Reaction products of 7-amino-4-hydroxynaphthalene-2-sulfonic acid reacted with 2-((1,3,5-triazin-2-yl)amino)ethan-1-ol, coupled with diazotised 3-amino-4-hydroxybenzenesulfonic acid, subsequently coupled with diazotised 2-amino-5-methoxybenzenesulfonic acid, chelated with copper, 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:

from 1991-04-05 to 1991-05-16

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Test Concentrations: 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L.
- Sampling method: The test sample solutions were diluted in dechlorinated tap water at nominal concentrations of 3.2 and 10 mg/L. The test samples were measured at 0, 24 and 96 hours in fresh and old solutions.
- Sample storage conditions before analysis: Room temperature

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct dispersion in water. 5.0 g of test material were dispersed in water and the volume adjusted to 1 liter to give a stock solution of 5.0 g/L. Appropriate dilutions were made from this stock solution to give the test series of 3.2; 5.6; 10; 18; 32; 56 and 100 mg/L.
- Controls: Blank control performed with the test solution without test substance.
- Stability of test concentrations: Ensured by daily renewal of test media and verified by chemical analysis at 0, 24 and 96 hours. Verification of test concentration was done.
- Evidence of undissolved material: no

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Name: Rainbow trout (Oncorhynchus mykiss)
- Source: Parkwood Trout Farm, Wigmore, Kent, U.K.
- Mean standard length: 4.6 cm (SD: 0.5 cm)
- Mean weight: 1.55 g (SD: 0.65 g)

ACCLIMATION
- Acclimation period: Stock held since 7 February 1991 and acclimatised to test conditions from 8 – 15 April.
- Acclimation conditions: Same as test.
- Feeding frequency during acclimation: Commercial trout pellets daily. Discontinued 24h prior to test

FEEDING DURING TEST
No

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Total hardness approximately 50 mg/L as CaCO3.

Test temperature:

Temperature: 14 ± 1 °C

pH:

pH: range from 7.0 to 7.8

Dissolved oxygen:

Dissolved Oxygen >= 10.0 mg O2/L

Nominal and measured concentrations:

- Nominal concentrations: 0 (control) 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L
- Measured concentrations:
- At 0 hours: Non detected 3.16, 5.73, 9.67, 17.9, 31.7, 55.9 and 102 mg/L corresponding to 98.6, 102.3, 96.7, 99.7, 99.0, 99.9 and 102.2 % of the nominal concentration.
- At 24 hours: 3.35, 5.77, 10.0, 18.1, 31.8, 56.5 and 102 mg/L corresponding to 104.8, 103.0, 100.5, 100.5, 99.4, 101.0 and 102.0 % of the nominal concentration.
- At 96 hours: 3.27, 5.54, 9.96, 18.6, 31.3 and 56.6 mg/L corresponding to 102.1, 98.9, 99.6, 103.2, 97.9 and 99.2 % of the nominal concentration.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Volume: 20 L
- Renewal rate of test solution (frequency/flow rate): 24 hours
- Aeration: via narrow bore glass tubes
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Loading: 0.78 g body weight/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water, dechlorinated by addition of sodium thiosulphate.
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH, temperature and oxygen were measured each 24 hours
- Total hardness approximately 50 mg/L as CaCO3
- Temperature: 14 ± 1°C
- Dissolved Oxygen >= 10.0 mg O2/L

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16h light : 8h dark

EFFECT PARAMETERS MEASURED: Mortality, absence of respiratory movement and response to a physical stimulation after 24 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: No range finding test was performed.

Reference substance (positive control):

no

Duration:

3 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

6 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 54-210 mg/L

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

27 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 19-37 mg/L

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 15-26 mg/L

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 14-22 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural Observations: Marked reactions to exposure (other than death) were lethargy, loss of equilibrium and the presence of moribund fish.
- Behavioural abnormalities: See Table 5 below.
- Observations on body length and weight: not observed
- Other biological observations: not observed
- Mortality of control: no
- Other adverse effects control: not observed
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

No test with reference substance was performed.

Sublethal observations / clinical signs:

VALIDATION OF THE TEST METHOD:

To confirm that the „Method of Analysis" was sufficiently accurate for the purposes of this study a range of synthetic test samples was prepared, accurately, fortified at known concentration of the test material, and analysed as per the method of analysis.

