth. The vessels were prepared seven days before addition of the test animals, and were acclimatized during this period to the test conditions. Water levels did not change by more than 10% during the test period.

During the 26 day duration of the study (with exception of the period from introduction of the larvae (Chironomus riparius) until immediately after application of the test item) the water in the water-sediment systems was gently aerated through a glass Pasteur pipette, fixed above the sediment layer. The larvae were randomly assigned to the test vessels 24-hours before the addition of the test treatments and were fed three times a week (until day 24) with finely ground fish food. The test substance was prepared as a stock solution and 10 mL of an appropriate stock was added just below the water surface in each test vessel and gently mixed (without disturbing the sediment) to ensure homogeneous distribution. The number of emerged adults and their sex was recorded daily from Day 10 after application until Day 26 (6 days after emergence of the last test animals in the controls). Only the number of fully emerged male and female midges was counted. The number of visible pupae that failed to emerge was counted separately in each test beaker. Any other symptom of toxicity was also recorded.

Water temperature, pH and dissolved oxygen concentration were recorded before test item application and then weekly until study termination (dissolved oxygen concentrations and the water temperature were measured three times per week). Water hardness and concentration of ammonium were measured in one test beaker of the control and the highest test concentration at the start and end of the study. The concentration of the test substance was also determined. From each application solution two samples were taken after dosing the test system on Day 0. Water, pore water (obtained by centrifuging the wet sediment at 6500 g for 10 minutes) and sediment samples from the test systems were taken on Day 0 (approximately one hour after the test item application), on Day 7 and at test termination on Day 26. The test systems of the middle and the highest test concentration (nominal 25 and 100 µg/L) and the control were sampled (control on Days 0 and 26 only).

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

26 d

Dose descriptor:

LOEC

Effect conc.:

50 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

development rate

Remarks:

and reduced emergence ration, reduced development rates of females and all midges pooled and high number of successfully emerged but dead adults.

Effect concentrations 2

Key result

Duration:

26 d

Dose descriptor:

NOEC

Effect conc.:

25 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

development rate

Remarks:

and reduced emergence ration, reduced development rates of females and all midges pooled and high number of successfully emerged but dead adults.

Details on results:

Physical parameters monitored:
The temperature of the test vessels varied between 19.6 and 21.1°C, pH from 7.7 to 7.9 at test initiation and 8.3 at termination and the dissolved oxygen between 6.8 and 7.4 mg/L at study initiation and 8.3 to 8.9 mg/L at study termination. The total hardness was 3.4 mmol/L at initiation and 3.8 at termination and the ammonium concentration was 0.38 mg/L at initiation and 0.03-0.05 mg/L at study end. Light intensity was 760-990 Lux with 16 hours illumination and a 30 minute transition period between light and darkness.

Analytical findings:
Measured concentrations of cyromazine in the application solution samples ranged from 101 to 103%. The mean measured concentrations of cyromazine in the water columns one hour after the test item application corresponded to 85 and 113% of the nominal test concentrations of 25 and 100 µg/L, respectively.
Seven days after the test item application the concentrations in the water columns corresponded to 55 and 72% of the nominal initial concentrations of 25 and 100 µg/L (12.9 and 71.6 µg/L, respectively). At study termination the test concentrations corresponded to 51 and 77% of the initial nominal test concentrations (12.8 and 76.5 µg/L, respectively).

In the pore water, the concentrations of cyromazine were below the limit of detection of 3 µg/L In the sediment, the concentrations of cyromazine were below the limit of quantification at Day 0 and increased to 226 and 211 µg/kg dry sediment at Day 7 and to 74 and 203 µg/kg dry sediment at Day 26, respectively. Based on these analytical findings all results will therefore be based on nominal values.

Mortality and symptoms of toxicity:
At the lowest test concentration of 6.3 µg/L, the emergence ratio was statistically significantly lower than in the control. However, since the emergence ratios at of 12.5 and 25 µg/L did not differ statistically significantly from the control, the statistically significant difference between the test concentration of 6.3 µg/L and the control was not considered to be biologically significant. The emergence ratios at the test concentrations of 50 and 100 µg/L were statistically significantly lower than in the control.

For the males, at test concentrations of 12.5, 50 and 100 µg/L, the development rates of the males were statistically significantly lower than in the control. However, at 12.5 µg/L the reduction was not considered to be biologically significant because no significant effects were evident at a concentration of 25 µg/L. For males and females pooled the development rates up to and including the test concentration of 25 µg/L did not differ significantly from the control. For the females, at test concentrations of 50 and 100 µg/L, the development rates of female midges were statistically significantly reduced compared with the control. At 50 µg/L, 33 successfully emerged adult midges (41% of the introduced larvae) were found to be dead on the water surface.

