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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:

October from 14 to 20, 2005

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:

- Concentration: at the start of the test, all the concentrations were analitically measured. At the end of the test, only the concentration of the undiluted filtrate was analyzed due to the absence of a toxic effect.
- Samples: just before the start of the test, duplicate samples from each test medium and one replicate for control (without daphnids); after 48 hours (stability samples), duplicate samples from each test medium and one replicate for control.

Vehicle:

no

Details on test solutions:

Due to the low water solubility of the test item, a supersaturated dispersion with a loading rate of 100 mg/l was prepared by weighing 100.3 mg of the test item into 1000 ml of test water. No auxiliary solvent or emulsifier was used. The test item was mixed into the test water as homogeneously as possible using ultrasonic treatment for 15 minutes and intense stirring for 96 hours at room temperature in the dark to dissolve a maximum concentration of the test item in the dispersion.

Then, the dispersion of the test item was filtered through a membrane filter just before the preparation of the test media.
The undiluted filtrate of the dispersion with the maximum concentration of dissolved test item was used as the highest concentrated test medium. Additionally, aliquots of the filtrate were diluted with test water to prepare the test media with lower test item concentrations.

The test concentrations/dilutions were: the undiluted filtrate, as well as the dilutions 1:2.2, 1:4.6, 1:10 and 1:22. Additionally, a control (test water without test item) was tested in parallel.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: the study was performed with young daphnids of a clone of the species Daphnia magna Straus. A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield/UK in 1992.
- Breeding: the clone was bred in the testing laboratory 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 of parental stock: the daphnids used for 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:

no

Total exposure duration:

48 h

Test temperature:

20 °C

pH:

The pH values ranged from 7.8 to 7.9.

Dissolved oxygen:

At the beginning and the end of the test period, the dissolved oxygen concentrations in the test media and the control were at least 8.3 mg/l.

Nominal and measured concentrations:

0.09, 0.20, 0.48, 0.99 and 2.54 mg/l, measured at the start of the test
Highest concentration (undiluted): 2.1 mg/l, geom. mean.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml glass beakers filled with 50 ml of test medium.
- Type: the test vessels were covered with glass plates to reduce the loss of water and to avoid the entry of dust into the solutions.
- Aeration: the test water was aerated until oxygen saturation was reached. During the test period the test water was not aerated.
- No. of organisms per vessel: 20 daphnids were used per concentration.
- No. of vessels per concentration: four replicates of five daphnids each.
- No. of vessels per control: four replicates of five daphnids each.
- Biomass loading rate: the loading rate was less than one daphnia per 5 ml of test solution.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: analytical grade salts were dissolved in purified water to obtain the following nominal concentrations CaCI2 × 2H20 2.0 mmol/l (= 294 mg/l), MgSO4 × 7H20 0.5 mmol/l (= 123 mg/l), NaHCO3 0.75 mmol/l (= 65 mg/l), KCI 0.075 mmol/l (= 5.8 mg/l), water hardness 2.5 mmol/l (= 250 mg/l as CaCO3), alkalinity 0.8 mmol/l, ratio of Ca : Mg = 4: 1 (based on molarity), 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: between approximately 570 and 740 Lux

EFFECT PARAMETERS MEASURED
The daphnids were observed for immobility after 24 and 48 hours of exposure; those organisms not able to swim within 15 seconds after gentle agitation of the test beaker were considered immobile.

RANGE-FINDING STUDY
- Test solution: the test item precipitated immediately after addition to test water at a concentration of 100 mg/l. Thus, the test item was not completely soluble at the test concentration of 100 mg/l and no homogeneous dispersion could be prepared. Therefore, a filtrate of a supersaturated dispersion of the test item with the loading rate of 100 mg/l was prepared and was used as stock solution for the preparation of the test media with lower test item concentrations.

Reference substance (positive control):

yes

Remarks:

potassium dichromate, tested at least once a year

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 2.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

In the control and at all test item concentrations no immobilized test organisms were observed during the test period of 48 hours. The highest test item concentration corresponds to the loading rate of 100 mg/l.
Therefore, the 48-hour NOEC and the 48-hour EC0 to Daphnia magna were at the loading rate of 100 mg/l (2.1 mg/l). The 48-hour NOEC and the 48-hour EC0 might even be higher, but concentrations above the loading rate of 100 mg/l were not tested due to the limited water solubility of the test item in the test water.
The 48-hour EC50 and the 48-hour EC100 were clearly higher than the loading rate of 100 mg/l. These values could not be quantified, since the test item had no toxic effect at the concentration of 2.1 mg/l, and thus above the water solubility limit of the test item in test water.
In conclusion, test item had no acute toxic effects on Daphnia magna up to its solubility limit in test water under the present conditions of the test.
No remarkable observations were made concerning the appearance of the test media. The test media were clear solutions coloured by the test item throughout the whole test duration.

APPEARANCE OF TEST SOLUTION
Over the whole exposure period, the test solution appeared to be clear; noticeable colouration caused by the test item (violet).

MEASURED CONCENTRATIONS
The analytically measured test item concentrations in the analyzed test media samples (dilutions 1:22, 1:10, 1:4.6, 1:2.2 and the undiluted filtrate) amounted to 0.09, 0.20, 0.48, 0.99 and 2.54 mg/l at the start of the test. At the end of the test, only the concentration of the undiluted filtrate was analyzed due to the absence of a toxic effect. The measured concentration of test item in the undiluted filtrate decreased slightly to 1.72 mg/l after 48 hours.
The mean measured test item concentration was 2.1 mg/l, which was calculated as the geometric mean of the test item concentration in the undiluted filtrate measured at the start and the end of the test period.

