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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 30 August 2002. Experimental completion date: 13 September 2002

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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/Batch number: 6558
Expiry: October 2002
Purity/Composition: 99.9%
Date received: 15 October 2001

Analytical monitoring:

yes

Details on sampling:

Duplicate samples, of 50 ml, were taken from the control and the test concentration at 0 hours (main stock solutions) and after 48 hours (replicates pooled) for analysis.

Vehicle:

no

Details on test solutions:

Method of preparation
The test substance, 20 mg of ST463 was dispersed in test medium, 1L of Elendt M4, 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 based on preliminary investigations inot the solubilit and stability of ST463 conducted in the acute fish study.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna (Straus) used in this study were cultured in-house and were obtained from a strain originating from the Institute National de Recherche Chimique Applique (IRChA), France.

Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 500 to 800 ml of Elendt M4 culture medium with a 16 hour light: 8 hour dark photoperiod at 19 to 20°C.
Cultures were fed daily with a suspension of the unicellular green alga, Chlorella vulgaris. Culture conditions ensure that cultures reproduce by parthenogenesis.

The day before the start of the study, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by the gravid (egg-bearing) adult Daphnia were
removed from the culture vessels and held in a separate holding vessel; these animals, which were less than 24 hours old, were used in the test.

The test organisms were maintained and the tests conducted in a reconstituted medium Elendt M 4. The medium was prepared using analytical grade reagents and deionised water.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

Cultures were maintained at 21 to 22 °C

pH:

ca. 7.7

Dissolved oxygen:

ca. 7.3

Nominal and measured concentrations:

Nominal concentration: 100% saturated solution
Mean measured concentration: 6.8 µg/L (0.0068 mg/L)

Details on test conditions:

Experimental design:
Four replicate vessels were prepared for the control and the test group. The test vessels were 250 ml capacity glass jars.

Five first instar Daphnia were placed without conscious bias into each glass jar, containing 100 ml of prepared test medium to give a loading of 20 ml test solution per organism. The jars were loosely covered to minimise evaporative losses.

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

Selection of 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 to conduct the definitive test at the limit of solubility.

Nominal test concentration: 100% saturated solution
Mean measured test concentration: 6.8 µg/L (Equivalent to 0.0068 mg/L)

The mean measured concentration of 6.8 µg/L was higher than the value achieved under the same conditions in the solubility trial (5.9 µg/L), conducted in a related acute fish study. The replication of such a low limit of solubility with a method of preparation involving prolonged stirring was thought to contribute to this variation. A difference in media type and temperature may have also affected the solubility.

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:
Daphnia were exposed to the test and control conditions for a period of 48 hours without renewal of test media.

Environmental conditions:
Cultures were maintained at 21 to 22 °C, in darkness and without supplementary aeration or feeding during the 48 hour exposure period .
The temperature, pH and dissolved oxygen levels were recorded in stock solutions at 0 hours and in
each test vessel at 48 hours

Criterion of effect:
Daphnia were considered to be immobile if they were unable to swim within approximately 15 seconds following gentle agitation of the test vessel.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 6.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

6.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Measured concentrations ranged from 7.5 to 7.7 µg/L at 0 hours and 5.6 to 6.4 µg/L at 48 hours.

Analysis of the immobility data gave the following results:
Time 24-48 (hours): EC50: >6.8 µg/L (Equivalent to >0.0068 mg/L)
Highest test concentration resulting in 0% immobilisation: 6.8 µg/L (Equivalent to 0.0068 mg/L)
Lowest test concentration resulting in 100% immobilisation: >6.8 µg/L (Equivalent to >0.0068 mg/L)
“No-observed effect concentration”: 6.8 µg/L (Equivalent to 0.0068 mg/L)
Control immobilisation: 0 %

Individual pH, temperature and dissolved oxygen values remained within acceptable limits during thestudy

Cumulative immobilisation data for Daphnia magna exposed for 48 hours to ST463

Nominal concentration (% saturated solution)	Mean measured concentration (µg/l)	Cumulative immobilised Daphnia magna (initial population: 5 per replicate)
		24 hours						48 hours
		R1	R2	R3	R4	Total	%	R1	R2	R3	R4	Total	%
Control	ND	0	0	0	0	0	0	0	0	0	0	0	0
100%	6.8	0	0	0	0	0	0	0	0	0	0	0	0

R1 -R4: replicates 1, 2, 3 and 4.

ND: None detected.

Validity criteria fulfilled:

yes

Conclusions:

The 48 hour EC50 (immobilisation) value for ST463 with Daphnia magna was determined to be >6.8 µg/L (Equivalent to >0.0068 mg/L).
The ‘No-observed effect concentration’ (NOEC) was 6.8 µg/L (Equivalent to 0.0068 mg/L).

Executive summary:

The potential acute toxicity of ST463 to Daphnia magna was assessed under static exposure conditions.

The study was conducted in compliance with the following guidelines:

- EC Methods for Determination of Ecotoxicity; Annex to Directive 92/69/EEC (OJ. No. L383A, 29.12.02) Part C, Method 2 “ Acute toxicity to Daphnia”

- OECD Guideline for Testing of Chemicals No. 202, Part I “ Daphnia Acute Immobilisation Test and Reproduction Test”.

Groups of twenty, first instar Daphnia, less than 24 hours old, were exposed to ST463 dispersed in Elendt M4 medium for 48 hours at a nominal concentration of 100% saturated solution. The numbers of immobilised daphnids were recorded for the test and control group after 24 and 48 hours and the

following values were determined. All results are expressed in terms of mean measured concentration.

Time 24-48 (hours): EC50: >6.8 µg/L (Equivalent to >0.0068 mg/L)

Highest test concentration resulting in 0% immobilisation: 6.8  µg/L (Equivalent to 0.0068 mg/L)

Lowest test concentration resulting in 100% immobilisation: >6.8  µg/L (Equivalent to >0.0068 mg/L)

“No-observed effect concentration”: 6.8  µg/L (Equivalent to 0.0068 mg/L)

Control immobilisation: 0 %

Measured concentrations ranged from 7.5 to 7.7 µg/L at 0 hours and 5.6 to 6.4 µg/L at 48 hours.

Description of key information

The potential acute toxicity of ST463 to Daphnia magna was assessed under static exposure conditions.

Groups of twenty, first instar Daphnia, less than 24 hours old, were exposed to ST463 dispersed in Elendt M4 medium for 48 hours at a nominal concentration of 100% saturated solution. The numbers of immobilised daphnids were recorded for the test and control group after 24 and 48 hours and the following values were determined. All results are expressed in terms of mean measured concentration.

Time 24-48 (hours): EC50: >6.8 µg/L (Equivalent to >0.0068 mg/L)

Highest test concentration resulting in 0% immobilisation: 6.8  µg/L (Equivalent to 0.0068 mg/L)

Lowest test concentration resulting in 100% immobilisation: >6.8  µg/L (Equivalent to >0.0068 mg/L)

“No-observed effect concentration”: 6.8  µg/L (Equivalent to 0.0068 mg/L)

Control immobilisation: 0 %

Measured concentrations ranged from 7.5 to 7.7 µg/L at 0 hours and 5.6 to 6.4 µg/L at 48 hours.

The mean measured test concentration was 6.8 µg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

6.8 µg/L