PIGMENTADDITIV RL

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:

1989-02-21 to 1989-06-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to relevant guidelines and compliant with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

(adopted April 4, 1984)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

(adopted April 25, 1984)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: see section "Principles of of method if other than guideline".

Principles of method if other than guideline:

"Letale Wirkung beim Zebrabärbling Brachydanio rerio" (LC0, LC50, LC100; 48 - 96 STUNDEN). Empfehlung des Umweltbundesamtes zur Durchführung der Prüfung nach §4 Abs.1 Nr.6 der Verordnung über Anmeldeunterlagen und Prüfnachweise nach dem Chemikaliengesetz Texte 16/84, Umweltbundesamt.

GLP compliance:

yes

Test material

Reference

Name:

Unnamed

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Sampling for analytical determination of the actual test item concentration was scheduled for the beginning and the end of each 24-hour interval of the semistatic test.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was made into a paste using Tween 80 as solubilizing agent. Water for dilution was added and the mixture homogenized using an Ultra-Turrax and ultrasonic bath. The mixture was then poured into the chamber under stirring with a glass rod. The chambers content was stirred for approximately 2 hours with a KPG-stirrer and a glass rod.
- Eluate: dilution water according to ISO/DIS 7346/1.
- Controls: one negative control, one control with an emulsifying agent.
- Chemical name of vehicle (emulsifier): Tween 80
- Concentration of vehicle in test medium: 0.095 ml/l
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The test batches were discolored.

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Source: West Aquarium, Bad Lauterberg, Germany
- Date of hatching: 1989-12-20/22
- Length at study initiation: 2.7 - 3.4 cm (Mean body length of 10 representative fish from the test batch. These fish were not used for the experiment).
- Feeding during test: no
- Food type: Tetra Min, Tetra Werke, Melle, Germany

ACCLIMATION
- Acclimation period: yes
- Acclimation conditions (same as test or not): yes
- Food type: Tetra Min, Tetra Werke, Melle, Germany
- Amount/frequency: twice daily ad libitum.
- Population density: <= 1 g fish/l water.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Hardness:

According to ISO/DIS 7346/1.

Test temperature:

Treatment group: 21.8 - 22.4°C
Control group: 21.3 - 22.3 °C

pH:

Treatment group: 7.6 - 8.3
Control group: 7.8 - 8.3

Dissolved oxygen:

Treatment group: 4.5 - 9.3 mg/l
Control group: 6.3 - 9.0 mg/l

Nominal and measured concentrations:

Nominal concentrations: 500 mg/l.
Measured concentrations: not applicable because of difficulties with regard to the analytical measurements (based on the low water solubility of the test item).

Details on test conditions:

TEST SYSTEM
- Test vessel: test chambers
- Material, size, headspace, fill volume: glass, length 30 cm, width 22 cm, height 24 cm, 10 l
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): The test chamber stood in a water bath made out of Hostalit Z^R with a Plexiglas viewing panel.
- Renewal rate of test solution (frequency/flow rate): every 24 hours.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to ISO/DIS 7346/1, preparation was carried out in a unit consisting of two Hostalen Z^R- lined steel vessels, each with a capacity of 1700 liters.
- Oxygen saturation: The prepared dilution water was aerated until oxygen saturation.
- Intervals of water quality measurement: At inspection times, i.e. after 2-4 and 24 hours at each part of the test (semi-static test, fish were transferred in new test chambers every 24 hours).

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12h
- Light intensity: 700 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Effect parameters: mortality, changes in appearance and behavior.
- Observation intervals: after 2-4 and 24 hours for every part of the test (semi-static test: fish were transferred in new test chambers every 24 hours).

TEST CONCENTRATIONS
- Range finding study: yes
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: no, however due to the dark water coloration observational recoding was limited.
- Mortality of control: no
- Other adverse effects control: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test water of the treatment groups was discolored.
- Effect concentrations exceeding solubility of substance in test medium: yes

Any other information on results incl. tables

Sublethal observations / clinical signs:

PRELIMINARY TEST (STATIC)

Prior the semi-static acute toxicity test a static test system was applied with the same nominal concentration of 500 mg/l as in the semi-static test. However, because of the low water solubility of the test item precipitation occurred during the experiment. The water was discolored and sediment deposits of the test item were observed. The body surface of the test organisms was dyed in a dark color. No mortality of fish was recorded but a change of startle response was observed.

MEASUERD TEST CONCENTRATIONS (SEMI-STATIC)

Nominal conc. (mg/l)	Measured conc. (mg/l)
	0 h	24 h	48 h	96 h
500	203,0	58	na	na

The analyzed samples of the concentrations depicted in the table above were taken in the first part of the experiment, i.e. at 0h and 24h. As the measured test item concentration was less than 50% of the nominal concentration and was decreasing over time, effect concentrations were based on nominal test concentrations.

Applicant's summary and conclusion

Validity criteria fulfilled:

no

Remarks:

The dissolved oxygen content was less than 60% of the air saturated value at the end of the third and fourth part of the experiment (semi-static). However, no mortality in fish occurred.

Conclusions:

In this 96-hour semi-static acute toxicity study in zebra fish (Brachydanio rerio) neither lethality nor changes in appearance or behavior could be observed in the treatment group. The LC50-96h is therefore >500 mg/l (based on the nominal concentration).

Executive summary:

The 96-hours acute toxicity study in zebra fish (Brachydanio rerio) was conducted in a semi-static test system because of the low water solubility of the test item. With regard to a range finding study, a limit test was performed at nominal 500 mg/l test item. In order to gain a better suspension of the test item in the test water, TWEEN 80 was used as the emulsifying agent. The concentrations tested were 500 mg/l test item, a negative control (0 mg/l) and a control group with the emulsifying agent TWEEN 80 using the concentration of 0.095 ml/l (number of fish per group: 10). After the preparation of the test medium, the batches showed a discoloration.

Due to a water solubility of the test item well below the limit of detection (5 mg/L), the actually dissolved fraction could not be analyzed. In order to determine the amount of test item dispersed in the test chamber, water samples were taken after 2-4 hours and 24 hours in the first treatment section of the experiment (semi-static test, fish were transferred in new test chambers every 24 hours). The amount of undissolved test item measured by gravimetric determination was 203 mg/l (0h) and 58 mg/l (24h) in the test batch with a nominal concentration of 500 mg/l. As the measured concentrations were less than 50% of the nominal concentration, the determined LC50 was based on the nominal value.

In this 96-hour semi-static acute toxicity study in zebra fish (Brachydanio rerio) neither lethality nor changes in appearance or behavior could be observed in the treatment group. The LC50-96h is therefore >500 mg/l (based on nominal concentration).

The study was performed compliant with GLP and according to the OECD guideline 203 (adopted April 4, 1984) and EU Method C.1 (Acute Toxicity for Fish) (adopted April 25, 1984).