Calcium bis[4-[[3-[[2-hydroxy-3-[[(4-methoxyphenyl)amino]carbonyl]-1-naphthyl]azo]-4-methylbenzoyl]amino]benzenesulphonate]

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:

2017-06-26 to 2017-06-30

Reliability:

1 (reliable without restriction)

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

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The concentration of Pigment Red 247 was analytically verified via HPLC-DAD in the saturated solution and the control at the start of the exposure (0 hours).
The measured concentration of the limit concentration was below the lowest calibration standard (LCL) at the start of the exposure (0 hours). Therefore analytical monitoring at the end of the exposure (96 hours) was waived and the test item concentrations were considered to be below the LCL in the aged media, too.

Vehicle:

no

Details on test solutions:

Test method
A static test procedure with one application at the start of the study was performed.

Test concentration
The saturated solution was tested in a limit test as threshold concentration. Taking into account the aquatic toxicity data of the test item the test was performed without a range-finding test. The threshold concentration (TC) was based on the toxicity data of the test item of an alga toxicity test
(ErC50 (0-72 h) > 0.243 mg/L, EyC50 (0-72 h) > 0.243 mg/L, based on the geometric mean measured test item concentration of the saturated solution) SCHEERBAUM, 2017.


Preparation of the saturated solution
The saturated solution (10.0 mg/L test item were weighed out) was prepared with dilution water.
The dispersion was stirred for 15 minutes (1100 rpm, room temperature) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL filtrate were discarded. The filtration was interrupted for ca. 15 minutes to allow for adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. During filtration the filter was always kept covered. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). Tyndall effect was negative.
The following filtrate, i.e. the saturated solution, was used in the test.


Control
7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: All fish used in the test were gained at NOACK LABORATORIEN from a single brood stock. (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin)
- Length at study end (length definition, mean, range and SD): Average body length: 2.61 cm
- Weight at study end (mean and range, SD): Average body weight: 0.163 g
- Method of breeding:Holding was performed at the test facility at 23 +/- 2 °C and diffuse light (0.1 - 10 µmol photons • m-2 • s-1, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. No disease treatments were administered throughout holding and testing. Food was provided 3 times per week. The amount of food was 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started.
- Feeding during test: No feeding during test


ACCLIMATION
- Acclimation period: Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within these days before the study starts were used in the test.
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Food: Sera Vipan; SERA GMBH, D-52518 Heinsberg
- Feeding frequency: 3 times per week.
- Health during acclimation (any mortality observed): No mortality observed, no disease treatments were administered throughout holding and testing.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Total Hardness at day 0: 69 mg CaCO3/L

Test temperature:

Please refer to "Any other information on materials and methods"

pH:

Please refer to "Any other information on materials and methods"

Dissolved oxygen:

Please refer to "Any other information on materials and methods"

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Type (delete if not applicable):
- Material, size, headspace, fill volume: Glass aquaria of 3 L were used, (dimensions: 11.5/13/20 cm), 2.5 L test volume
- Aeration: Gentle aeration was provided
- Renewal rate of test solution (frequency/flow rate): A static test procedure with one application at the start of the study was performed.
- Control: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates
- Reference item: No reference item is recommended for this test according to the guideline.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control and pH-control (replicates): 1
- Biomass loading rate: 0.456 g fish per L test solution


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove possible chlorine.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
Alkalinity: 0.60 mmol/L (measured quarterly)
Acidity: 0.10 mmol/L (measured quarterly)
Conductivity: 151 µS/cm (measured quarterly)



- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH-value, temperature and oxygen saturation were measured in all test vessels at the start and end of the
exposure and every 24 hours. Total hardness was determined at the start of the exposure out of the control. During the test the water temperature was recorded continuously (once per hour) with a data logger. The light intensity on the surface of the test aquaria was measured at the start of the exposure.




OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: A photoperiod of 16 h light / 8 h dark corresponding to natural daylight hours occurred during the course of the study.
- Light intensity: 7 - 750 Lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). Observations were made after 2, 24, 48, 72 and 96 h.


