Tetrasodium 4,4'-bis[[4-morpholino-6-(p-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

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:

Experiment start date - 25 June 2008; Experiment end date - 07 August 2008; Study completion date - 20 October 2008.

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:

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: FAT 66042/A TE
Batch No.: 0582900
Purity: >80 %
Expiration date: 25-Feb-2013
pH: 9.5 at concentration of 10 g/L
Aggregate state / physical form at room temperature: solid
Storage conditions: At room temperature at about 20 °C, away from direct sunlight.

Analytical monitoring:

yes

Details on sampling:

The analytically determined concentration of the test item in the test medium at the start and the end of the test. For the 48-hour stability samples, the contents of the respective replicates were combined prior to sampling. All samples were stored deep-frozen (at about -20 °C) immediately after sampling. From the control, only one of the duplicate samples was analysed from each of the sampling times.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna Straus.
- Source: a clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield / UK in 1992. Since that time, the clone has been bred in the laboratories of RCC 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. For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control twice a year. The result of the latest positive control test indicated that the sensitivity of the test organisms was within the historical range of the RCC laboratory.
- Age at study initiation: young daphnids, 6-24 hours old, not first brood progeny.
- Feeding during test: Daphnids were not fed during the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

The test was performed in a temperature-controlled room with continuous monitoring of the room temperature. The water temperature was maintained at 20 °C.

pH:

The pH values of the test medium and control were 7.8.

Dissolved oxygen:

At the beginning and end of the test period, the dissolved oxygen concentration in the test medium and control was at least 8.4 mg/l.

Nominal and measured concentrations:

A single nominal concentration of the test item of 100 mg/l was tested.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml glass beakers
- Fill volume: Beakers filled with 50 ml of test medium.
- Type: The test vessels were covered with glass plates to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions.
- Aeration: During the test period, the test water was not aerated.
- Renewal rate of test solution: No renewal.
- No. of organisms: 20 daphnids were used divided into four replicates of five daphnids each.
- Volume of test solution provided for each daphnia: 10 ml.
The daphnids were randomly distributed to the test vessels at initiation of the test.

TEST MEDIUM / WATER PARAMETERS
- Dilution water: reconstituted test water according to ISO 6341 was used in the study. It consisted of analytical grade salts dissolved in purified water at the following nominal concentrations:
CaCl2 x 2H2O: 2.0 mmol/l, 296 mg/l
MgSO4 x 7H2O: 0.5 mmol/l, 123 mg/l
NHCO3: 0.75 mmol/l, 65 mg/l
KCl: 0.075 mmol/l, 5.58 mg/l
Water Hardness: 2.5 mmol/l, 250 mg/l as CaCO3
Alkalinity: 0.8 mmol/l
The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity.
- Aeration: Test water was aerated prior to the start of the study until oxygen saturation was reached.

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light to 8-hour dark cycle with a 30 minute transition period was used.
- Light intensity: between approximately 500 to 630 Lux.

At the start and at the end of the test, the pH values, the dissolved oxygen concentrations and the water temperature were determined at the single test concentration and in the control. The appearance of the test medium was visually recorded at the start of the test and after 24 and 48 hours.

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).

CONTROL
A control was tested in parallel (test water without addition of the test item).

TEST CONCENTRATIONS
The test medium was prepared by dissolving 100.1 mg of test item completely in 1000 mL of test water under intense stirring for 15 minutes at room temperature. The test medium was prepared just before introduction of the daphnids (i.e., start of the test).

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

The 48-hour NOEC and the 48-hour EC0 of test item to Daphnia magna were determined to be at least 100 mg/l.
The 48-hour NOEC and the 48-hour EC0 might even be higher but concentrations above 100 mg/l were not tested, in accordance with the test guidelines.
The 48-hour EC50 and the 48-hour EC100 were clearly higher than 100 mg/l. These values could not be quantified due to the absence of toxicity at the test concentration of 100 mg/l. No remarkable observations were made concerning the appearance of the test medium. The test medium was a clear solution throughout the whole test duration. In the control and at the test concentration of 100 mg/l, no immobilized test organisms were observed during the test period of 48 hours.

