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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:
May 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Principles of method if other than guideline:
The study also followed the Guidance document on aquatic toxicity testing of difficult test substances and mixtures. OECD series on testing and assessment number 23, December 14, 2000.
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Final test: A water soluble fraction prepared as a supernatant of an ultra-centrifuged dispersion with a nominal loading rate of 100 mg/l (100%) and a range of subsequent dilutions containing 10, 18, 32 and 56%.

- Sampling method:
Limit test: Samples for analyses were taken at the start and at the end of the 48-h test period.
Final test: During the final test no samples for analysis were taken as in either of the previous tests chemical analysis showed no detectable levels of the test substance in any of the samples taken from the water soluble fraction tested.

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- The batch of EA-3098 tested was a white powder of unknown purity. The water solubility was at the highest less than 0.004 g/l. Preparation of test solutions started with weighed amounts of test substance that were quantitatively added to volumetric flasks. In the range-finding test the solutions were treated with ultra-sonic waves for 10 minutes and then stirred for 30 minutes.

In the limit and the final test, preparation started with quantitatively adding weighted amounts of test substance to Milli-Q water. These solutions were then stirred for 72 hours.Subsequently they were ultra-centrifuged at relative centrifugal fields of up to 108,800 g and the salts for the ISO-medium were added to the supernatents to obtain the final test solutions.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain: not stated
- Source: In-house laboratory culture with a known history
- Age at study initiation (mean and range, SD): young daphnids with an age < 24 hrs, from parental daphnids of more than two weeks old
- Weight at study initiation (mean and range, SD): not stated in report
- Length at study initiation (length definition, mean, range and SD): not stated in report

- Method of breeding: Acyclical parthenogenesis under specified breeding conditions
Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week
Temperatyre of medium: 18-22°

- Feeding during test:
- no feeding during test

ACCLIMATION
- Acclimation period: at least third generation
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: fresh algae
- Feeding frequency: daily
- Health during acclimation (any mortality observed): no

QUARANTINE (wild caught)
- not applicable
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Not applicable
Hardness:
250 mg CaCO3/L
Test temperature:
Start of test: 20°C.
The temperature measured continuously in a temperature control vessel varied between 20 and 21°C during the test.
pH:
8.1 - 8.2
Dissolved oxygen:
0 hr: O2 = 9.4 - 9.5 mg/l
48 hr: O2 = 9.0 mg/l
Salinity:
Not applicable as freshwater study
Nominal and measured concentrations:
Nominal: 10, 18, 32, 56 and 100 mg/l prepared as water soluble fraction.
Measured: Measured concentrations of all three peaks were below their limits of detection.
Details on test conditions:
RANGE-FINDING TEST:
A range-finding test was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
- The solutions were treated with ultra-sonic waves for 10 minutes and then stirred for 30 minutes before filtering through a 0.45 µm filter.
- Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to solutions containing 0, 0.1, 1, 10 and 100 % of the filtrate prepared at 100 mg/l
- Dissolved oxygen concentrations and pH were only measured in the blank-control and the highest test concentration.
- No sampling for determination of actual test concentrations was performed other than initial samples taken from the filtrate prepared at 100 mg/l.

LIMIT TEST:
A limit test was performed based in the results of the range-finding test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
- Twenty daphnids per concentration (in quadruple, 5 per vessel) were exposed to a water soluble fraction prepared as a supernatant of an ultra-centrifuged dispersion (48,000 x g) with a nominal loading rate of 100 mg/l or an untreated control.
- Samples for analyses were taken at the start and at the end of the 48-h test period. The pellet of the centrifuged solution was dissolved in methanol, applying ultra-sonic treatment for 20 minutes.

FINAL TEST (additional test):
Test concentrations:
EA-3098: A water soluble fraction prepared as a supernatant of an ultra-centrifuged dispersion with a nominal loading rate of 100 mg/l (100%) and a range of subsequent dilutions containing 10, 18, 32 and 56%.
Controls: Test medium without test substance or other additives (blank-control).

Test procedure and conditions:
Test duration: 48 h
Test type: Static
Test vessels: 100 ml, all-glass
Medium: ISO
Number of daphnids: 20 per concentration
Loading: 5 per vessel containing 80 ml of test solution
Light: 16 h photoperiod daily
Feeding: No feeding
Aeration: No aearation of the test solutions
Introduction of daphnids: Within 15 minutes after preparation of the test solutions.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (including mortality): At 24 h and at 48 h

pH and dissolved oxygen: At the beginning and at the end of the test, for the test concentration and the control.

Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.









Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 56 - < 100 other: % of its water-soluble fraction
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(loading rate)
Basis for effect:
mobility
Remarks on result:
other: The EC50 of EA-3098 for the mobility of Daphnia magna was between 56 and 100 % of its water-soluble fraction, i.e. at a highest level of 0.0001 g/l.
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
56 other: % of water soluble fraction
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(loading rate)
Basis for effect:
mobility
Details on results:
RANGE-FINDING TEST:
Table 1 shows the responses recorded during the range-finding test.
No immobility was observed at any of the test solutions during the test period. Chemical analysis of samples taken from 100 % filtrate revealed that concentrations of all three major components were below their limits of detection. The expected EC50 was above the maximum solubility level of EA-3098 in water. However, the test solutions were only stirred for 30 minutes. Since no detectable concentrations were found it was decided to stir for 72 h instead. This period was considered to be sufficient to reach a saturated solution. Further it was decided to use ultra-centrifugation instead of filtration through a 0.45 µm filter to exclude possible adsorption of the dissolved components of EA-3098 to the filter.

