4-[[5-(anilino)carbonyl-2-methoxyphenyl]azo]-3-hydroxynaphthalene-2-carboxamide

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:

Experimental start date: 12 April 2002. Experimental completion date: 28 September 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: ST463
Chemical name: 4-[[5-(anilino )carbonyl-2-methoxyphenyl]azo]-3-hydroxynaphthalene-2-carboxamide
Appearance: Magenta powder
Storage conditions: Room temperature in darkness
Lot number: 6558
Expiry: October 2002
Purity: 99.9%
Date received: 15 October 2001

Analytical monitoring:

yes

Details on sampling:

Duplicate samples, 50 ml, were taken from the control and the test concentration at 0 and 72 hours (fresh media), 24 and 96 hours (expired media) for analysis.

Vehicle:

no

Details on test solutions:

Method of preparation: The test substance, 400 mg of ST463, was dispersed in test medium, 20L of softened dechlorinated water, to give a stock solution of 20 mg/L. This stock solution was then stirred for 48 hours in the dark. Once stirred, the solution was filtered through GF/F glass fibre filter paper, size 0.7 µm, to
achieve 100% saturated solution.

Due to the photosensitivity of the test material, stirring was conducted in darkness by covering with black plastic sheeting, and the weighing of the test substance and filtering of the solution was conducted under subdued lighting to reduce the amount of exposure to light.

This method of test solution preparation was decided upon based on preliminary investigations into the solubility and stability of ST463.

DILUTION WATER
The water used for the study was laboratory tap water filtered, dechlorinated and softened by passage
through an Elga® water purification system. The water passes through a high grade activated carbon
filter to remove chlorine and any organic contaminants. A proportion of the supply then passes
through a water softener before final reverse osmosis (RO) treatment to produce a highly purified
water supply. These two supplies are then re-mixed in the ratio of approximately 2 parts 4R05 water
to 1 part carbon filtered water to give a laboratory supply with the desired water hardness.
Chlorine levels ranged from to 0.01 to 0.03 mg/L and free chlorine levels ranged from 0.00 to 0.02
mg/1 throughout the seven days of the acclimatisation period and during the exposure period and the
hardness level, calculated from daily measurements during the same period, was between 151 and
164 mgCaC03/L.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Name
Rainbow trout (Oncorhynchus mykiss).

Source
H.T.F, UK.

Acclimatisation
The stock of fish was obtained from the supplier on 10 September 2002 and acclimatised to test conditions from 12 September 2002. Temperature remained within the range 15 to 16°C and
dissolved oxygen within the range 8.3 to 9.4 mg02/L in the 12-day period immediately before the study with a 16 hour light: 8 hour dark photoperiod. The fish were fed daily to repletion with
commercial fry pellet but food was not given during the 24 hour period immediately before exposure or during the exposure period itself. No medication was given during the acclimatisation period and mortalities were recorded as <1 % in the 7 days before the test.

A representative sample of stock fish was measured at the start of the study. The mean standard length was 4.1 cm (SD = 0.2 cm) and the mean weight was 0.54 g (SD = 0.13 g).

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

The hardness level, calculated from daily measurements, was between 151 and 164 mgCaCO3/L.
Hardness of diluent water on Day 0: 156 mg CaC03/L
Hardness of highest test concentration on Day 0: 169 mg CaC03/L

Test temperature:

The treatment and control groups were maintained at 14 - 15°C throughout the exposure period.

pH:

7.6 (0 hours), 7.6 (24 hours), 7.6 (48 hours), 7.5 (72 hours), 7.7 (96 hours). Mean 7.6

Dissolved oxygen:

8.2. (0 hours), 8.8 (24 hours), 8.7 (48 hours), 8.8 (72 hours), 8.8 (96 hours). Mean 8.7 mgO2/L

Nominal and measured concentrations:

Nominal: 100 % saturated solution
Mean measured concentration: 2.7 µg/L (0.0027 mg/L)

Details on test conditions:

Experimental design:
A single test concentration was prepared plus one control. Ten fish were added to each vessel.

Fish were placed without conscious bias into glass aquaria containing control or prepared test medium, as appropriate. Each vessel contained 10 litres of medium to a depth of 18.5 cm
(approximate dimensions of vessel: 20 x 30 x 20 cm ). This provided a loading of 0.54 g bodyweight/litre (static volume).

Due to the photosensitivity of the test material, exposure tanks were covered with black plastic and the renewal of medium, fish observations, chemistry sampling and environmental measurements were all conducted under subdued lighting to reduce the amount of test material exposure to light.

Test concentrations:
The test material was expected to have little or no toxic effect at such a low limit of solubility.
Therefore a preliminary range finding test was not considered necessary.
In agreement with the Sponsor, the decision was taken by the Study Director to conduct the definitive
test at the limit of solubility .
Nominal concentrations: 100% saturated solution
Mean measured concentration: 2.7 µg/L (Equivalent to 0.0027 mg/L)

The mean measured concentration of 2.7 µg/L was lower than the value achieved under the same conditions in the solubility trial (5.9 µg/L). The replication of such a low limit of solubility with a method of preparation involving prolonged stirring was thought to contribute to this variation.

Nominal exposure concentrations quoted in this report refer to the test material as received; no allowance has been made for a purity of less than 100%.