The results have been detailed below:

Table 1: Validation of method of analysis: Recoveries

Fortification (mg/L)	Recoveries
	mg/L	(%)	Mean (%)	Remark: The method of analysis was determined to be sufficiently accurate for the purpose of this study, and the test sample results have not been corrected for recovery.
3.42	3,48	101.9
3.36	3,55	105.5	103.7
5.98	6,30	105.4
5.89	6,19	105.1	105.3
10.7	10,6	99.5
10.5	10,4	99.3	99.4
19.2	19,3	100.5
18.9	18,9	100.1	100.3
34.2	34,2	100.1
33.6	33,7	100.2	100.2
59.8	59,4	99.3
58.9	58,9	100.0	99.7
107	106	98.9
105	105	100.3	99.6

 

Limit of quantification

The limit of quantification of the „Method of Analysis“has been determined to be 0.22 mg/L.

Limit of Detection:

The limit of detection of the “method of analysis” has been determined to be 0.04 mg/L.

 

 

Stability

To determine whether or not the test material is stable when dissolved/suspended in the dechlorinated tap water, a range of synthetic test samples were prepared and analysed initially and then after storage at ambient temperature in light and dark conditions over 24 hour period (equivalent to the period of media renewal)

The results obtained have been detailed below:

Table 2: Validation of method of analysis: Stability

Nominal concentration (mg/L)	3.2	5.6	10	18	32	56	100
Concentration found initially (mg/L)	3.48	6.30	10.6	19.3	34.2	59.4	106
Concentration found initially, expressed as % of nominal	108.8	112.5	106.0	107.2	106.9	106.1	106.0
Concentration found after storage at ambient temperature in light for 24 hours (mg/L)	3.45	5.97	10.6	19.2	33.9	59.3	105

 

Stability

To determine whether or not the test material is stable when dissolved/suspended in the dechlorinated tap water, a range of synthetic test samples were prepared and analysed initially and then after storage at ambient temperature in light and dark conditions over 24 hour period (equivalent to the period of media renewal)

The results obtained have been detailed below:

Table 2: Validation of method of analysis: Stability

 

Nominal concentration (mg/L)	3.2	5.6	10	18	32	56	100
Concentration found initially (mg/L)	3.48	6.30	10.6	19.3	34.2	59.4	106
Concentration found initially, expressed as % of nominal	108.8	112.5	106.0	107.2	106.9	106.1	106.0
Concentration found after storage at ambient temperature in light for 24 hours (mg/L)	3.45	5.97	10.6	19.2	33.9	59.3	105
Concentration expressed as % of initial	98.9	94.8	99.8	99.4	99.0	99.9	99.8
Concentration found after storage at ambient temperature in dark for 24 hours (mg/L)	3.43	5.95	10.7	19.2	33.8	59.2	106
Concentration expressed as % of initial	98.5	94.4	100.8	99.4	98.8	99.7	100.1
The test samples have been considered to be sufficiently stable for the purposes of this study.

 

Table 3: Results:

Enviromental Measurements:

pH: range from 7.0 to 7.8

Temperature: 13 – 14 °C

O₂saturation: 10.0 – 13.0 mg/L

 

 

Time (h)	LC50	95% confidence limits (mg/L)
3	>100	-
6	>100	-
24	110	54-210
48	27	19-37
72	20	15-26
96	18	14-22

 

NOEC at 96h: 10 mg/L

Table 4: Cumulative mortality data for rainbow trout exposed for 96h to KAYARUS SUPRA RUBINE BLN

Concentration (mg/L)	Cumulative mortality (initial population 10)
	3h	6h	24h	48h	72h	96h
Control	0	0	0	0	0	0
3.2	0	0	0	0	0	0
5.6	0	0	0	0	0	0
10	0	0	0	0	0	0
18	0	0	0	5	7	7
32	0	0	0	6	8	8
56	0	0	2	7	8	10
100	0	0	5	10	10	10

 

Behavioural Observations:

Marked reactions to exposure (other than death) were lethargy, loss of equilibrium and the presence of moribund fish.

