Reaction mass of Cobaltate(1-), bis[6-(amino-κN)-5-[2-[2-(hydroxy-κO)-4-nitrophenyl]diazenyl-κN1]-N-methyl-2-naphthalenesulfonamidato(2-)]-, sodium (1:1) and disodium [6-amino-5-[(2-hydroxy-4-nitrophenyl)azo]-N-methylnaphthalene-2-sulphonamidato(2-)][6-amino-5-[(2-hydroxy-4-nitrophenyl)azo]naphthalene-2-sulphonato(3-)]cobaltate(2-)

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 starting date: 29 November 1994 and Experimental termination date: 01 December 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

limit test: 3 concentrations tested.

GLP compliance:

yes

Specific details on test material used for the study:

Code number: FAT 20036/F
Batch-No: 1
Purity: ca. 97.7 %
Appearance: Solid
Solubility: 80 mg/L (in water)
Storage: room temperature
Expiration date: 07/99

Analytical monitoring:

yes

Details on sampling:

All test medium samples for the analytical determinations were taken from the approximate center of the test vessels (without mixing the medium) and kept at -18 °C to -25 °C until analysis.
The concentrations of the test substance at the beginning and at the end of the test were analysed by the PE Analytical Services K, Textile Dyes Division.

Vehicle:

yes

Details on test solutions:

The nominal test concentrations were chosen as 45, 100 and 220 mg test article/I.
For the preparation of the test concentrations only the supernatant of the stock solution was taken.
All test concentrations contained the vehicle Tween 80 (Polyoxyethylensorbitanmonooleat) in a concentration of 5 ppm.

Test organisms (species):

Daphnia magna

Details on test organisms:

The organism used in this test system is the species Daphnia magna. The Daphnia were bred in the laboratories of the Product Ecology TF, Textile Dyes Division under standardised conditions.

*Breeding:
- Cultures of Daphnia magna were held in glass vessels containing approx. 3.0 litre of reconstituted water at 18 - 22°C. Water was renewed partially three times/week. Daphnia's were fed with a suspension of green algae (Scenedesmus subspicatus) supplemented by a TETRAMIN-extract in such quantites that the food was consumed within 24h.

*Pretreatment:
- 24 hours before the start of the exposure reproductive, Daphnia were separated from the young Daphnia by sieving all individuals through a 1000 µm sieve.

For each test concentration, 20 Daphnia divided in two groups of 10 animals were used.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

None

Post exposure observation period:

Not available

Hardness:

197 mg/l CaCO3

Test temperature:

21 ± 1 °C

pH:

7.8 - 8.2

Dissolved oxygen:

Not available

Salinity:

Not applicable

Nominal and measured concentrations:

The nominal test concentrations were chosen as 45, 100 and 220 mg test article/L.
For the preparation of the test concentrations, only the supernatant of the stock solution was used.

Details on test conditions:

- Test procedure: static
- Duration: 48 hours
- 12 hours light/12 hours dark.
The Daphnia were exposed to the test substance in 250 mL beakers containing 100-200 mL test medium with 10 test animals per beaker. All tests were carried out in duplicate.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

131 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 131 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 131 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

131 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 131 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

131 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No immobilization and abnormal behaviour of the Daphnia was observed in the controls and in the test concentrations. Therefore, the NOEC and EC0 value after 48 hours were determined to be 131 mg/l, the EC50 and the EC100 value could not be calculated, but are clearly greater than 131 mg/L.

Results with reference substance (positive control):

The quality of the Daphnia will be checked at least every 3 months by determining the EC50 value (24 h) for potassium dichromate (range: 0.8 -1.5 mg/l). Last reference control at September 09, 1994 ; EC50 : 1.16 mg/l.

Reported statistics and error estimates:

No data

The test concentrations were prepared from a 1.0 g/l stock solution. Although, the test substance is soluble in pure (deionised) water (about 80 g/l), a part of the test substance in the stock solution settled down and build a sediment at the bottom of the flask. This effect was possibly caused by a reaction of the test substance with the water hardness compounds in the test water. For the preparation of the test concentrations only the supernatant of the stock solution was taken ( limit of the solubility ).

The nominal concentrations tested were 45, 100 and 220 mg test article/L, a blank and a vehicle control without any test material. In the test concentrations of 100 and 220 mg/l, nominal a sediment at the bottom of the test vessel was observed after 24 hours.

Because of the low solubility of the test article, the actual test conentrations in the analysed test media were below 45 - 74 % of the nominal values. Therefore, the reported results are related to the actual mean values of the analysed test concentrations of the test article measured after beginning and at the end of the test.

No immobilization and abnormal behaviour of the Daphnia was observed in the controls and in the test concentrations. Therefore, the NOEC and EC0 value after 48 hours were determined to be 131 mg/l, the EC50 and the EC-JOO value could not be calculated, but are clearly greater than 131 mg/l.

