Disodium [6-[(2-anilino-1-naphthyl)azo]-2,4-dinitrophenolato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]naphthalene-1-sulphonato(3-)]chromate(2-)

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:

Preliminary test November from 26th to 30th, 2001; main test January from 14th to 18th, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

adopted 17 July, 1992

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

GLP compliance:

not specified

Analytical monitoring:

no

Test organisms (species):

Poecilia reticulata

Details on test organisms:

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

BREEDING
- Illumination: 14 hours 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.
- Feed: Sera micropan, Sera vipan (Sera apanristic GmbH, D-52525 Heinsberg, Germany) daily. The fish were last fed 24 h belore the start of the definitive test.
- Validity: during 7 days before the definitive test, mortality in the batch of which fish were used for the test was less than 5 %.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

Control: 20.5 - 22 °C
Test vessels: 20.5 - 22 °C

pH:

Control: 8.2 - 8.7
Test vessels: 8.1 - 8.7

Dissolved oxygen:

Control: 7.2 - 7.3 mg/l
Test vessels: 6.9 - 7.4 mg/l

Nominal and measured concentrations:

Nominal 0.25, 0.5, 1.2 and 4 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 1 l aquaria, all-glass.
- Aeration: aerated.
- No. of organisms per vessel: 7 fish per test concentration.
- No. of vessels per concentration: 1 replicate.
- No. of vessels per control: 1 replicate.

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: tap water from Schlieren, aerated.
- Preparation of medium: at least 1 h before the test the test vessels were filled with the medium to allow equilibration of pH, oxygen and temperature.

OTHER TEST CONDITIONS
- Adjustment of pH: pH was not adjusted.
- Photoperiod: 14 h photoperiod a day.
- Light: light was supplied by overhead white fluorescent tubes.

MEASUREMENTS
Oxygen, pH, temperature: prior to the addition of fish to the test medium and after 2 ,24, 48, 72 and 96 h of exposure

EFFECT MEASURED
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. The median lethal concentration (LC50 value) is the concentration killing 50 % of the fish after a cedain period of exposure. Any mortality above 1 fish per sample is considered to be significant.

TEST CONCENTRATIONS
- Test concentrations: 1, 10 and 100 mg/l.
- Number fish per concentration: 3 fish.
- Results used to determine the conditions for the definitive study: 100 % mortality was observed at 100 and 10 mg/l after 2 and 24 hours of exposure, respectively. 2 fish died at 1 mg/l after 48 hours. No mortality was observed in the control after 96 hours of exposure. Based on these data nominal concentrations were chosen for the definitive test.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

After 96 hours of exposure a total of 7 and 2 of 7 fish were dead at the nominal concentrations of 4 and 2 mg/l, respectively. 1 fish died at 1 mg/l after 72 hours of exposure. No mortality was observed at 0.5 and 0.25 mg/l and in the control after 96 hours of exposure.
Other toxic effects than mortality, e.g. loss of coordination, hypoactivity and swimming on the back, were observed at 4 and 2 mgll, respectively.

Sublethal observations / clinical signs:

Incidence of mortality observed.

Nominal concentration (mg/l)	Number of dead fish observed at
	2 hrs	24 hrs	48 hrs	72 hrs	96 hrs
control	0	0	0	0	0
0.25	0	0	0	0	0
0.5	0	0	0	0	0
1	0	0	0	1	0
2	0	1	1	0	0
4	0	6	0	0	1

Cumulative mortality.

Nominal concentration (mg/l)	Cumulative mortality observed at
	2 hrs	24 hrs	48 hrs	72 hrs	96 hrs
control	0	0	0	0	0
0.25	0	0	0	0	0
0.5	0	0	0	0	0
1	0	0	0	1	1
2	0	1	2	2	2
4	0	6	6	6	7

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

Nominal concentration (mg/l)	Number of affected fish*	Time of exposure (hrs)	Body-length (mm)	Body-weight (mg)	Symptoms**
4	6/1	24	24 28 26 26 30 21	119 270 167 89 201 96	dead, at bottom
2	1/6	24	21	83	dead, at bottom
2	1/6	24	-	-	swimminq on the back
4	1/1	48	-	-	slow movements
2	1/5	48	24	133	dead, at bottom
4	1/1	72	-	-	slow movements
1	1/6	72	29	154	dead, at bottom
4	1/0	96	27	212	dead, at bottom

*Number of affected fish / number of surviving fish.

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

Conclusions:

LC50 (96h): 2.1 mg/l (nominal)

Executive summary:

The acute lethal toxicity (LC50) of test item to Poecilia reticulata (Guppy) was investigated under static exposure conditions over a period of 96 h. 7 fish per concentration were exposed to the test substance. Nominal concentrations of the substance as selected by a range finding test were 0.25, 0.5, 1.2 and 4 mg/l, respectively. Individual test concentrations were prepared by dilution of a stock solution. No chemical analyses of test concentrations were conducted.

After 96 h of exposure a total of 7 and 2 of fish were dead at the nominal concentrations of 4 and 2 mg/l, respectively. 1 fish died at 1 mg/l after 72 h of exposure. No mortality was observed at 0.5 and 0.25 mg/l and 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 4 and 2 mg/l, respectively.

Based on these data, nominal lethal concentration (LC50) of test item to Poecilia reticulata was calculated to be 2.1 mg/l.

The no-observed-effect concentration (NOEC) was 0.5 mg/l. One fish died at 1 mg/ and is considered to represent a significant effect since already in the range finding test 2 of a total of 3 fish died at 1 mg/l.

100 % mortality (LC100) was observed at the nominal concentration of 4 mg/l.

Conclusion

LC50 (96h): 2.1 mg/l (nominal)

Description of key information

LC50 (96h): 2.1 mg/l (nominal)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

2.1 mg/L

Additional information

The acute lethal toxicity (LC50) of test item was investigated, under static exposure conditions, over a period of 96 h. The experiment was performed in accordance with the OECD guideline 203. After 96 h of exposure a total of 7 and 2 of fish were dead at the nominal concentrations of 4 and 2 mg/l, respectively. 1 fish died at 1 mg/l after 72 h of exposure. No mortality was observed at 0.5 and 0.25 mg/l and 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 4 and 2 mg/l, respectively. Based on the observations, the nominal lethal concentration (LC50) of test item to Poecilia reticulata was calculated to be 2.1 mg/l.

Similar results were obtained in a second experiment conducted on Brachydanio rerio, Hamilton-Buchanan. Also in this case, the study was conducted according to the OECD guideline 203. In order to identify the LC50, the cumulative percentage mortality was plotted against the concentration on logarithmic-probability paper and a line was fitted, by eye. The maxlmum concentration producing no mortality was 0.9 mg/l (meas. geom. mean); while, the minimun concentration producing 100 % mortality was 2.3 mg/l (meas. geom. mean). The LC50 (96h) was estimated to be 2 mg/l (graphically estimated).

In addition, an old test is available: it was performed in accordance with internal procedures of the testing facility. Unfortunately, the reliability of the data cannot be judged because of the lacking of details about test material and testing procedures and conditions. The LC50 after 48 hours was indicated to be 1.4 mg/l (nominal).

REFERENCE

See attachment