Sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From July 29 to September 04, 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

the complete read across justification is detailed in section 13. source study has reliability 1.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

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 (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.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

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:

Nominal concentrations: 0, 0.939, 2.07, 4.55, 10 mg/l.

Details on test conditions:

Test procedure: semi-static test
Duration: 96 hours
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

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

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.

Sublethal observations / clinical signs:

Determined concentrations of test substance during the test

test item nominal [mg/l]	sampling [h]	conc. test substance		mean fresh
		[mg/l]	% of nominal	[%]
0	0 h fresh	n.d.	-	-
	24 h aged	n.d.	-
	24 h fresh	n.d.	-
	48 h fresh	n.d.	-
	72 h fresh	n.d.	-
0.939	0 h fresh	0.919	98	87
	24 h aged	0.843	90
	24 h fresh	0.886	94
	48 h fresh	0.747^1	80
	72 h fresh	0.726^1	77
2.07	0 h fresh	2.104	102	90
	24 h aged	2.046	99
	24 h fresh	1.988	96
	48 h fresh	1.691	82
	72 h fresh	1.621	78
4.55	0 h fresh	4.03	89	84
	24 h aged	3.92	86
	24 h fresh	3.96	87
	48 h fresh	3.69	81
	72 h fresh	3.51	77
10.0	0 h fresh	10.4	104	-
	24 h aged	9.88	99

Limit of quantification (LOQ) = 0.800 mg/l

¹⁾ < LOQ

n.d. = not detectable

LC50 values of fish exposed to the test item (Weibull analysis)

nominal conc. test substance [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

Validity criteria fulfilled:

yes

Conclusions:

LC50 (96 h) = 3.70 mg/l.
NOEC (mortality) (96 h) = 2.07 mg/l.
No sublethal effects were observed up to and including 0.939 mg/l after 96 h.

Executive summary:

Method

Acute toxicity to fish exposed to test substance for 96 h, under semi-static conditions. The content of substance in test solutions was analytically determined to confirm the correct application of the test item. Nominal concentrations used: 0, 0.939, 2.07, 4.55 and 10 mg/l.

Results

LC50 values of fish exposed to the test item

nominal conc. test substance [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 mean measured content of substance in fresh test solutions was between 84 % and 104 % of nominal. The measured content of substance in aged test solutions was between 86 % and 99 % of nominal. Since the measured concentrations of substance in aged test item solutions were between 80 and 120 % of nominal concentrations, the biological endpoints were evaluated using nominal test item concentration.

The LC50 (96h) was found to be 3.70 mg/l, equivalent to ca. 3 mg a.i./l.

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 %.

Description of key information

LC50 (96 h) = 3.70 mg/l.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

3.7 mg/L

Additional information

No information was available on the substance itself, thus the assessment relied on a read across approach. Solvent Orange 062 and Similar Substance 01 are chromium complexes with 2 ligands. In both cases, the 2 ligands have the same chemical structure, i.e. a phenyl ring connected via diazo bond to a phenyl pyrazole. These ligands show minor differences in the position of substituents, i.e. hydroxy and nitro gorups, as well as in the presence of a sulfonate group in Similar Substance 01.

All information about the read across process is inserted in section 13.

In the key study, the acute lethal toxicity of Similar Substance 01 (purity > 80 %) to fish was investigated under semi-static exposure conditions over a period of 96 h at nominal concentrations of 0.939, 2.07, 4.55 and 10 mg/l with rainbow trout according to OECD guideline 203. There was evidence that the concentration of test substance was satisfactorily maintained throughout the test, thus effect levels were expressed using nominal test item concentrations.

All fish were dead at the nominal concentrations of 10 mg/l after 4 h of exposure; 5/7 fishes were dead at the nominal concentration of 4.55 mg/l after 96 h; no substance-related mortality was observed at the lower concetrations after 96 h of exposure. Toxic effects other than mortality in terms of reduced motility and dark pigmentation were observed starting from the concentration of 2.07 mg/l.

On these bases, median lethal concentration (LC50) at 96 h was estimated to be 3.70 mg/l.

In a previous study on rainbow trout exposed to Similar Substance 01 (purity ca. 80 %), concentrations of 2, 10 and 20 mg/l were tested. At 2 mg/l, no fishes died after 48 h, while all fishes were dead after 24 h at 10 and 20 mg/l. Therefore, LC50(48 h) was estimated to be 6.3 mg/l.

Overall, based on these findings,it was concluded that the substance interacts with aquatic environment and it is toxic for freshwater fish, showing LC50 values below 10 mg/l. Following a precautionary approach, the lowest LC50 value obtained in studies with Similar Substance 01, i.e. 3.70 mg/l, was taken into account in the decision on classification under CLP Regulation (EC 1272/2008).