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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December from 05 to 07, 1990
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:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
adopted April 4th, 1984.
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Analytical measurements of the test substance were performed in samples of test media of the nominal test concentrations 31.3, 125.0 and 500.0 mg test substance/l and control at the start and the end of the test by sampling freshly prepared test media and media, which were incubated during test duration under the same conditions as in the test itself (but without Daphnia).
All samples are kept stored at -20 °C to enable additional analyses. After delivery of the final test report all samples will be discarded.
Details on test solutions:
A stock solution was prepared by dissolving the test substance in test water (0.5 g/l). The stock solution was diluted in a series of sequential dilutions with test water to prepare the nominal concentrations.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna.
- Strain/clone: clone of Daphnia magna Straus.
- Source: bred in the testing laboratory.
- Age at the start of the test: 6 - 24 hours old.

ACCLIMATION
- Acclimation conditions: the parental Daphnia were maintained in the same reconstituted water as in the test for at least 24 hours prior to the start of the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
18.1 - 19.6 °C
pH:
7.6 - 8.1
Dissolved oxygen:
≥ 8 mg/l
Nominal and measured concentrations:
Nominal: 31.3, 62.5, 125, 250 and 500 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml - glass beaker.
- Fill volume: 50 ml.
- No. of organisms per vessel: groups of 5 animals.
- No. of organisms per concentration: 20.
- No. of vessels per concentration: 4.
- No. of vessels per control: 4.

TEST MEDIUM
- Preparation of test medium: reconstituted water aerated to saturation was used in the test (according to the EEC Directive). In deionized water with a conductivity lower than 0.1 μs/cm analytical grade salts were dissolved to following nominal concentrations: 294 mg/l of CaCl2.2H2O, 123 mg/l of MgSO4.7H2O, 65 mg/l of NaHCO3, 5.8 mg/l of KCl.
- Hardness: 2.5 mmol/l as CaCO3.
- Alkalinity: 0.8 mmol/l.
- Ca : Mg ratio: 4 : 1
- Na : K ratio: 10 : 1

OTHER TEST CONDITIONS
- Photoperiod: 16 hrs light / 8 hrs darkness.
- Light intensity: about 200-600 Lux.

EFFECT PARAMETERS MEASURED
The immobility or mortality of the Daphnia was determined by visual control after 24 and 48 hours: those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile, respectively dead.
The 48-hour EC 10, -50 and -90 and their 95 %-confidence limits were calculated by the Probit Analysis. After 24 hours test duration only the EC 50 could be calculated by the Moving Average Interpolation.

PARAMETERS MEASURED
At the start of the test, the pH-values and the dissolved oxygen concentrations were determined in the control and in the highest concentration tested, at the end of the test these parameters in all test concentrations and control. Water temperature was measured in one control beaker at the start and the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.
- Range finding study: the test concentrations were based on the results of a range-finding test which was though not performed in compliance with GLP.
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
64.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other:
Remarks:
95 % CL: 53.8 - 78.3 mg/l.
Details on results:
The 24-hour EC50 of the test substance was calculated to be 240.6 mg test substance/l with 95 % confidence limits from 204.5 to 283.0 mg test substance/l.
After 48 hours test duration the toxicity of the test substance has highly increased. The 48-hour EC50 was calculated to be 64.9 mg test substance/l with 95 % confidence limits from 53.8 to 78.3 mg test substance/l. The calculated 48-hour EC10 was 35.5 mg test substance/l (95 % confidence limits: 26.4 47.8 mg/l), the 48-hour EC90 amounted to 118.8 mg test substance/l (95 % confidence limits: 89.0 - 158.5 mg/l). The 48-hour EC100 was determined to be 250 mg test substance/l.

In the control no immobility or mortality of the test animals was observed.

MEASURED CONCENTRATION
The test substance concentrations in the analysed test media were in the range of 95.8 - 112.3 % of the nominal values during test duration. The test substance was sufficiently stable under test conditions. Therefore, all reported results are related to the nominal concentrations of the test substance.

Influence of the substance on the mobility of Daphnia magna.

Nominal conc.
of test substance
No of Daphnia
tested
No of immobilized Daphnia after: % of immobilized Daphnia after:
24 h 48 h 24 h 48 h
Control 20 0 0 0 0
31.3 mg/l 20 1 1 5 5
62.5 mg/l 20 1 10 5 50
125.0 mg/l 20 1 18 5 90
250.0 mg/l 20 15 20 75 100
500.0 mg/l 20 10 20 50

100

Quality measurements (dissolved oxygen and pH) of the test media at the start and end of the acute toxicity test are presented in the table below.

Nominal conc.
of test substance
Start (0 hours) End (0 hours)
Oxygen  pH Oxygen  pH
Control 9.3 7.9 8.5 8.1
31.3 mg/l

*

*

8.5 8.0
62.5 mg/l

*

* 8.6 7.9
125.0 mg/l

*

* 8.5 7.9
250.0 mg/l * * 8.4 7.9
500.0 mg/l 9.3 7.6 8.0 7.9

* = not measured

Results obtained for the concentrations of test item in test medium

Nominal concentration [mg/l] Sampling period Conc. of test item found [mg/l]* % of nominal
31.3
B
30.0
95.8
31.3
E
32.4
103.4
125.0
B
128.2
102.6
125.0
E
140.0
112.0
500.0
B
510.8
102.2
500.0
E
561.6
112.3
Control sample
B
--- **
---
Control sample
E
--- **
---

* Mean value of a repeated test

** Counts were below lowest calibration point

B = Beginning of experiment

E = End of experiment

Validity criteria fulfilled:
yes
Remarks:
mortality of the control did not exceed 10 % at the end of the test, dissolved oxygen concentration was more than 60 % of air saturation value
Conclusions:
EC50 (48h): 64.9 mg/l (nominal)
Executive summary:

The acute toxicity of test item 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 April 04, 1984 and the EEC Directive 84/449, L 251, Part C 2.

