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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 17 September 2002. Experimental Completion date: 20 September 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition 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/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 control and test stock solution at 0 hours and from culture vessels at 72 hours (replicates pooled) for analysis. The samples were not filtered to remove algal cells before analysis.

Additional samples were taken from flasks containing the “no algae” cultures at 72 hours (replicates pooled).

Vehicle:

no

Details on test solutions:

Method of Preparation: The test substance, 20 mg of ST463 was dispersed in test medium, 1L of algal medium, 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.

Additional flasks containing the test substance at a nominal concentration equivalent to 1.7 µg/L, but without the presence of algal cells, were also prepared in order to obtain information on the extent of adsorption/absorption of the test substance by the algal cells.

Test organisms (species):

other: Selenastrum capricornutum

Details on test organisms:

Name:
Selenastrum capricornutum , Strain No. CCAP 278/4.

Source:
Algal cultures were obtained from the Culture Collection of Algae & Protozoa, Institute of Freshwater Ecology, Cumbria, UK.

Pre-culture:
Sterile nutrient medium was inoculated from a master culture and cultured under continuous illumination (8480 - 8720 lux) in an orbital incubator at 22 - 23°C, to give an algal suspension in log phase growth, characterised by a cell density of 2.1 x 10E6 cells/ml.

Diluent water:
Algal pre-culture was maintained, and the study conducted, in sterile nutrient medium as recommended in EU Method A.3.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

The temperature was maintained at 23 ± 1°C.

pH:

The pH of the test cultures was 7.6 at 0 hours and from 8.7 to 9.0 at 72 hours

Nominal and measured concentrations:

Nominal: 100 % saturated solution
Mean measured concentration: 1.7 µg/L

Details on test conditions:

Experimental design:
A single test concentration was prepared plus one untreated control, both with six replicates.

Although it was known from preliminary tests that the test material was unstable in aqueous solution under laboratory lighting, the cultures were exposed to normal test lighting conditions (6000 - 10000 lux). This was necessary to ensure the protocol validity requirement of 16 times growth was achieved in the control cultures.

Test concentrations:
A preliminary range finding study was not considered necessary because toxicity of the test material was unexpected at such a low limit of solubility discovered in previous preliminary work. In agreement with the Sponsor, the decision was taken to conduct the definitive
at the limit of solubility.
Nominal test concentration: 100% saturated solution
Mean measured test concentration: 1.7 (µg/L (Equivalent to 0.0017 mg/L).

The initial measured concentration of 2.5 µg/L was lower than the value achieved under the same conditions in the solubility trial (5.9 µg/L), conducted in a related GLP compliant study(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%.

Culture conditions:
Conical flasks (250 ml) each containing 100 ml of test or control culture were loosely stoppered and placed without conscious bias in a Gallenkamp illuminated orbital incubator. The cultures were incubated, without medium renewal, for 72 hours under continuous illumination of approximately 8120 - 9060 lux provided by fluorescent tubes. The temperature was maintained at 23 ± 1°C. Gaseous exchange and suspension of the algal cells were ensured by the action of the orbital shaker, oscillating at 130 cycles per minute.

MEASUREMENT OF GROWTH:
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined by direct counting using a Coulter ® Multisizer II particle counter.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

All results are expressed in terms of mean measured concentration. These results are based on mean measured concentrations, which ranged from 2.4 to 2.5 µg/L at 0 hours and were below the limit of detection (1.0 µg/L) at 72 hours in this study. Stability tests conducted as part of another related GLP compliant study indicate the substantial loss of parent material observed throughout the definitive test was due to photodegradation. Lighting was necessary for this study to ensure a sufficiently high algal growth rate was achieved to satisfy protocol validation criteria.

Due to the photosensitivity of the ST463, the concentration of the test substance in both cultures with and without the presence of algae was below the limit of detection (1.0 µg/L). Therefore an
assessment on whether the presence of algal cells had affected the stability of the exposure solutions could not be made.

EC50 (0 - 72 hours): >1.7 µg/L (Equivalent to >0.0017 mg/L)
“ No-observed effect concentration” (NOEC): 1.7 µg/L (Equivalent to 0.0017 mg/L)

The test was considered valid because cell concentrations in control cultures increased by a factor of at least 16 within 72 hours ( mean cell density of controls at 0 and 72 hours: 1.2 x 10E4 and 3.9 x 10E6 cells/ml, respectively).

The pH of the control cultures was 7.7 at 0 hours and from 7.7 to 8.8 at 72 hours, and therefore did not remain constant to within 1 unit over the 72 hour test period . This is believed to be a consequence of high growth rate.

OBSERVATIONS

All test and control cultures were inspected microscopically at 72 hours. No abnormalities were detected in any of the cultures examined.

No cultures showed any signs of contamination by foreign algal cells or protozoa.

Validity criteria fulfilled:

yes

Conclusions:

ST463 did not inhibit the growth of Selenastrum capricornutum, Strain No. CCAP 278/4 at concentrations tested in excess of 1.7 µg/L (Equivalent to >0.0017 mg/L) under the conditions of this
test.
The EC50 (0 - 72 hours) was >1.7 µg/L (Equivalent to 0.0017 mg/L).

Executive summary:

A study was conducted to assess the potential inhibitory effect of ST463 on the growth of the unicellular green alga Selenastrum capricornutum, Strain No. CCAP 278/4.

The study was conducted in compliance with the following guidelines:

- EC Methods for Determination of Ecotoxicity: Annex to Directive 92/69/EEC (O.J. No. L383 A, 29.12.92 ) Part C, Method 3 “ Algal Inhibition Test” .

- OECD Guideline for Testing of Chemicals No. 201 “ Alga, Growth Inhibition Test”.

Six algal cultures, with an initial cell count of approximately 1 x 10⁴ cells/ml, were exposed to ST463 at a mean measured test concentration of 1.7 µg/L. These cultures, together with one untreated control group of six replicates, were incubated in a Gallenkamp illuminated orbital incubator under

continuous illumination at 23 ± 1 °C for 72 hours. Cell numbers were counted daily to monitor growth.

The following values were derived from the data:

EC50 (0 - 72 hours):       >1.7 µg/L (Equivalent to >0.0017 mg/L)

“ No-observed effect concentration” (NOEC):       1.7 µg/L (Equivalent to 0.0017 mg/L)

These results are based on mean measured concentrations, which ranged from 2.4 to 2.5 µg/1 at 0 hours and were below the limit of detection (1.0 µg/1) at 72 hours in this study.

Description of key information

A study was conducted to assess the potential inhibitory effect of ST463 on the growth of the unicellular green alga Selenastrum capricornutum, Strain No. CCAP 278/4.

Six algal cultures, with an initial cell count of approximately 1 x 10⁴cells/ml, were exposed to ST463 at a mean measured test concentration of 1.7 µg/L. These cultures, together with one untreated control group of six replicates, were incubated in a Gallenkamp illuminated orbital incubator under continuous illumination at 23 ± 1 °C for 72 hours. Cell numbers were counted daily to monitor growth.

The following values were derived from the data:

EC50 (0 - 72 hours):       >1.7 µg/L (Equivalent to >0.0017 mg/L)

“ No-observed effect concentration” (NOEC):       1.7 µg/L (Equivalent to 0.0017 mg/L)

These results are based on mean measured concentrations, which ranged from 2.4 to 2.5 µg/1 at 0 hours and were below the limit of detection (1.0 µg/1) at 72 hours in this study.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.7 µg/L

EC10 or NOEC for freshwater algae:

1.7 µg/L