2-amino-6-methyl-4-propyl-1,2,4-triazolo[1,5-a]pyrimidin-5(4H)-one

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP, available as unpublished report, minor restrictions in design and/or reporting but otherwise adequate for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The 24, 48, and 96 hour LC50 values were determined with rainbow trout using a continuous flow system.

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

Analytical measurements were made of the level of the test substance in each fish exposure concentration.

Vehicle:

yes

Details on test solutions:

The amount of DMSO used in the preparation of the stock solution was kept to the minimum and never exceed a level of 100 mg/L in any test solution. At this concentration is very unlikely to have any effects whatsoever on the test organisms. The reported 96 hour LC50 value of DMSO to Rainbow Trout is of the order of 33 g/L (Willford W.A. Toxicity of Dimethyl Sulphoxide (DMSO) to fish. Investigations in Fish control No. 2. United States department of the Interior Resource, publication 37. Washington DC, April 1967).

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

- Common name: Rainbow Trout
- Source: the fish were obtained from the Samaki Trout Farm, Wiltshire.
- Keeping: Batches of fish were kept in stock tanks for a period of 4 weeks at a temperature of 10 °C prior to testing.
- Acclimation: The fish were acclimatized for a period of 2 days in the test vessel at a temperature of 13°C ± 0.5 °C, before commencement of the test.- Weight: The fish ranged in weight from 11.2 to 19.0 g with a mean weight of 14.84 g.
- Length: The range in length was 95 mm to 110 mm with a mean length of 101.7 mm.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Run 1 and 2: 54.0 mg/L (mean; as CaCO3)
Run 3 and 4: 62.29 mg/L (mean; as CaCO3)

Test temperature:

13 ± 0.5 °C

pH:

7.5 - 7.7

Dissolved oxygen:

93.9 - 98.0 % saturation

Nominal and measured concentrations:

Nominal concentrations:
- Run 1: 15, 22, 33 mg/L
- Run 2: 150, 220, 330 mg/L
- Run 3: 68, 100 mg/L
- Run 4: 47 mg/L
A control was run simultaneously with each set of experiments.

Mean analytical concentrations in mg/L (nominal percent):
- Run 1: 10.25 (68.3%), 19.2 (87.3%), 26.2 (79.4%) mg/L
- Run 2: 121.2 (80.8%), 112.5 (51.13%), 292.5 (88.64%) mg/L
- Run 3: 63.2 (92.94%), 71.0 (71%) mg/L
- Run 4: 43.7 (92.98%) mg/L

Details on test conditions:

FLOWTHROUGH CONDITIONS:
The apparatus used in this study was a continuous flow through system (Maddock B.G. and Williams B.R.H. A continues Flow Apparatus for Assessing the Toxicity of Substances to Aquatic Animals. ICI Brixham Laboratory report BL/A/1579. May 1974). The dilution water was supplied from a 20000 gallon reservoir. Twenty litre glass vessels were used to hold the test fish (n=10 per concentration) and the test solutions were renewed at a rate of 200 mL/minute. A complete exchange of the test solution occurred within 3 hours. The dilution ratio of the stock concentrates to freshwater was 1:100 in this study.

APPLICATION OF TEST SUBSTANCE:
The concentrated stock solutions were fed to the system by a series of peristaltic pumps and a further series of these pumps was used to supply freshwater. The concentrated solutions of PP 796 and freshwater were fed to glass splash heads where mixing occurred before the test solution passed into the fish test vessels.

EFFECT PARAMETERS MEASURED:
The individual times of death of the fish in minutes and symptoms of distress throughout the test.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL: 36 - 44 mg/L

Details on results:

MORTALITY:
No deaths occurred during the 96 hours test period at exposure concentrations of 15.0, 22.0 and 33.0 mg/L test substance. The 96-h no effect level for symptoms of distress was 15 mg/L test substance.