Medium renewal:
Fish were exposed to the test or control conditions for a period of 96 hours with daily batchwise renewal of the test medium to ensure the maintenance of satisfactory environmental conditions and near nominal exposure levels.

Environmental conditions:
The treatment and control groups were maintained at 14 - 15°C throughout the exposure period.
Supplementary aeration was provided through narrow bore glass tubes in this study. A photoperiod of 16 hours light : 8 hours dark was maintained and daily records of pH, dissolved oxygen and temperature were kept for the control and test vessel. The fish were not fed during the 96 hour exposure period.

Criteria of effect
The criteria of death employed in this study were (i) absence of respiratory movement and (ii) absence of response to physical stimulation of the caudal peduncle.
In addition to observations on mortality at 2, 4, 24, 48, 72 and 96 hours, subjective assessments were made on the type and incidence of sub-lethal effects compared with control fish.

Reference substance (positive control):

no

Key result

Duration:

96 d

Dose descriptor:

LC50

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 2.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

All results are expressed in terms of mean measured concentration.

Measured concentrations ranged from 2.8 to 2.9 µg/L at 0 hours (fresh), 3.3 to 3.5 µg/L at 24 hours (expired), 1.8 to 2.3 µg/L at 72 hours (fresh) and 2.2 to 2.3 µg/L at 96 hours (expired).


Analysis of the mortality data gave the following results:
Time 2 - 96 (hours): LC50: > 2.7 µg/L (Equivalent to >0.0027 mg/L)
Highest test concentration resulting in 0% mortality: 2.7 µg/L (Equivalent to 0.0027 mg/L)
Lowest test concentration resulting in 100% mortality: >2.7 µg/L (Equivalent to >0.0027 mg/L)
“No-observed effect concentration”: 2.7 µg/L (Equivalent to 0.0027 mg/L)

Environmental parameters (pH, T°C and mg02/ l) remained within acceptable limits throughout the study.

Observations
There were no marked sub- lethal reactions to exposure throughout the 96 hour exposure period of this study.

Sublethal observations / clinical signs:

Cumulative mortality data for rainbow trout exposed for 96 hours to ST463

Nominal concentration (% saturated solution)	Mean measured concentration (µg/l)	Cumulative mortality (initial population: 10)
		2 h	4 h	24 h	48 h	72 h	96h
Control	ND	0	0	0	0	0	0
100	2.7	0	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

The 96 hour LC50 value for ST463 with rainbow trout was >2.7 pg/1 (Equivalent to >0.0027 mg/L).
The ‘no-observed effect concentration’ for ST463 with rainbow trout was 2.7 µg/L (Equivalent to 0.0027 mg/L).

Executive summary:

A study was performed to assess the potential toxicity of ST463 to rainbow trout ( Oncorhynchus mykiss) under semi-static conditions.

The study was conducted in accordance with the following guidelines:

- EC Methods for Determination of Ecotoxicity; - Annex to Directive 92/69/EEC (O.J. No. L383A, 29.12.92) Part C, Method 1 “ Acute toxicity for Fish” .

- OECD Guideline for Testing of Chemicals No. 203 “ Fish, Acute Toxicity Test”.

A group of ten juvenile fish were exposed to a single concentration, nominally 100% saturated solution, of ST463 dispersed in water. Observations were made on the numbers of dead fish and the incidence of sub-lethal effects after 2, 4, 24, 48, 72 and 96 hours exposure. The following values

were determined:

Time 2 - 96 (hours):       LC50: > 2.7 µg/L (Equivalent to >0.0027 mg/L)

Highest test concentration resulting in 0% mortality:       2.7 µg/L (Equivalent to 0.0027 mg/L)

Lowest test concentration resulting in 100% mortality:       >2.7 µg/L (Equivalent to >0.0027 mg/L)

“No-observed effect concentration”:       2.7 µg/L (Equivalent to 0.0027 mg/L)

Measured concentrations ranged from 2.8 to 2.9 µg/L at 0 hours (fresh), 3.3 to 3.5 µg/L at 24 hours (expired), 1.8 to 2.3 µg/L at 72 hours (fresh) and 2.2 to 2.3 µg/L at 96 hours (expired).

The mean measured test concentration was 2.7 µg/L.

Description of key information

A study was performed to assess the potential toxicity of the test substance to rainbow trout ( Oncorhynchus mykiss) under semi-static conditions.

A group of ten juvenile fish were exposed to a single concentration, nominally 100% saturated solution, of test substance dispersed in water. Observations were made on the numbers of dead fish and the incidence of sub-lethal effects after 2, 4, 24, 48, 72 and 96 hours exposure. The following values

were determined:

Time 2 - 96 (hours):       LC50: > 2.7 µg/L (Equivalent to >0.0027 mg/L)

Highest test concentration resulting in 0% mortality:       2.7 µg/L (Equivalent to 0.0027 mg/L)

Lowest test concentration resulting in 100% mortality:       >2.7 µg/L (Equivalent to >0.0027 mg/L)

“No-observed effect concentration”:       2.7 µg/L (Equivalent to 0.0027 mg/L)

Measured concentrations ranged from 2.8 to 2.9 µg/L at 0 hours (fresh), 3.3 to 3.5 µg/L at 24 hours (expired), 1.8 to 2.3 µg/L at 72 hours (fresh) and 2.2 to 2.3 µg/L at 96 hours (expired).

The mean measured test concentration was 2.7 µg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

2.7 µg/L