Disodium 3,3'-[[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

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:

From September 11 to 23, 2006

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:

2004

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Just before the start of the test: duplicate samples from the test medium (without daphnids) and duplicate samples from the control (without daphnids).
- After 48 hours: duplicate samples from the test medium and duplicate samples from the control.
- Stability samples: for the 48-hour stability samples, the contents of the respective replicates were combined prior to sampling.
- Sample storage: all samples were deep-frozen (at about -20 °C) immediately after sampling. Based on preexperiments for investigation of the storage stability (without GLP), the test item was found to be stable in the test water under these storage conditions.

Details on test solutions:

The test medium was prepared by mixing 50.3 mg of test item into 500 ml of test water using ultrasonic treatment for 15 minutes and intense stirring for approximately 30 minutes at room terperature. The test medium was prepared just before introduction of the daphnids (i.e., start of the test).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: the study was performed with a clone of the species Daphnia magna Straus, definet by the supplier as clone 5.
- Source: a clone of the species was originally supplied by the University of Sheffield /UK in 1992.
- Breeding conditions: the clone has been bred in the testing laboratories in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests.
- Age at the start of the test: at the start of the test, the organisms used in the test were 6-24 hours old and were not first brood progeny.
- Feeding during test: none.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

140 mg/l as CaCO3

Test temperature:

20 °C

pH:

7.8

Dissolved oxygen:

At least 8.2 mg/l

Nominal and measured concentrations:

100 mg/l, nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml glass beakers.
- Fill volume: 50 ml of test medium.
- Aeration: the test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, the test water was not aerated.
- No. of organisms per vessel: 5 daphnids each.
- No. of vessels per concentration: 4 replicates.
- No. of vessels per control: 4 replicates.
- Biomass loading rate: at least 2 ml of test solution per daphnia.

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: the reconstituted test water was prepared according to ISO 6341. Hardness was lowered at minimum recommended by OECD. Analytical grade salts were added to purified water.
- CaCl2 x 2H2O: 1.12 mmol/l (i.e. 165 mg/l)
- MgSO4 x 7H2O: 0.28 mmol/l (i.e. 69 mg/l)
- NaHCO3:: 0.42 mmol/l (i.e. 36 mg/l)
- KCl: 0.042 mmol/l (i.e. 3.2 mg/l)
- Water hardness: 1.4 mmol/l (i.e. 140 mg/l, as CaCO3)
- Alkalinity: 0.45 mmol/l
- Ratio Ca : Mg: 4 : 1 (based on molarity)
- Ratio Na : K: 10 : 1 (based on molarity)

OTHER TEST CONDITIONS
- Photoperiod: a 16-hour light to 8-hour dark photoperiod (with a 30 minute transition period).
- Light intensity: light intensity during the light period was between approximately 470 and 640 Lux.

EFFECT PARAMETERS MEASURED
The daphnids were observed for immobility after 24 and 48 hours of exposure (daphnids not being able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobilized). The NOEC and EC0 were determined directly from the raw data.

POSITIVE CONTROL
Positive control was tested at lest once a year in the testing laboratory. Last test results demonstrated the sensitivity of the test organisms.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 78 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

ln the control and at the nominal concentration of the test item of 100 mg/l (mean measured concentration of 78 mg/l), no immobilized test organisms were observed during the test period of 48 hours.
Therefore, the 48-hour NOEC and the 48-h EC0 to Daphnia magna were determined to be at least 100 mg/l (mean measured 78 mg/l). The 48-h NOEC and 48-h EC0 might even be higher, but higher concentrations of the test item could not be tested due to the limited solubility of the test item in the test water. The 48-h EC50 and EC100 were clearly higher than 100 mg/l (mean measured 78 mg/l). These values could not be quantified due to the absence of toxicity of test item at the tested concentration.

MEASURED CONCENTRATIONS
At the start of the test, the measured concentration of the test item in the test medium was 82 % of the nominal concentration. At the end oithe test, 74 % of the nominal concentration was found in the test medium. The slight decrease of the test item concentration in the test medium during the test period was caused by precipitation and sedimentation of a part of the test item. At the end of the test, a part of the test item lay at the bottom of the test vessels. Thus, the maximum solubility of the test item in the test medium under the test conditions was obviously exceeded. The biological results were based on the nominal concentration of 100 mg/l and the mean measured concentration of thetest item in the test medium of 78 mg/l (calculated as geometric mean of the concentrations measured at the start and the end of the test).

TEST CONDITIONS
At the start of the test and after 24 hours, the test medium was colored (yellow) by the test item and a turbidity of the test medium was observed. After 48 hours, a part of the test item lay on the bottom of the test vessels. At the beginning and the end of the test period, the dissolved oxygen concentration in the test medium and in the control was at least 8.2 mg/l. The pH value of the test medium and the control was 7.8. The water temperature was 20 °C at the start and the end of the test.

