trisodium 4-hydroxy-6-(sulfonatomethylamino)-5-(2-(2-sulfatoethylsulfonyl)phenylazo)naphthalene-2-sulfonate

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:

From January 26, 1998 to February 18, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Prepared test media of all concentrations and the control at the start of the test (Day 0) and at the last test media preparation on Day 3.
For the determination of the stability of the test substance, additional samples were taken in duplicate out of all test media and the control after one day at the end of the first test medium renewal and at the end of the last test medium renewal.
All samples were taken from the approximate center of the aquaria without mixing of the test media, and were deep frozen immediately after sampling.

Vehicle:

no

Details on test solutions:

A concentrated stock solution with a nominal concentration of 5.0 g/L was prepared at each test medium renewal by dissolving the test article in test water (15.00-15.001 g at 3 liters). Requisite volumes of this intensively stirred stock solution were added to the test water in aquaria and afterwards the test media were intensively mixed. The following nominal concentrations were tested: 46, 100, 220, 460, and 1000 mg test substance/L. Additionally control was tested in parallel.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The test fish were obtained from Zoohaus Schaub, CH-4410 Liestal, Switzerland. The fish were held in the laboratories of RCC for more than two weeks and were acclimated one week prior to the test start. During the holding and acclimation the fish were fed with a commercial fish diet (TETRA MIN Hauptfutter, supplied by TETRA-Werke). During the four weeks prior to the test no fish died in the test fish batch and all fish were healthy. The mean body length of the fish at the start of the test was 2.6 ± 0.12 cm, the mean body wet weight 0.18 ± 0.06 g.

Test type:

semi-static

Water media type:

other: Reconstituted water, in purified water analytical grade salts

Limit test:

no

Total exposure duration:

96 h

Hardness:

2.5 mmol/L = 250 mg/L

Test temperature:

21-23 °C

pH:

7.1-8.1

Dissolved oxygen:

8.0 mg/L (or higher, thus higher than 60% oxygen saturation)

Salinity:

CaCl2 x 2H2O = 294 mg/L; MgSO4 x 7H2O = 123 mg/L; NaHCO3 = 65 mg/L; KCl = 5.8 mg/L. Alkalinity = 0.8 mmol/L.

Nominal and measured concentrations:

The nominal test concentrations were: 46, 100, 220, 460, and 1000 mg test substance/L.

Details on test conditions:

Since the test substance is not stable in water over the test period of 96 hours (determined in a pre-experiment without GLP), a semi-static test procedure was chosen with a test medium renewal every day to keep the test concentrations as constant as possible. A flow-through test was not possible, since a concentrated homogeneous stock solution of the test substance in water could not be prepared.

One glass aquarium with approximately 8 litre test medium was used for each test concentration and the control. The test vessels were labelled with the RCC project number. At the start of the test 10 fish were introduced into each aquarium in a random order. During this semi-static test, each day the surviving test fish were placed in a clean aquarium with freshly prepared test medium of the corresponding test concentration. The test media were slightly aerated during the test period. The fish were not fed during the test.
The test fish were observed after approximately 4, 24, 48, 72 and 96 hours test duration for symptoms of intoxication and mortality. Dead fish were removed at least once daily. The LC50 and 95 % confidence interval at the observation dates were calculated as far as possible by moving average interpolation.

The water temperature, pH-values and dissolved oxygen concentrations were measured once every day during the test in the freshly prepared and old test media. Light conditions: Photoperiod of 16 hours light and 8 hours darkness, light intensity at light period approximately 50-200 Lux.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

609 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

169 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

In the control and at the test concentrations up to and including 220 mg/L (nominal) all fish survived until the end of the test and no symptoms of intoxication were observed. At the 460 mg/L (nominal) three test fish showed strong intoxication symptoms, but all ten test fish survived during the test period. At the highest test concentration of 1000 mg/L (nominal) two fish showed strong intoxication symptoms already after 4 hours of exposure. The number of fish with intoxiciation symptoms increased during the test, and the mortality rate increased at this test concentration from 20 % on day 2 to 100 % after 4 days. At all test concentrations the test media were clear solutions, but strongly reddish coloured by the test substance.

Analytical analysis:
- The analytically determined mean test substance concentrations in the freshly prepared test media ranged from 85 to 99 % of the nominal values.
- In the stock solution samples 104-105 % of the nominal value were found.
- During the test medium renewal periods of approximately 24 hours the test substance concentration in the test solutions had slightly decreased to 64-90 % of the nominal concentrations.
- The total mean measured test substance concentrations (calculated as the average over all measurements per test concentration) ranged from 77 to 91 % of the nominal values. Therefore, the reported biological results are related to the total mean measured test article concentrations (calculated as the average over all measurements per test concentration) ranged from 77 to 91 % of the nominal values:

Nominal test concentration: 46 mg/L – total mean measured test article concentration: not analysed
Nominal test concentration: 100 mg/L – total mean measured test article concentration: not analysed
Nominal test concentration: 220 mg/L – total mean measured test article concentration: 169 mg/L
Nominal test concentration: 460 mg/L – total mean measured test article concentration: 400 mg/L
Nominal test concentration: 1000 mg/L – total mean measured test article concentration: 906 mg/L

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 96-h LC50, NOEC and LOEC were determined to be 609, 169 and 400 mg/L, respectively (based on mean measured concentrations).

Executive summary:

A study was conducted to determine the toxicity of the test substance to Zebra fish (Danio rerio) according to OECD Guideline 203 and EU Method C.1, in compliance with GLP. The toxicity was investigated in a 96 h semi-static test with daily test medium renewal. The recorded effects were mortality and symptoms of intoxication. The nominal test concentrations studied were 0, 46, 100, 220, 460 and 1000 mg/L. The analytically determined mean concentrations in the freshly prepared test media ranged from 85 to 99 % of nominal values. In the stock solution samples, 104-105% of the nominal values were found. During the test medium renewal periods of approximately 24 h, the test substance concentrations slightly decreased to 64-90% of nominal values. The losses could have been due to an adsorption of the test substance onto glass surfaces, particularly in the low-level samples. The total mean measured test substance concentrations (calculated as the average over all measurements per test concentration) ranged from 77 to 91% of nominal values. Therefore, the reported biological results were related to total mean measured concentrations. Under the study conditions, the 96 h LC50, NOEC and LOEC were determined to be 609, 169 and 400 mg/L, respectively (based on measured concentrations) (Memmert, 1998).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

609 mg/L