Normally, less than 10% of the emerged midges were observed dead on the water surface, mainly due to flying problems at high densities. This unusually high number was assumed to be caused by an unknown effect of the test item. Since these dead midges did not show any observable signs of developmental deformations, they were taken into account for the calculation of the emergence ratios and the development rates. Note that a separate statistical analysis of data excluding the successfully emerged, but dead midges resulted in the same NOEC- and LOEC-values for emergence ratio and development rate.

The 26-day EC10 for the development rates of the males and females were calculated to be 24 and 38 µg/L respectively (EC10 for the pooled sample was 36µg/L) and the 26-day EC50 was calculated to be 63 µg/L for both sexes. No other symptoms of toxicity were apparent. Based on emergence and development of Chironomus riparius the 26-day NOEC was 25µg cyromazine/L and the 26-day LOEC was 50µg/L due to the reduced emergence ratio of the midges, the reduced development rates of the females and of all midges pooled, respectively and the high number of successfully emerged, but dead adults at this concentration.

Any other information on results incl. tables

Table 1

 

Effect of cyromazine on the emergence rate of Chironomus riparius (male and female) when added to the water column

Parameter assessed	Test item concentration (µg/L)
	Control		6.3		12.5		25		50		100
Mean  number of emerged midges [%]	90.0		76.3		88.8		80.0		42.5		0.0
Emergence	1.254	±	1.065	±	1.235	±	1.109	±	0.707	±	0.000	±
ratio (mean ± SD)	0.071		0.077		0.082		0.051		0.155		0.000
% of control	-		84.9*		98.5		88.4		56.4*		0.0*

*Statistically different from the control (Dunnett-test, a = 0.05)

 

 

Table 2

 

Effect of cyromazine on the development rate of Chironomus riparius when added to the water column

Nominal cyromazine concentration (µg/L)t	Mean development rate (day^-1) ± SD						% of control
	Male		Female		Pooled		Male	Female	Pooled
Control	0.0736	±	0.0629	±	0.0676	±	-	-	-
	0.0021		0.0017		0.0017
6.3	0.0705	±	0.0635	±	0.0673	±	95.8	100.8	99.5
	0.0018		0.0010		0.0007
12.5	0.696 ± 0.0012		0.0611	±	0.0658	±	94.5*	97.1	97.3
			0.0026		0.0019
25	0.0716	±	0.0633	±	0.0684	±	97.3	100.6	101.1
	0.0019		0.0029		0.0025
50	0.0695	±	0.0567	±	0.0619	±	94.4*	90.1*	91.6*
	0.0037		0.0061		0.0028
100	0.000 ± 0.000		0.000 ± 0.000		0.000 ± 0.000		0.0*	0.0*	0.0*

*Statistically different from the control Dunnett-test, a = 0.05

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

When cyromazine was added to a water sediment system (water column) and the effect on Chironomus riparius
studied the 26-day NOEC was 25 µg/L and the LOEC 50 µg/L.

Executive summary:

The effect of cyromazine on the emergence ratio and development time/rate of Chironomus riparius relative to an untreated control was studied in a water-sediment test system following the Proposal for a BEA-Guideline: “Effects of plant protection products on the development of sediment-dwelling larvae of Chironomus riparius in a water-sediment system” (1995), and the OECD Guidelines for Testing of Chemicals, Proposal for a new Guideline 219, Draft Document: “Chironomid Toxicity Test Using Spiked Water" (2001).

 

First-instar larvae of Chironomus riparius were exposed for a period of 26 days until full maturation of the larvae to adult midges. The test parameters of the study were development time/rate of the midges and emergence ratio as the number of fully emerged male and female midges. The test item was applied to the water column in static water-sediment systems. The nominal initial test item concentrations in the overlaying water columns were 6.3, 12.5, 25, 50 and 100 µg/L of cyromazine. A control (water-sediment systems without test item application) was tested in parallel.

 

Seven days after the test item application the concentrations in the water columns corresponded to 55 and 72% of the nominal initial concentrations of 25 and 100 µg/L (13.9 and 71.6 µg/L, respectively). At study termination the test concentrations corresponded to 51 and 77% of the initial nominal test concentrations (12.8 and 76.5 µg/L, respectively).

 

Based on emergence and development of Chironomus riparius the 26-day NOEC was 25 µg cyromazine/L and the 26-day LOEC was 50 µg/L due to the reduced emergence ratio of the midges, the reduced development rates of the females and of all midges pooled, respectively and the high number of successfully emerged, but dead adults at this concentration.