The analytically determined concentration of the test item in the test medium at the start and the end of the test was 105 and 104 % of the nominal value, respectively. Under the conditions of the test, the test item was stable during the test period of 48 hours.

Effect of test item on the Mobility of Daphnia magna

Nominal test item conc. (mg/l)	No daphnids tested	Immobilized daphnids after 24 hrs		Immobilized daphnids after 48 hrs
		N.	%	N.	%
Control	20	0	0	0	0
100	20	0	0	0	0

Dissolved Oxygen Concentrations, pH Values, and Temperature in the Treatments

Nominal test item conc. (mg/l)	Start (0 hours)			End (48 hours)
	pH	Oxygen (mg/l)	Temperature (°C)	pH	Oxygen (mg/l)	Temperature (°C)
Control	7.8	8.4	20	7.8	8.4	20
100	7.8	8.4	20	7.8	8.4	20

Analytical results;

The R2 fit of the calibration curve used was 0.9997. This reflects the linearity of the HPLC-system within the calibration range of 10.3 - 124 mg test item/L. Concurrent with the sample analysis, recoveries of spiked test water samples at a relevant concentration (101 mg test item/L) were performed in duplicate. The average recovery rate was found to be 106% of the spiked values (n = 2). No correction for the recovery rate was made. The limit of quantification for the test item in the injected solution (LOQ) was derived from the lowest calibration solution, which fits into the calibration curve: the value is 10.3 mg test item/L. The biological control samples and an analysed analytical blank (test water) did not significantly affect the HPLC-chromatogram at the retention time of the test item. The average recoveries found in the treatment samples at test start and at test end were 105 % and 104 % of the nominal concentrations.

Validity criteria fulfilled:

yes

Remarks:

In "control group" not more that 10 % of the daphnids were immobilised and the dissolved oxygen was higher than 3 mg/l in control and test vessels

Conclusions:

In the control and at the test item concentration of 100 mg/L, no immobilized test organisms were observed during the test period of 48 hours. The EC50 (48 h) of FAT 66042/A was determined to be greater than 100 mg/L.

Executive summary:

The acute toxicity of the test item to Daphnia magna was determined in a 48-hour static test according to the EU Commission Directive 92/69/EEC, Part C.2 (1992) and the OECD Guideline for Testing of Chemicals, 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 a nominal concentration of 100 mg/l. Thus, the only nominal concentration tested was 100 mg/l. Additionally, a control group was tested in parallel. The analytically determined concentration of the test item in the test medium at the start and the end of the test was 105 and 104 % of the nominal value, respectively.

Under the conditions of the test, the test item was stable during the test period of 48 hours. In the control and at the test item concentration of 100 mg/l, no immobilized test organisms were observed during the test period of 48 hours. The summary of the biological results (based on nominal test item concentration) is as follows:

EC0 and NOEC (48h) ≥100 mg/l

EC50 (48 h) >100 mg/l

Description of key information

In the control and at the test item concentration of 100 mg/l, no immobilized test organisms were observed during the test period of 48 hours.

The summary of the biological results (based on nominal test item concentration) is as follows:

EC0 and NOEC (48h) ≥ 100 mg/l

EC50 (48 h) > 100 mg/l

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

The acute toxicity of the test item to Daphnia magna was determined in a 48-hour static test according to the EU Commission Directive 92/69/EEC, Part C.2 (1992) and the OECD Guideline for Testing of Chemicals, 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 a nominal concentration of 100 mg/l. Thus, the only nominal concentration tested was 100 mg/l. Additionally, a control group was tested in parallel. The analytically determined concentration of the test item in the test medium at the start and the end of the test was 105 and 104 % of the nominal value, respectively.

Under the conditions of the test, the test item was stable during the test period of 48 hours. In the control and at the test item concentration of 100 mg/l, no immobilized test organisms were observed during the test period of 48 hours. The summary of the biological results (based on nominal test item concentration) is as follows:

EC0 and NOEC (48h) ≥100 mg/l

EC50 (48 h) >100 mg/l