Results with reference substance (positive control):

The latest result of the positive control test in February 2005 (48-hour EC50: 0.53 mg/l) showed that the toxic performance was valid and within the historical range of the testing laboratory (from 1996 to 2005: 48-hour EC50: 0.53 - 1.1 mg/l).

Effect of test item on the mobility of Daphnia magna.

Dilution / treatment	Initially measured conc (mg/l)	N. of daphnids tested	Immobilized daphnids		% immobilization
			24 hrs	48 hrs	24 hrs	48 hrs
Control	--	20	0	0	0	0
1 : 22	0.09	20	0	0	0	0
1 : 10	0.20	20	0	0	0	0
1 : 4.6	0.48	20	0	0	0	0
1 : 2.2	0.99	20	0	0	0	0
Undiluted filtrate (LR 100 mg/l)	2.54 (2.1*)	20	0	0	0	0

LR: loading rate

* Mean of measured concentration (calculated as the geometric mean of the test item concentration in the undiluted filtrate measured at the start and the end of the test period).

Dissolved oxygen concentrations, pH values and temperature in the test media and the control.

Dilution / treatment	Start (0 hr)			End (48 hrs)
	pH	Oxygen (mg/l)	Temperature (°C)	pH	Oxygen (mg/l)	Temperature (°C)
Control	7.8	8.7	20	7.8	8.5	20
1 : 22	7.8	8.6	20	7.8	8.3	20
1 : 10	7.8	8.6	20	7.8	8.3	20
1 : 4.6	7.8	8.7	20	7.8	8.3	20
1 : 2.2	7.9	8.7	20	7.8	8.5	20
Undiluted filtrate (LR 100 mg/l)	7.9	8.7	20	7.9	8.6	20

LR: loading rate

Conclusions:

The test item had no acute toxic effects on Daphnia magna up to the solubility limit in test water (i.e. 2.1 mg/l) under conditions of the test.

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 202 (2004).

Due to the low water solubility of the test item, a supersaturated dispersion of the test item with a loading rate of 100 mg/l was continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered and the undiluted filtrate with the maximum concentration of dissolved test item was used as the highest test medium concentration and as stock solution for dilutions in a geometric series differing by a constant factor of 2.2. Additionally, a control was tested in parallel. The analytically measured test item concentrations in the analyzed test media samples (dilutions 1:22, 1:10, 1:4.6, 1:2.2 and the undiluted filtrate) amounted to 0.09, 0.20, 0.48, 0.99 and 2.54 mg/l at the start of the test. At the end of the test, only the concentration of the undiluted filtrate was analyzed due to the absence of a toxic effect. The measured concentration of in the undiluted filtrate decreased slightly to 1.72 mg/l after 48 hours. The mean measured test item concentration was 2.1 mg/l, which was calculated as the geometric mean of the test item concentration in the undiluted filtrate measured at the start and the end of the test period.

In the control and at all test item concentrations, no immobilized test organisms were observed during the test period of 48 hours. The highest test item concentration corresponds to the loading rate of 100 mg/l.

The 48-hour NOEC and the 48-hour EC₀ of the substance to Daphnia magna were at the loading rate of 100 mg/l (2.1 mg/l). The 48-hour NOEC and the 48 hour EC₀ might even be higher, but concentrations above the loading rate of 100 mg/l were not tested according to the test guidelines. The 48-hour EC₅₀ and the 48-hour EC₁₀₀ were clearly higher than the loading rate of 100 mg/l. These values could not be quantified, since the test item had no toxic effect on the daphnids up to the highest test item concentration which could be dissolved in the test water under the conditions of the test.

Conclusion

The test item had no acute toxic effects on Daphnia magna up to the solubility limit in test water (i.e. 2.1 mg/l) under conditions of the test.

Description of key information

No acute toxic effects up to solubility limit in test water (i.e. 2.1 mg/l) under conditions of the test.

Additional information

Short term toxicity to aquatic invertebrates was assessed by data on Direct Blue 267 as well on a structural analogue, i.e. Similar Substance 01.

Direct Blue 267 was tested in a 48-hour static test according to the OECD guideline 202 (2004). Due to the low water solubility of test item, a supersaturated dispersion of test item with a loading rate of 100 mg/l was prepared; the dispersion was filtered and the undiluted filtrate. The analytically measured test item concentrations in the analysed test media samples (dilutions 1:22, 1:10, 1:4.6, 1:2.2 and the undiluted filtrate) amounted to 0.09, 0.20, 0.48, 0.99 and 2.54 mg/l at the start of the test. At the end of the test, only the concentration of the undiluted filtrate was analysed due to the absence of a toxic effect. The measured concentration of in the undiluted filtrate decreased slightly to 1.72 mg/l after 48 hours. The mean measured test item concentration was 2.1 mg/l, which was calculated as the geometric mean of the test item concentration in the undiluted filtrate measured at the start and the end of the test period. In the control and at all test item concentrations, no immobilized test organisms were observed during the test period of 48 hours. The test item had no acute toxic effects on Daphnia magna up to the solubility limit in test water (i.e. 2.1 mg/l, measured geometric mean) (Büche, 2005).

Comparable results were obtained with Similar Substance 01. After 24 hours of exposure, the total rate of immobilisation of daphnia was 100 % at 1000 mg/l, 75 % at 560 mg/l, 20 % at 320 mg/l, 30 % at 180 mg/l, 15 % at 100 mg/l and at 56 mg/l and 5 % at 32 mg/l. At the concentrations of 18 mg/l and lower, no immobilisation of daphnia was seen. The No Observed Effect Concentration for test item was found to be 18 mg/l after 24 hours of exposure (Bogers, 1990).

In the report it is indicated that the colour of the test concentrations increased from clear light to deep dark blue; this indication let to suspect that a non-clear solution was tested.