TEST CONCENTRATIONS
- Range finding study: None

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: Not observed
- Mortality of control: 0%
- Other adverse effects control: Not observed
- Abnormal responses: Not observed
- Any observations (e.g. precipitation):

Results with reference substance (positive control):

No reference substance tested

Reported statistics and error estimates:

Evaluation
After 96 hours, 0 % mortality occurred in both the control and the limit concentration. Hence, the LC0 , LC50 and LC100 were determined from these raw data. The NOEC and LOEC were determined from the recorded observations without any statistical calculations.

Software
All data were computer-generated and rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures.

 

Sublethal observations / clinical signs:

The test solution was visibly clear throughout the period of exposure.

 

All effect levels are given based on the nominal loading of the saturated solution of Pigment Red 247.The LC₀after 96 hours was 10.0 mg/L.

 

Observations in the Test Vessels

Nominal loading of the saturated solution of the test item [mg/L]

Effect *

Number of fish effected at observation time [hours]

2

24

48

72

96

10.0

(1)

7/7

7/7

7/7

7/7

7/7

Control

(1)

7/7

7/7

7/7

7/7

7/7

              *) The numbers in brackets correspond to the following observations: 

                         (1)      = Normal behaviour                          

 

Cumulative Mortality[%]in the Test Vessels

Nominal loading of the saturated solution of the test item [mg/L]		Cumulative mortality at observation time [hours]
		2		24		48		72		96
10.0		0		0		0		0		0
Control		0		0		0		0		0

Validity criteria fulfilled:

yes

Conclusions:

All effect levels are given based on the nominal loading of the saturated solution of the test item Pigment Red 247. The saturated solution of nominal 10.0 mg/L was found to have no effect on the vitality of zebrafish after 96 hours.
In conclusion, the LC0 corresponds to the nominal loading of the saturated solution 10.0 mg/L. The LC50 and the LC100 correspond to the nominal loading of the saturated solution > 10.0 mg/L. The measured initial concentration was below the lowest calibration level (< 0.3 mg/L).

Executive summary:

The acute toxicity of the test itemPigmentRed 247 (batch no.: MA566.004) to fish (zebrafish) was determined according to the OECD Guideline for Testing of Chemicals No. 203 (1992)and EU Council Regulation No. 440/2008/C.1 from 2017-06-26 to 2017-06-30 at the test facility.

 

A static test was performed as a limit test with the saturated solution (nominal loading: 10.0 mg/L) of the test item. The test had a duration of 96 hours. 7 test organisms were exposed to the test concentration and the control, respectively. The water quality parameters pH-value, temperature and oxygen saturation, measured after 0, 24, 48, 72 and 96 h, were determined to be within the acceptable limits.

 

The concentrations of the test item Pigment Red 247 were analytically verified via HPLC-DAD from the freshly prepared test media at start of the exposure.

 

The measured concentration of the limit concentration was below the lowest calibration standard (LCL) at the start of the exposure (0 hours). Therefore analytical monitoring at the end of the exposure (96 hours) was waived and the test item concentrations were considered to be below the LCL in the aged media, too.

 

All effect levels are given based on the nominal loading of the saturated solution of the test item Pigment Red 247. The saturated solution of nominal 10.0 mg/L was found to have no effect on the vitality of zebrafish after 96 hours.The results are summarized in the following table:

 

 

LC-Values(4 – 96 hours) of the Test Item

                  Based on the nominal concentration of the saturated solution of the test item [mg/L]

 

LC100= Lowest test item concentration with 100 % mortality after 96 h	> 10.0 mg/L
LC50	> 10.0 mg/L
LC0= Highest test item concentration with 0 % mortality after 96 h	10.0 mg/L

                     n.a. = not applicable

Description of key information

The acute toxicity of the test item Pigment Red 247 to fish (zebrafish) was determined according to the OECD Guideline for Testing of Chemicals No. 203 (1992)and EU Council Regulation No. 440/2008/C.1.

A static test was performed as a limit test with the saturated solution (nominal loading: 10.0 mg/L) of the test item. The test had a duration of 96 hours. 7 test organisms were exposed to the test concentration and the control, respectively. The water quality parameters pH-value, temperature and oxygen saturation, measured after 0, 24, 48, 72 and 96 h, were determined to be within the acceptable limits.

All effect levels are given based on the nominal loading of the saturated solution of the test item Pigment Red 247. The saturated solution of nominal 10.0 mg/L was found to have no effect on the vitality of zebrafish after 96 hours.