Results with reference substance (positive control):

The latest result of positive control test showed an EC50 (3h) of 0.67 mg/l; the value resulted to be within the historical range, i.e 0.53 - 1.1 mg/l
.

Results obtained for the concentrations of the test item

	Sampling time	Measured concentration
		Test item (mg/l)	% of nominal	Average test item (mg/l)	Average % of nominal
Test item, 100 mg/l (nominal)	At start	80	80	82	82
	At start	83	83
	At 48 hrs	74.6	75	74	74
	At 48 hrs	73.4	73
Average over the test duration (geonetric mean)				78	78

Test conditions

	Start of the experiment			End of the experiment
Test vessel	pH	Oxygen (mg/l)	Temperature (°C)	pH	Oxygen (mg/l)	Temperature (°C)
Control	7.8	8.4	20	7.8	8.4	20
Test item, 100 mg/l	7.8	8.3	20	7.8	8.2	20

Validity criteria fulfilled:

yes

Remarks:

In the control not more that 10 % of the daphnids were immobilised; the dissolved oxygen concentration at the end of the test was more than 3 mg/l in control and test vessels

Conclusions:

EC50 (48h) > 78 mg/l (meas. geom. mean)

Executive summary:

The acute toxicity of the test item to Daphnia magna was determined in a 48-h static test, according to the EU Commission Directive 92/69/EEC, part c.2 (1992) and the OECD Guideline No. 202 (2004).

A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test organisms up to and including the concentration of 100 mg/l. Thus, the only nominal conceniration tested was 100 mg/l and a control. In pre-experiments it was noticed that a part of the test item precipitates in the test water during the test period, presumably due to formation of complexes with ions of the test water. Therefore, the hardness of the test water used in the main study was lowered to the minimum hardness of 140 mg/l (as CaCO₃) recommended by the OECD test guideline to minimize precipitation of the test item.

At the start of the test, the measured concentration of the test item in the test medium was 82 % of the nominal concentration. At the end of the test 74 % of the nominal concentration was found in the test medium. The slight decrease of the test item concentration in the test medium during the test period was caused by precipitation and sedimentation of a part of the test item. At the end of the test, a part of the test item lay at the bottom of the test vessels. Thus, the maximum solubility of the test item in the test medium under the test conditions was obviously exceeded. The biological results were based on the nominal concentration of 100 mg/l and the mean measured concentration of the test item in the test medium of 78 mg/l (calculated as geometric mean of the concentrations measured at the start and the end of the test).

In the control and at the nominal concentration of 100 mg/l (mean measured concentration of 78 mg/l), no immobilized test organisms were observed during the test period of 48 hours.

Therefore, the 48-h NOEC (highest concentration tested without toxic effects after the test period of 48 hours) and the 48-h EC0 of test item to Daphnia magna were determined to be at least 100 mg/l (mean measured 78 mg/l). The 48-h NOEC and the 48-h EC0 might even be higher, but higher concentrations of the test item could not be tested due to the limited solubility of the test item in the test water. The 48-h EC50 and EC100 were clearly higher than 100 mg/l (mean-measured 78 mg/l). These values could not be quantified due to the absence of toxicity of test item at the tested concentration.

Conclusion

EC50 (48h) > 78 mg/l (meas. geom. mean)

Description of key information

Not harmful/toxic to aquatic invertebrates up to the limit of solubility in the test medium (NOEC (48h) ≥ 78 mg/l (meas. geom. mean)).

Additional information

The acute toxicity of the test item to Daphnia magna was determined in a 48-h static test, according to the OECD Guideline No. 202 (2004). The only nominal concentration tested was 100 mg/l and a control. In pre-experiments it was noticed that part of the test item precipitates in the test water during the test period, presumably due to formation of complexes with ions of the test water. Therefore, the hardness of the test water used in the main study was lowered to the minimum hardness of 140 mg/l (as CaCO₃) recommended by the OECD test guideline to minimize precipitation of the test item.

At the start of the test, the measured concentration of the test item in the test medium was 82 % of the nominal concentration. At the end of the test, 74 % of the nominal concentration was found in the test medium. The slight decrease of the test item concentration in the test medium during the test period was caused by precipitation and sedimentation of a part of the test item. At the end of the test, a part of the test item lay at the bottom of the test vessels. Thus, the maximum solubility of the test item in the test medium under the test conditions was obviously exceeded.

In the control and at the nominal concentration of 100 mg/l (mean measured concentration of 78 mg/l), no immobilized test organisms were observed during the test period of 48 hours. Therefore, the 48-h NOEC was determined to be at least 100 mg/l (mean measured 78 mg/l). The 48-hour NOEC and the 48-h might even be higher, but higher concentrations of the test item could not be tested due to the limited solubility of the test item in the test water.