Reported statistics and error estimates:

The original LC50 values were obtained with the geometric mean survival period (GMSP). In an amendment the probit method of Finney (1971) was used. There was little variation between the two sets of values, the original values obtained with GMSP being all within the 95% confidence intervals of the probit method.

Sublethal observations / clinical signs:

Percentage mortalities

Nominal conc. (mg/L)	Percentage mortalities
	24 h	48 h	72 h	96 h
0 (control)	0	0	0	0
15	0	0	0	0
22	0	0	0	0
33	0	0	0	0
47	0	0	30	100
68	10	0	100	100
100	40	100	100	100
150	100	100	100	100
220	100	100	100	100
330	100	100	100	100

LC50 values (mg/L) GMSP method vs Probit.

	GMSP data	Probit values
24-h LC50	88	99 (86* - 114)
48-h LC50	56	57 (51 – 63)
72-h LC50	-	51 (45 – 57)
96-h LC50	40	40 (36 – 44)

 *95% lower and upper confidence intervals

Fish survival and Geometric Mean Survival Periods (GMPS)

Concentration (mg/L)	Time (minutes from ‘0’)	Survivors	GMPS (minutes)
15   (run No. 1)	5760	10	-
22   (run No. 1)	5760	10	-
33   (run No. 1)	5760	10	-
47   (run No. 4)	3980	9	4803
	4250	7
	4600	6
	4920	4
	5300	2
	5380	1
	5440	0
68  (run No. 3)	1260	9	1937.5
	1620	8
	1740	7
	2040	4
	2190	3
	2220	2
	2280	0
100  (run No. 3)	750	9	1329.7
	960	8
	1140	7
	1260	6
	1500	4
	1620	2
	1710	1
	1740	0
150  (run No. 2)	365	9	708.4
	540	7
	810	4
	840	3
	855	2
	915	0
220  (run No. 2)	240	9	504.8
	300	8
	365	7
	540	3
	810	1
	840	0
330  (run No. 2)	120	9	256.8
	140	8
	240	6
	265	5
	300	4
	365	1
	385	0

Validity criteria fulfilled:

not applicable

Conclusions:

The 96 hour LC50 was 40.0 mg/L under the conditions of the test.

Executive summary:

The short-term toxicity to freshwater fish was determined in a 96 h acute toxicity test. In this study groups of 10 Rainbow Trout (Salmo gairdnerii) were exposed to nominal test concentrations of 0, 15, 22, 33, 47, 68, 100, 150, 220, and 330 mg/L for 96 hours under continuous flow through conditions. Test concentrations were analytical verified. Measured concentrations were determined to be 10.25, 19.2, 26.2, 43.7, 63.2, 71.0, 121.2, 112.5, and 292.5 mg/L (mean). Observations for mortalities and symptoms of toxicity were made every few minutes. No deaths occurred during the 96 hours test period at exposure concentrations of 33.0, 22.0 and 15.0 mg/L. A “no effect” level, the concentration at which no symptoms of distress were observed throughout the 96 hour test period, was found to be 15 mg/L. The 96 -h LC50 value was determined at 40 mg/L (nominal).

Description of key information

The 96-h LC50 value is 40 mg/L in fish (Salmo gairdnerii).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

40 mg/L

Additional information

The short-term toxicity to freshwater fish was determined in a non-GLP pre-guideline study. In this study groups of 10 Rainbow Trout (Salmo gairdnerii) were exposed to nominal test concentrations of 0, 15, 22, 33, 47, 68, 100, 150, 220, and 330 mg/L for 96 hours under continuous flow through conditions. Test concentrations were analytically verified. Mean measured concentrations were determined to be 10.25, 19.2, 26.2, 43.7, 63.2, 71.0, 121.2, 112.5, and 292.5 mg/L. Observations for mortalities and symptoms of toxicity were made regularly throughout the test. No deaths occurred during the 96 hours test period at exposure concentrations of 15.0 22.0 and 33.0 mg/L. No symptoms of toxicity were observed up to a test concentration of 15 mg/L. The 96-h LC50 value was 40 mg/L (nominal).