Immobilization of Daphnia

Number of immobilized Daphnia after 24 and 48 hours

Actual cone, oftest substance(mg/l)	N° of animals	N° of response 24 h	N° of response 24 h	% immobility 24 h	% immobility 48h
Blank	10	0	0	0	0
Vehicle	10	0	0	0	0
27	10	0	0	0	0
58	10	0	0	0	0
131	10	0	0	0	0

Analysis of the test substance

Analytical determination of test substance concentrations

	Strat concentrations		end concentrations
Nominal conc of test substance (mg/l)	mg/l	% of nominal	mg/l	% of nominal	Actual mean conc. mg/l
Blank	<3.0	/	<3.0	/	<3.0
Vehicle	<3.0	/	<3.0	/	<3.0
45	24	53	30	67	27
100	47	47	68	68	58
220	99	45	162	74	131

Method : Spectrophotometric Method AAK 61/94 ; SOP FO 3.31-2.3

Detection limit : 3.0 mg/l ( related to the nominal concentrations )

Measurements

The oxygen concentration, the temperature and the pH was controlled at the beginning and at the end of the test.

Start:

Oxygen: saturated

pH: 8.0 ( in the highest test concentration )

Temperature: 21 °C

End:

Nominal conc. (mg/l)	Blank	Vehicle	45	100	220
Oxygen %	99	99	98	97	95
pH	7.7	7.8	7.7	7.7	7.7
Temperature °C	20.5	20.5	20.5	20.5	20.5

ANALYTICAL DETERMINATION OF TEST ARTICLE IN DAPHNIA WATER

RESULTS

Designation of the sample: Content of test article

Test no. G 258 14: in daphnia water

Start	blank	29.11.1994	/	<3.0  mg/l
end	blank	01.12.1994	/	<3.0  mg/l
Start	vehicle	29.11.1994	/	<3.0  mg/l
End	Vehicle	01.12.1994	/	<3.0  mg/l
Start/A	45 mg/l	29.11.1994	24.4 mg/l
Start/B	45 mg/l	29.11.1994	23.8 mg/l	24  mg/l
Ende/A	45 mg/l	01.12.1994	30.2  mg/l
Ende/B	45 mg/l	01.12.1994	30.5  mg/l	30  mg/l
Start/A	100 mg/l	29.11.1994	45.9  mg/l
Start/B	100 mg/l	29.11.1994	47.7  mg/l	47  mg/l
Ende/A	100 mg/l	01.12.1994	69.3  mg/l
Ende/B	100 mg/l	01.12.1994	67.2  mg/l	68  mg/l
Start/A	220 mg/l	29.11.1994	99.8  mg/l
Start/B	220 mg/l	29.11.1994	98.3  mg/l	99  mg/l
Ende/A	220 mg/l	01.12.1994	161.2  mg/l
Ende/B	220 mg/l	01.12.1994	163.4  mg/l	162  mg/l

Validity criteria fulfilled:

yes

Conclusions:

No immobilization and abnormal behaviour of the Daphnia was observed in the controls and in the test concentrations. Therefore, the NOEC and EC0 value after 48 hours were determined to be 131 mg/l, the EC50 and the EC 100 value could not be calculated, but are clearly greater than 131 mg/l.

Executive summary:

The acute toxicity of FAT 20036/F to Daphnia magna was determined in a 48-hour static test according to the OECD Guideline for Testing of Chemicals, Section 2, No. 202, adopted 4 April 1984 ( issued 1993 ) and EU-Guideline 92/69, EEC Nr. L 383 A, C. 2, (29th Dec. 1992). The study was performed under Good Laboratory Practice (GLP) conditions.

The test concentrations were prepared from a 1.0 g/L stock solution. Although the test substance is soluble in pure (deionised) water (about 80 g/l, most of the test substance in the stock solution settled down and build a sediment at the bottom of the flask. This effect was possibly caused by a reaction of the test substance with the water hardness compounds in the test water. For the preparation of the test concentrations only the supernatant of the stock solution was taken ( limit of the solubility).

The nominal concentrations tested were 45, 100 and 220 mg test article/l, a vehicle and a blank control without any addtions. In the nominal test concentrations of 100 and 220 mg/l a sediment at the bottom of the test vessel was observed after 24 hours.

Because of the low solubility of the test article in the test media, the actual test concentrations in the analysed test media were in the range of 45 - 74 % of the nominal values. Therefore, the reported results are related to the actual mean values of the analysed test concentrations of the test article measured at the beginning and at the end of the test.

No immobilization and abnormal behaviour of the Daphnia was observed in the controls and in the test concentrations. Therefore, the NOEC and EC0 value after 48 hours were determined to be 131 mg/l, the EC50 and the EC 100 value could not be calculated, but are clearly greater than 131 mg/l.

Results (24hrs)

EC0: 131 mg/L

EC50: >131 mg/L

EC100: > 131 mg/L

Results (48hrs)

EC0: 131 mg/L

EC50 : >131 mg/L

EC100 > 131 mg/L

NOEC 131 mg/L

Conclusion:

FAT 20036/F has been tested up to the limit of solubility in the test water. According to Directive 93/21/EEC ( adaptation to Directive 67/548/EEC ) the ecotoxicological classification of FAT 20036/F is not toxic to Daphnia magna after 48 hours.

Description of key information

A study was conducted according to OECD guideline 202 to evaluate acute toxicity potential of FAT 20036/F to aquatic invertebrates (Daphnia magnia).

According to the results of the test, the NOEC and EC0 value after 48 hours were determined to be 131 mg/l, the EC50 and the EC 100 value could not be calculated, but are clearly greater than 131 mg/l.

Based on this data, the test substance FAT 20036/F can be considered as non-toxic to aquatic invertebrates.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

131 mg/L