The nominal concentrations tested were 31.3, 62.5, 125, 250 and 500 mg test substance/l and a control (test water without any additions).

The 24-hour EC50 of the test substance was calculated to be 240.6 mg test substance/l with 95 % confidence limits from 204.5 to 283.0 mg test substance/l. After 48 hours test duration the toxicity of the test substance has highly increased. The 48-hour EC50 was calculated to be 64.9 mg test substance/l with 95 % confidence limits from 53.8 to 78.3 mg test substance/l. The calculated 48-hour EC10 was 35.5 mg test substance/l (95 % confidence limits: 26.4 47.8 mg/l), the 48-hour EC90 amounted to 118.8 mg test substance/l (95 % confidence limits: 89.0 - 158.5 mg/l). The 48-hour EC100 was determined to be 250 mg test substance/l.

In the control no immobility or mortality of the test animals was observed.

The test substance concentrations in the analysed test media were in the range of 95.8 - 112.3 % of the nominal values during test duration. The test substance was sufficiently stable under test conditions. Therefore, all reported results are related to the nominal concentrations of the test substance.

Conclusion

EC50 (48h): 64.9 mg/l (nominal) (95 % conf. limits: 53.8 - 78.3 mg/l)

Description of key information

EC50 (48h): 64.9 mg/l (nominal)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
64.9 mg/L

Additional information

The acute toxicity of test item to Daphnia magna was determined in a 48-hour static test according to the OECD Guideline 202. The nominal concentrations tested were 31.3, 62.5, 125, 250 and 500 mg test substance/l. The 24-hour EC50 of the test substance was calculated to be 240.6 mg test substance/l with 95 % confidence limits from 204.5 to 283.0 mg test substance/l. After 48 hours test duration the toxicity of the test substance has highly increased. The 48-hour EC50 was calculated to be 64.9 mg test substance/l with 95 % confidence limits from 53.8 to 78.3 mg test substance/l. The calculated 48-hour EC10 was 35.5 mg test substance/l (95 % confidence limits: 26.4 47.8 mg/l), the 48-hour EC90 amounted to 118.8 mg test substance/l (95 % confidence limits: 89.0 - 158.5 mg/l). The 48-hour EC100 was determined to be 250 mg test substance/l.

In the control no immobility or mortality of the test animals was observed. The test substance concentrations in the analysed test media were in the range of 95.8 - 112.3 % of the nominal values during test duration. The test substance was sufficiently stable under test conditions.

A second test on the substance under registration is available. The composition of the tested substance is comparable to those of the lot tested in the abovementioned experiment, however the results obtained are significantly different.

The acute toxicity of the substance to Daphnia magna was determined in a 48-hr test according to the OECD Guideline 202. 20 daphnids (divided in 4 groups of 5 daphnids each) were exposed to 5 concentrations of the substance, i.e. 0.1, 1, 10, 100 and 1000 mg/l, over a period of 48 hours. The EC50 at 24 hours resulted to be 12.9 mg/l, while at 48 hours the EC50 resulted to be 1.20 mg/l.

Also in this case, after 48 hours test duration the toxicity of the test substance has highly increased; however, the magnitude of the effects resulted to be significantly higher respect to those recorded in the key study. Based on the available information, no clear explanation can be given about the differences obtained in the toxicity impact: as mentioned, the composition of the tested substances is comparable and the testing conditions are very similar. From the mobility reading point of view, the colouration of the test item may be a discriminating; however, there are no information or indication in the test report, which may let rise a suspect of an adverse impact.

In order to clarify the potential for short-term toxicity to aquatic invertebrates, the available data on the structural analogous Similar Substance 01 have been taken into account. Similar Substance 01 shares with Direct Black 168 the same scaffold chemical structure base; the differences are considered to be not able to impact the study outcomes, thus the read across can be considered as reliable and appropriated to evaluate the endpoint. Details on read-across approach are given in the IUCLID section 13.

The toxicity of the substance to Daphnia magna was assessed in the acute immobilization test, according to the OECD Guideline 202. Twenty daphnids (divided in 4 groups of 5 daphnids each) were exposed to 9 concentrations of the substance, ranging from 10 to 1000 mg/l (in a geometric progression with a dilution factor of 1.8), over a period of 48 hours. The number of daphnids that were floated in the water surface (immobilised) were measured at 24 h and 48 h in order to calculate the EC0 and EC100. The concentration-immobilization percentage was presented in a graph and the EC50 was calculated from the graph. The substance was dissolved in the water completely and the test solutions were all homogenised. However, at the concentrations 580 mg/l and 1000 mg/l it was not possible to measure the immobilised daphnids due to black coloration of the test solutions. No immobilization in the control group was recorded, i.e. 0 % at 24 h and 48 h. The recovery rate of the measured concentration was over 100 % in all the cases; it was higher than 120 % in 3 cases (32 mg/l, 100 mg/l and 180 mg/l). The EC50 value at 48 hours was found to be 76 mg/l (nominal).