Chromate(2-), [3-[4,5-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonato(3-)][2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]-, sodium

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Jul 2014 - 04 Sep 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

EC Number:

274-386-6

EC Name:

Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

Cas Number:

70209-87-9

Molecular formula:

C32H21CrN10O11S.2Na

IUPAC Name:

Disodium-[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-chromate(2-)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

After sampling, the test medium samples (0.5 mL) were mixed with 0.5 mL methanol and stored deep-frozen (below -18 °C) until analysis. At the analytical laboratory, the samples were thawed and shaken well using a Vortex mixer. If necessary, the samples were further diluted with methanol/water (1:1, v/v) prior to analysis by HPLC-PDA.

Test solutions

Vehicle:

no

Details on test solutions:

Each day the necessary amount of test item for preparing the stock solution was weighed on a weighing scoop and transferred to a volumetric flask. Test medium was added up to the bench mark and the solution was homogenised by shaking. The lower test solutions were prepared by dilution of the stock solution. Each solution was applied to the respective aquaria.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Species: Oncorhynchus mykiss (Walbaum)
Size: 4 - 6 cm
Producer: Fischzucht Störk, D-88343 Wagenhausen, Germany
Acclimatisation:
Fish for the test were obtained from the supplier on 15 July 2014 (acclimatisation > 12 days) and held under testing conditions in 300 L containers with continuous renewal of water (5 - 10 % per day) and permanent aeration of water. The fish were held under identical conditions for the entire pre-experimental period. The last feeding was performed 24 hours prior test start.
Holding water:
Water composed of dechlorinated drinking water and deionised water. The pH-value of the aerated water was between 6.0 - 8.5. The dissolved oxygen was above 60 % saturation and the total hardness was between 140 - 250 mg/L (as CaCO3), corresponding to 7.8 - 14°dH.
Light: 12 to 16 hours daily.
Temperature: The water temperature was 15 to 17 °C.
Oxygen: The concentration of oxygen was controlled 3 times a week. The air saturation value was ≥ 60 %.
Feeding: The fish were fed each day with granular rearing food with approx. 2 % of their body-weight. The food was obtained from the fish supplier.
Mortality: Following a 48-hour settling-in period, mortality was recorded. No mortality above 5 % was observed throughout the acclimatisation period.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Hardness:

8°dH corresponding to 143 mg CaCO3/L

Test temperature:

15.2 – 16.9 °C

pH:

7.69 – 8.47

Dissolved oxygen:

≥ 97 % of air saturation

Nominal and measured concentrations:

see table below

Details on test conditions:

Test procedure: semi-static test
Duration: 96 hours
Temperature: 15.2 – 16.9 °C
Oxygen concentration: ≥ 97 % of air saturation
pH-value: 7.69 – 8.47
Hardness: 8°dH corresponding to 143 mg CaCO3/L
Exposure to light: 16 hours daily
Feeding: none
Test vessels: 30 L glass aquaria, filled with 15 L
Loading: max. loading of 1 g fish/L
Aeration: continuous aeration of the test tanks with a membrane pump using a Pasteur pipette
Number of animals: 7 per concentration, no replicates

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

3.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL: 1.92-5.54 mg/L

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2.07 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Mortality:
In the control and at the test item concentrations up to and including 0.939 mg/L no mortality was observed within the period of the test. At 2.07 mg/L one fish died by accident after 72 hours and at 4.55 mg/L five fish were found dead after 96 hours. At the highest test item concentration of 10.0 mg/L all fish were found dead after 4 hours.
Observations:
No sublethal effects were observed in the control and at the test item concentration up to and including 0.939 mg/L after 96 h.
At 2.07 mg/L three fish showed reduced activity and dark pigmentation after 24 hours. After 72 hours one fish died by accident.
At 4.55 mg/L six fish showed reduced activity after 24 hours and one fish was dead. After 48 hours five fish showed reduced activity and three of them showed dark pigmentation. Two fish were dead. After 72 hours three fish showed reduced activity and four fish were dead. After 96 hours one fish showed reduced activity and one fish had lost equilibrium. Five fish were dead.
At 10.0 mg/L all fish were found dead after four hours.
All fish were weighed and measured. The average weight of the test organisms was 0.88 +/- 0.25 g; the average length was 46 +/- 3 mm.

Reported statistics and error estimates:

The NOEC (mortality) was established based on the highest test item concentration at which the mortality is not higher than the allowed control mortality.
The LC50-values after 4, 24, 48, 72 and 96 h were calculated using Weibull analysis.For evaluation, the statistical programme ToxRat Professional 2.10 was used.

Any other information on results incl. tables

Sublethal observations / clinical signs:

LC₅₀-values of fish exposed to the test item

test item nominal [mg/L]
Time [h]	NOEC mortality [mg/L]	LC50^1) [mg/L]	95 % confidence limit of LC50 [mg/L]
4	4.55	8.83	-
24	4.55	5.99	-
48	2.07	5.42	3.65 – 244
72	2.07	4.13	2.45 – 7.23
96	2.07	3.70	1.92 – 5.54

1) calculated using Weibull analysis

- not calculable

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

According to the results of the test, the LC50 (96 h) of the test item was determined to be 3.70 mg test item/L (nominal). The corresponding NOEC (mortality) (96 h) was 2.07 mg test item/L (nominal).
No sublethal effects were observed up to and including 0.939 mg test item/L (nominal) after 96 h.

Executive summary:

Analogue substance was tseted for acute toxicity to fish following OECD 203.

LC₅₀-values of fish exposed to the test item

FAT 20060/F TE nominal [mg/L]
Time [h]	NOEC mortality [mg/L]	LC50^1) [mg/L]	95 % confidence limit of LC50 [mg/L]
4	4.55	8.83	-
24	4.55	5.99	-
48	2.07	5.42	3.65 – 244
72	2.07	4.13	2.45 – 7.23
96	2.07	3.70	1.92 – 5.54

1)         calculated using Weibull analysis

-          not calculable

The content of FAT 20060/F TE in test solutions was analytically determined to confirm the correct application of the test item. The mean measured content of FAT 20060/F TE in fresh test solutions was between 84 % and 104 % of nominal. The measured content of FAT 20060/F TE in aged test solutions was between 86 % and 99 % of nominal. Since the measured concentrations of FAT 20060/F TE in aged test item solutions were between 80 and 120 % of nominal concentrations, the biological endpoints were evaluated using nominal test item concentration.

The total hardness (as CaCO3) of the test water was determined to be 8°dH; the mean pH-value of the control was determined to be 8.24 +/- 0.20. The mean temperature of the untreated control and all test item concentrations was measured to be 16.1 +/- 0.5 °C and the oxygen saturation was determined to be 99 +/- 1 %.