Anthraquinone acid dye

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:

15 December 2008 and 30 January 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols with no deviations from standard test guidelines and/or minor methodological deficiences which do not affect the quality of the relevant results

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The test material concentration in the test samples was determined spectrophotometrically using an external standard. The method was developed by the Department of Analytical Services, Harlan Laboratories Ltd, Shardlow, UK. A volume of test sample was diluted with methanol to give a final theoretical concentration of 10 mg/l. Standard solutions of test material were prepared in methanol at a nominal concentration of 10 mg/l.

- Sample storage conditions before analysis:
Room temperature in the dark
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

For the purpose of the definitive test the test material was dissolved directly in dechlorinated tap water.

An amount (20 g) of test material was dissolved in dechlorinated tap water and the volume adjusted to 20 litres to give the test concentration of 1000 mg/I. To ensure adequate mixing and homogeneity the test vessels were stirred using a flat bladed mixer for approximately 1 minute.

This method of preparation was conducted in triplicate to give replicates R1 to R3.

The concentration and stability of the test material in each replicate test vessel was verified by chemical analysis at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 hours (old media)

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Strain:
- Source: Obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Age at study initiation (mean and range, SD): nda
The fish had a mean standard length of 6.0 cm (sd = 0.6) and a mean weight of 3.05 g (sd = 0.86) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 1.5 g bodyweight/litre (static volume). This loading rate was in excess of the maximum value stated in the protocol of 0.8 g/l. This deviation was considered not to have adversely affected the results of the test as the fish showed no mortalities or sub-lethal effects of exposure and was considered not to have affected the validity or integrity of the test.

- Feeding during test: feeding was discontinued prior to the test commencing


ACCLIMATION
- Acclimation period: January 12 to January 26, 2009
- Acclimation conditions (same as test or not): as test
- Type and amount of food:
- Feeding frequency: The stock fish were fed commercial trout pellets which was discontinued 48 hours prior to the start of the definitive test.
- Health during acclimation (any mortality observed): There was less than 1% mortality in the 7 days prior to the start of the test and

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Hardness:

120 mg/L CaCO3

Test temperature:

14-16 C, above the 12 +/- 2 C range given in protocol. Deviation not believed to have affected outcome.

Dissolved oxygen:

>/= 9.8 mg/L

Nominal and measured concentrations:

A single nominal test concentration of 1000mg/l was used for the study. Analysis of the test preparations at 0, 24, 48, 72 and 96 hours showed measured test concentrations to range from 87% to 110% of nominal. It was therefore considered justifiable to estimate the LC50 values in terms of the nominal test concentrations only

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): covered without additional aeration
- Material, size, headspace, fill volume: 20L glass tank
- Renewal rate of test solution (frequency/flow rate): Single pass water renewal system
- No. of organisms per vessel:10
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates):2
- Biomass loading rate: 1.5 g bodyweight/litre (static volume). This loading rate was in excess of the maximum value stated in the protocol of 0.8 g/l. This deviation was considered not to have adversely affected the results of the test as the fish showed no mortalities or sub-lethal effects of exposure and was considered not to have affected the validity or integrity of the test.



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated lab tap water (activated carbon); partly softened
- Total organic carbon: 0.21 mg C/L
- Particulate matter: 0.3 mg/L
- Metals: None exceeding prescribed concentration values
- Pesticides: None exceeding prescribed concentration values
- Chlorine: Chlorine of diluent at 0, 24, 48, and 72 hours ranged from 0.010 to 0.24 mg/L
- Alkalinity: 84 mg/L as Ca CO3
- Conductivity: Conductivity 339 uS/cm
- Intervals of water quality measurement:
- Chemical Oxygen Demand 0.691 mg/L


Lighting cycle 16 hours light:8 hours dark, 20 minute transition periods



OTHER TEST CONDITIONS
- Photoperiod:


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :


TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Basis for effect:

mortality (fish)

Effect concentrations 2

Duration:

6 h

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Basis for effect:

mortality (fish)

Effect concentrations 3

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Basis for effect:

mortality (fish)

Effect concentrations 4

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Basis for effect:

mortality (fish)

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Basis for effect:

mortality (fish)

Effect concentrations 6

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Basis for effect:

mortality (fish)

Details on results:

Acute toxicity of the test material indicates 96-hour LC50 >1000 mg/L. The No Observed Effect Concentration (NOEC) >/= 1000 mg/L

Any other information on results incl. tables

Sublethal observations / clinical signs:

There were no mortalities in 30 fish exposed to a test concentration of 1000 mg/I for a period of 96 hours. Inspection of the mortality data gave the following results:

Time (h)	LC50 (mg/I)	95% Confidence limits (mg/I)
3	> 1000	‑
6	> 1000	‑
24	> 1000	‑
48	> 1000	‑
72	> 1000	‑
96	> 1000	‑

The results of the definitive test showed the highest test concentration resulting in 0% mortality to be greater than or equal to 1000 mg/I, the lowest test concentration resulting in 100% mortality to be greater than 1000 mg/I and the No Observed Effect Concentration (NOEC) to be 1000 mg/I. The No Observed Effect Concentration is based

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave an 96-Hour
LC50 of greater than 1000 mg/l. Correspondingly the No Observed Effect Concentration was greater than or equal to 1000 mg/l.

Executive summary:

In an Acute toxicity to rainbow trout(Oncorhynchus mykiss)study(Harlan project number: 0959/0245) the test material

the substance was determined to have an LC50 of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) was greater than or equal to 1000 mg/l.

Analysis of the test preparations at 0, 24, 48, 72 and 96 hours showed measured test concentrations to range from 87% to 110% of nominal and so it was considered justifiable to estimate the LC50 values in terms of the nominal test concentrations only.

The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute

Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008, US Code of Federal

Regulations Title 40, Part 797, Section 1400; US EPA Pesticide Assessment Guidelines, Sub-Division E, Section 72-1

and US EPA Draft Ecological Effects Test Guideline, OPPTS 850.1075.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/l.