2,5-dichloro-4-[[2-(dibutylamino)-4-methyl-6-[[2-(4-sulphophenyl)ethyl]amino]-5-pyrimidinyl]azo]benzenesulphonic acid, sodium salt

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

Study period:

Limit test: from 19 to 23 August, 2002. Definitive test: from 02 to 06 September, 2002.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

adopted 17 July, 1992

GLP compliance:

not specified

Analytical monitoring:

no

Details on test solutions:

Due to the limited water solubility of the test substance, the nominal concentrations were prepared by direct addition. Most of the test material with the exception to the nominal concentration of 1 mg/l was lying on the bottom of the test vessels. No chemical analyses of the test substance concentrations were conducted.

Test organisms (species):

Poecilia reticulata

Details on test organisms:

TEST ORGANISM
- Common name: guppy.
- Breeding: parentaland young fish population was held in two 250 l glass aquaria.
- Length at study initiation: 1 - 3 cm.

BREEDING
- lllumination: 14 h a day
- Temperature: 23 ±.2 °C
- Medium: continuously aerated and filtered tap water from Schlieren. The bottom of the aquaria was covered with gravel (≤1.5 cm). Every month, one third of the total volume of the aquaria was replaced by fresh tap water.
- Type and amount of food during acclimation: Sera micropan, Sera vipan.
- Feeding frequency during acclimation: daily. The fish were last fed 24 h before the start of the definitive test.
- Health during breeding: during 7 days before the definitive test, mortality in the batch of which fish were used for the test was less than 5 %.
- Measurements: pH, conductivity, oxygen concentration and hardness of water were determined once a week. Temperature was controlled by a thermostatic heating element.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

Test item vessels: 20 - 22.5 °C
Control vessels: 20 - 22.5 °C

pH:

Test item vessels: 8.2 - 8.6
Control vessels: 8.0 - 8.5

Dissolved oxygen:

Test item vessels: 7.0 - 7.2 mg/l
Control vessels: 6.7 - 7.1

Nominal and measured concentrations:

1, 1 and 100 mg/l nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: 1 l aquaria, all-glass.
- No. of organisms per concentration: 7 fish per test concentration.
- Blank: a blank of test medium without test substance or other additives served as a control.

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: tap water from Schlieren, aerated.
- pH: 7.1 - 7.4
- Conductivitv (20 °C): 510 - 585 µS/cm
- CO3 - Hardness: 15.3 - 27.8 F
- CO3 - Hardness after Hever: 15.2 - 26.9 F
- Rest - Hardness: 2.4 - 4.1 F
- Total Hardness: 17.7 - 31.9 F
- KMnO4-consumption: 1.0 - 1.3 mg/l
- E. Coli 44 °C: 0 per 100 ml
- Colony count: none
- Chloride: 7.5 - 12.0 mg/l
- Sulfate: 17.5 - 21.0 mg/l
- Phosohate as PO4-P: < 0.002 - 0.038 mg/l
- Ammonia as NH4-N: < 0.01 mg/l
- Nitrite as NO2-N: < 0.002 mg/l
- Nitrate as NO3-N: 7.0 - 12.0 mg/l

OTHER TEST CONDITIONS
- Adjustment of pH: pH was not adjusted.
- Photoperiod: 16 h photoperiod a day, supplied by overhead white fluorescent tubes.

OBSERVATION
Observations of the fish were made after 2, 24, 48, 72 and 96 h of exposure. Mortality of the fish was recorded at the same time intervals. Dead fish were removed immediately from the test medium and weight and length recorded. Fish were considered to be dead when they showed no reaction after touching the caudal fin or no visible breathing movements.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: a succeeding screening test with 100, 10 and 1 mg/l was performed.
- Results used to determine the conditions for the definitive study: in a preceding limit test with 100 mg/l all 7 fish died alter 2 h of exposure. No mortality was observed in the control after 96 h of exposure.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1 - 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

All fish died at the nominal concentrations of 100 and 10 mg/l alter 2 and 24 h of exposure, respectively. After 96 h of exposure a total of 3 of 7 fish were dead at the nominal concentration of 1 mg/l. Other toxic effects than mortality, e.g. loss of coordination, hypoactivity and swimming on the back, were observed at 1 mg/l.
Due to the mortality of 43 % (3 of 7 fish) at the nominal concentration of 1 mg/l the nominal lethal concentration (LC50) cannot clearly be estimated. Due to the effects observed at 1 mg/l the LC0 was estimated to be <100 mg/l.
100 % mortality (LC100) was observed at 10 mg/l after 24 h of exposure.No mortality was observed in the control after 96 h of exposure.

Sublethal observations / clinical signs:

Incidence of mortality observed.

