reaction mass of sodium 1-amino-4-[[3,5-bis[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate and sodium 1-amino-4-[[3-[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

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:

November from 17 to 19, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted 4 April 1984

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

adopted 25 April 1984

Principles of method if other than guideline:

The study was conducted in accordance with ISO/IEC 17025.

GLP compliance:

no

Analytical monitoring:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Straus, 1820.
- Breeding: parental and young Daphnia were held in 250 I glass aquaria.
- Sze: < 2 mm

BREEDING
- Temperature: 20 ± 2 °C
- Illumination: 16 h per day.
- Aeration: continuously aerated reconstituted water (lSO 6341).
- Medium: distilled water (conductivity <1.5 µS/cm). The final test medium contained 25 ml/l of each of the following four stock solutions: CaCl2 x 2 H2O 11.76 g/l; MgSO4 x 7 H2O 4.93 g/l; NaHCO3 2.59 g/l; KCl 0.23 g/l. The sum of the Ca and Mg ions in this solution is 2.5 mmol/I. The ratio of Ca:Mg ions is 4:1 and of Na:K ions is 10:1. The total alkalinity of this solution is 0.8 mmol/l.
- Frequency of feeding: once a day, except weekends.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20 ± 0.5 °C

pH:

Test vessels: 7.0 - 7.7
Control: 6.9 - 7.5

Dissolved oxygen:

Test vessels: 6.4 - 6.7 mg/l
Control: 6.8 - 6.6 mg/l

Nominal and measured concentrations:

Nominal 1, 10 and 100 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml/flasks, all-glass, with 50 ml of test medium.
- Aeration: aeration vigorously over a period of 2 hours.
- No. of organisms per vessel: 20 daphnids.
- No. of vessels per concentration: 2 replicates
- No. of vessels per control: 3 replicates

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: reconstituted water (lSO 6341), not aerated.
- Conductivity: < 1.5 µS/cm
- Composition: CaCl2 x 2 H2O 11.76 g/l; MgSO4 x 7 H2O 4.93 g/l; NaHCO3 2.59 g/l; KCl 0.23 g/l.
- Ratio of Ca:Mg ions is 4:1 and of Na:K ions is 10:1.
- Total alkalinity: 0.8 mmol/l.

OTHER TEST CONDITIONS
- Photoperiod: 16 h photoperiod a day, supplied by overhead white fluorescent tubes.

EFFECT PARAMETERS MEASURED
Observations of immobile Daphnia were made atler 24 and 48 h of exposure.

PARAMETERS MEASURED
- Temperature: determined in a control flask at the start and at the end of the test.
- Oxygen, pH: determined in all vessels prior to the addition of Daphnia to the test medium and at the end of the test

VALIDITY OF THE TEST
- The test is considered valid if not more than 10 % of the Daphnia in the control have been immobilized or trapped at the surface of the water.
- The dissolved oxygen concentration at the end of the test should not be <60 % of the air saturation value at the temperature used.

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Alter 24 h of incubation no significant effects (<10 %) were observed at all test substance concentrations and in the blank control, respectively.
After 48 h of incubation the percentage of immobilization of the total of 40 individuals was 32.5 % at 100 mg/l. No significant effects (<10 %) were observed at 10 and 1 mg/l and in the blank control, respectively. lmmobilization at 48 h was significantly increased as compared to the effects after 24 h.

lmmobilization of Daphnia magna after 24 and 48 h of exposure to the test material

Nominal concentration (mg/l)	N.of daphnids per test vessel	lmmobile Daphnia		% immobile Daphnia
		24 hrs	48 hrs	24 hrs	48 hrs
Control	20	0	1	0	5
Control	20	1	1	5	5
Control	20	0	0	0	0
Test item 1 mg/l	20	0	1	0	5
Test item 1 mg/l	20	0	2	0	10
Test item 10 mg/l	20	1	1	5	5
Test item 10 mg/l	20	0	0	0	0
Test item 100 mg/l	20	1	7	5	35
Test item 100 mg/l	20	1	6	5	30

pH-values and oxygen concentrations at the start and at the end of the test

Nominal concentration (mg/l)	Mean pH values		Mean oxygen conc. (mg/l)
	24 hrs	48 hrs	24 hrs	48 hrs
Control	6.9	7.5	6.8	6.6
Control
Control
Test item 1 mg/l	7.0	7.6	6.6	6.6
Test item 1 mg/l
Test item 10 mg/l	7.0	7.6	6.5	6.7
Test item 10 mg/l
Test item 100 mg/l	7.0	7.7	6.4	6.7
Test item 100 mg/l

Validity criteria fulfilled:

yes

Remarks:

No more than 10 % of the Daphnia in the control were immobilized and the dissolved oxygen concentration at the end of the test was not less than 60 % of the air saturation value

Conclusions:

EC50 (48h) > 100 mg/l (nominal)

Executive summary:

The acute toxicity of test item to Daphnia magna was investigated under static exposure conditions over a period of 48 h. A screening test with nominal concentrations of 100, 10 and 1 mg/l, respectively, was performed.

40 individual Daphnia divided in 2 test vessels were exposed to each concentration of the test substance. The final test concentration was prepared by dilution of a stock solution of the test substance.

No chemical analysis of the test media was conducted. For the valuation of the test the nominal content of the test solution was used, assuming the test compound to be stable in water over 48 h.

Alter 24 hours of incubation no significant effects (<10 %) were observed at all test substance concentrations and in the blank control, respectively.

After 48 h of incubation the following percentage of immobilization of the total of 40 individuals was observed: 32.5 % at 100 mg/l. No significant effects (<10 %) were observed at 10 and 1 mg/l and in the blank control, respectively. Immobilization at 48 hours was significantly increased as compared to the effects after 24 hours.

The nominal median effect concentration (EC50) of test item to Daphnia magna for 24 and 48 hours was estimated to be higher then 100 mg/l.

Conclusion

EC50 (48h) > 100 mg/l (nominal)

Description of key information

Not harmful/toxic to aquatic invertebrates (EC50 (48h) > 100 mg/l (nominal)).

Additional information

The acute toxicity of Acid Blue 225 to Daphnia magna was investigated under static exposure conditions over a period of 48 hours. The experiment was conducted in accordance with the OECD guideline 202. A screening test with nominal concentrations of 100, 10 and 1 mg/l, respectively, was performed. No chemical analysis of the test media was conducted. For the valuation of the test the nominal content of the test solution was used, assuming the test compound to be stable in water over 48 hours. Alter 24 hours of incubation no significant effects (<10 %) were observed at all test substance concentrations and in the blank control, respectively. After 48 hours of incubation the following percentage of immobilization of the total of 40 individuals was observed: 32.5 % at 100 mg/l. No significant effects (<10 %) were observed at 10 and 1 mg/l and in the blank control, respectively. Immobilization at 48 hours was significantly increased as compared to the effects after 24 hours.