Table 5:  Behavioural Observations

A Chronological record of observations is given below ( unless stated no adverse reactions were observed)
Concentration mg/L	Abnormalities	3	6	24	48	72	96
Control	No abnormalities detected
3.2	No abnormalities detected
5.6	No abnormalities detected
10	No abnormalities detected
18	Lethargy					3/3	3/3
32	Lethargy Moribund fish Loss of equilibrium			3/10       1/10	1/4	1/2       1/2	1/2       1/2
56	Lethargy Moribund fish Loss of equilibrium			3/8 2/8	3/2	2/2	A/D
100	Lethargy			3/5	A/D
A/D All fish dead

 

Validity criteria fulfilled:

yes

Conclusions:

NOEC at 96h: 10 mg/L
Highest test concentration resulting in 0% mortality = 10 mg/L
Lowest test concentration resulting in 100% mortality = 56 mg/L
After 96 h the LC50 was determined to be 18 mg/L

Executive summary:

In an 96h acute fish toxicity test, Direct Red 83:1 was tested to Rainbow trout (Oncorhynchus mykiss) coming from Parkwood Trout Farm, Wigmore, Kent, U.K. Environmental Measurements:

pH: range from 7.0 to 7.8

Temperature: 13 – 14 °C

O₂saturation: 10.0 – 13.0 mg/L

Time (h)	LC50	95% confidence limits (mg/L)
3	>100	-
6	>100	-
24	110	54-210
48	27	19-37
72	20	15-26
96	18	14-22

 

NOEC at 96h: 10mg/l

 

Highest test concentration resulting in 0% mortality = 10 mg/L

Lowest test concentration resultin in 100% mortality = 56 mg/L

No Observed Effect Concentration (NOEC)= 10 mg/L

The No Observed Effect Concentration is based upon zero mortalities and the absence of any behavioural reactions to exposure at this concentration.

Verification of test concentrations showed that the measured test concentrations form analysis at 0, 24 and 96 hours was near nominal. Therefore it was considered justifiable to calculate the LC50 values in terms of nominal test concentrations only.

The 96h-LC50 was determined to be 18 mg/L

Behavioural Observations:

Lethargy, loss of equilibrium, presence of moribund fish.

ANALYTICAL METHOD 

Limit of quantification= 0.22 mg/L

Limit of detection= 0.04 mg/L Stability: To determine whether or not the test material is stable when dissolved/ suspended in the

dechlorinated tap water, a range of synthetic test samples were prepared and analysed initially and then after storage at ambient temperature in light and dark conditions over 24 hour period (equivalent to the period of media removal)

The test samples have been considered to be sufficiently stable for the purpose of this study.

 

 

 

Description of key information

A short-term toxicity test to fish was performed according to OECD 203. After 96 h the LC50 was determined to be 18 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

18 mg/L

Additional information

In an 96h acute fish toxicity test, Direct Red 83:1 was tested to Rainbow trout (Oncorhynchus mykiss) coming from Parkwood Trout Farm, Wigmore, Kent, U.K. Environmental Measurements:

pH: range from 7.0 to 7.8

Temperature: 13 – 14 °C

O₂saturation: 10.0 – 13.0 mg/L

Time (h)	LC50	95% confidence limits (mg/L)
3	>100	-
6	>100	-
24	110	54-210
48	27	19-37
72	20	15-26
96	18	14-22

 

NOEC at 96h: 10mg/l

 

Highest test concentration resulting in 0% mortality = 10 mg/L

Lowest test concentration resultin in 100% mortality = 56 mg/L

No Observed Effect Concentration (NOEC)= 10 mg/L

The No Observed Effect Concentration is based upon zero mortalities and the absence of any behavioural reactions to exposure at this concentration.

Verification of test concentrations showed that the measured test concentrations form analysis at 0, 24 and 96 hours was near nominal. Therefore it was considered justifiable to calculate the LC50 values in terms of nominal test concentrations only.

The 96h-LC50 was determined to be 18 mg/L

Behavioural Observations:

Lethargy, loss of equilibrium, presence of moribund fish.

ANALYTICAL METHOD 

Limit of quantification= 0.22 mg/L

Limit of detection= 0.04 mg/L Stability: To determine whether or not the test material is stable when dissolved/ suspended in the

dechlorinated tap water, a range of synthetic test samples were prepared and analysed initially and then after storage at ambient temperature in light and dark conditions over 24 hour period (equivalent to the period of media removal)

The test samples have been considered to be sufficiently stable for the purpose of this study.