Nominal conc. (mg/l)	Number of dead fish observed at different points in time after start of exposure
	2 h	24 h	48 h	72 h	96 h
Control	0	0	0	0	0
1	0	0	1	0	2
10	4	3	**	**	**
100	7	**	**	**	**

** All fish died, no further observations.

Specification of symptoms observed on affected fish and data of dead fish

Nominal conc. (mg/l)	N. of affected fish*	Time of score (h)	Body-lengh (mm)	Body-weight (mg)	Symptom**
100	7/0	2	21	118	Dead, at bottom
			23	108
			28	263
			25	149
			26	185
			20	86
			25	163
10	4/3	2	26	163	Dead, at bottom
			23	153
			25	172
			21	116
10	3/0	24	24	114	Dead, at bottom
			19	66
			20	94
1	1/6	48	21	83	Dead, at bottom
1	2/6	72	-	-	No activity
1	2/47	96	26	135	Dead, at bottom
			25	127

*Number of affected fish / number of surviving fish.

**e.g. hyper-/hypoactivity, immobility, swimming at the bottom/surface, darkened pigmentation/ discoloring.

pH-values

Nominal conc. (mg/l)	pH-values determined at different points in time after start of exposure
	2 h	24 h	48 h	72 h	96 h
0	8.0	8.5	8.5	8.4	8.5
1	8.2	8.2	8.3	8.4	8.4
10	8.2	8.6	**	**	**
100	8.4	**	**	**	**

** All fish died, no further observations.

Oxygen concentrations

Nominal conc. (mg/l)	Oxygen concentrations determined at different points in time after start of exposure (mg/l)
	2 h	24 h	48 h	72 h	96 h
0	7.1	7	7.1	7.0	7.2
1	6.8	7.1	6.9	6.8	6.9
10	6.7	6.9	**	**	**
100	7.0	**	**	**	**

** All fish died, no further observations.

Temperature

Nominal conc. (mg/l)	Temperature determined at different points in time after start of exposure (°C)
	2 h	24 h	48 h	72 h	96 h
0	22.5	21	20	22	22
1	22.5	21	20	22	22
10	22.5	21	**	**	**
100	22.5	**	**	**	**

** All fish died, no further observations.

Conclusions:

LC50 (96h): 1-10 mg/l (nominal)

Executive summary:

The acute lethal toxicity of test item to Poecilia reticulata (Guppy) was investigated under static exposure conditions over a period of 96 h. The only applied nominal test concentrations were 1, 10 and 100 mg/l. Due to the limited water solubility of the test substance, the nominal concentrations were prepared by direct addition. Most of the test material with the exception to the nominal concentration of 1 mg/l was lying on the bottom of the test vessels. No chemical analyses of the test substance concentrations were conducted. All fish died at the nominal concentrations of 100 and 10 mg/l after 2 and 24 h of exposure, respectively. After 96 h of exposure a total of 3 of 7 fish were dead at the nominal concentration of 1 mg/l. No mortality was observed in the control after 96 h of exposure. Other toxic effects than mortality, e.g. loss of coordination, hypoactivity and swimming on the back, were observed at 1 mg/l. Due to the mortality of 43 % (3 of 7 fish) at the nominal concentration of 1 mg/l the nominal lethal concentration (LC50) cannot clearly be estimated. Due to the effects observed at 1 mg/l the LC0 was estimated to be lower than 100 mg/l. 100 % mortality (LC100) was observed at 10 mg/l after 24 hours of exposure.

Conclusion

LC50 (96h): 1-10 mg/l (nominal)

Description of key information

LC50 (96h): 1 - 10 mg/l (nominal)

Additional information

The acute lethal toxicity of test item to Poecilia reticulata was investigated under static exposure conditions over a period of 96 hours. The applied nominal test concentrations were 1, 10 and 100 mg/l. Due to the limited water solubility of the test substance, the nominal concentrations were prepared by direct addition. Most of the test material with the exception to the nominal concentration of 1 mg/l was lying on the bottom of the test vessels. No chemical analyses of the test substance concentrations were conducted. All fish died at the nominal concentrations of 100 and 10 mg/l after 2 and 24 hours of exposure, respectively. After 96 hours of exposure a total of 3 of 7 fish were dead at the nominal concentration of 1 mg/l. Other toxic effects than mortality, e.g. loss of coordination, hypoactivity and swimming on the back, were observed at 1 mg/l. Due to the mortality of 43 % (3 of 7 fish) at the nominal concentration of 1 mg/l the nominal lethal concentration (LC50) cannot clearly be estimated. Due to the effects observed at 1 mg/l the LC0 was estimated to be lower than 100 mg/l. 100 % mortality (LC100) was observed at 10 mg/l after 24 hours of exposure.