LIMIT TEST:
Measured concentrations:
Chemical anaiysis revealed that concentrations of all three major components were below their limits of detection (Tables 4 - 6 of the Analytical Report - see attached background material). Hence, no detectable concentrations were present in spite of prolonging the stirring period to 72 h and using centrifugation instead of filtration. Analysis of the pellet after centrifugation showed that it contained all three major compounds.

Immobility:
Table 2 shows the responses recorded during the limit test.
Contrary to the range-finding test, 100% immobility was observed in the WSF prepared at 100 mg/l after 48 h. Hence, prolonging the stirring period to 72 h and using centrifugation instead of filtration increased the toxic potential of EA-3098 for D. magna.

FINAL TEST:
Immobility:
Table 3 shows the responses recorded during the final test.
Again 100% immobility was observed in the WSF prepared at 100 mg/l after 48 h, although the effect on mobility after 24 h was much lower compared to the limit test. No effect on mobility of D. magna was observed in any of the dilutions of the WSF.

DETERMINATION OF EFFECT CONCENTRATIONS:
Table 4 shows the effect parameters of EA-3098 for mobility of D. magna in relation to the water solubility level. No relation could be made with chemically identified components as none of three major components were detected.

EXPERIMENTAL CONDITIONS:
The test conditions remained within the limits prescribed by the protocol (pH: 6.0-8.5, not varying by more than 1 unit; oxygen: >7 mg/l at the start, >5 mg/l at the end of the test).
The temperature of the test medium was 20°C at the start of the test. The temperature measured continuously in a temperature control vessel varied between 20 and 21 °C during the test, and complied with the requirements put down in the protocol (18-22°C, constant within 2°C).

ACCEPTABILITY OF THE TEST:
1) In the control, no daphnids were immobilised and no more than 10% became trapped at the surface of the water.
2) The oxygen concentration was > 5 mg/l at the end of the test. Other test conditions (pH and temperature) were maintained within the limits prescribed by the guidelines.
3) The 24 h EC50 (based on the initial concentration) of potassium dichromate was within the range 0.6 mg/l to 1.7 mg/l.



Results with reference substance (positive control):
- Results with reference substance valid? Yes. The results from the positive control with potassium dichromate were within the normal range for the reference material.

- Mortality: not applicable. Immobilsation only determined.

- EC50/LC50:
24-h EC50: 0.70 mg/l with a 95% confidence interval between 0.63 and 0.83 mg/l.
48-h EC50: 0.51 mg/l with a 95% confidence interval between 0.47 and 0.58 mg/l
Reported statistics and error estimates:
No EC50 value could be calculated because the analytical support could not measure detectable test concentrations.

Table 1: Incidence of immobility of D. Magna in the range-finding test

 

Concentration EA-3098: % of a water soluble fraction prepared at 100 mg/l

Vessel number

Number D. magna exposed

Response at 24 h

Response at 48 h

Number immobile

Total %

Number immobile

Total %

0

A

B

5

5

0

0

0

 

0

0

0

0.1

A

B

5

5

0

0

0

0

0

0

1

A

B

5

5

0

0

0

0

0

0

10

A

B

5

5

0

0

0

0 (1)

0

0

100

A

B

5

5

0

0

0

0

0

0

Numbers of daphnids trapped at the surface are between parenthesis, these were re-immersed before scoring of moblility.

 

Table 2: Acute immobilisation of D. magna after 24 and 48 h in the limit test

 

Concentration EA-3098: % of a water soluble fraction prepared at 100 mg/l

Vessel number

Number D. magna exposed

Response at 24 h

Response at 48 h

Number immobile

Total %

Number immobile

Total %

Blank control

A

B

C

D

5

5

5

5

0

0

0

0

0

0

0

0

0

0

100

A

B

C

D

5

5

5

5

3

5

2

5

75

5

5

5

5

100

Table 3: Acute immobilisation of D. magna after 24 and 48 h in the final test

 

Concentration EA-3098: % of a water soluble fraction prepared at 100 mg/l

Vessel number

Number D. magna exposed

Response at 24 h

Response at 48 h

Number immobile

Total %

Number immobile

Total %

0

A

B

C

D

5

5

5

5

0 (1)

0 (1)

0

0

0 (1)

0

0

0

0

0

10

A

B

C

D

5

5

5

5

0

0 (4)

0

0

0 (20)

0

1

0

0

5

18

A

B

C

D

5

5

5

5

0 (2)

0

0

0

0 (10)

0

0

0

0

 0

32

A

B

C

D

5

5

5

5

0

0

0

0

0

0

0

0

0

0

56

A

B

C

D

5

5

5

5

0

0 (1)

0

0

0 (5)

0

0

0

0

0

100

A

B
C
D

5

5

5

5

0

0

0

1

5

5

5

5

5

100

Numbers of daphnids trapped at the surface are between parenthesis, these were re-immersed before scoring of moblility.

Table 4: Effect parameters

 

Parameter

Concentration EA-3098 (mg/l)

NOEC

<Solubility level (<0.1 mg/l)*

24 h EC50

>Solubility level (<0,1 mg/l)

48h EC50

Between 56 and 100% of the solubility level (<0.1 mg/l)

 

*No effect at 56% of the solubility level

Under the conditions of the test EA-3098 did not induce acute immobilisation of Daphnia magna as its water soluble fraction when tested as a solution exposed to EA-3098 prepared at 100 mg/l for a relative short stirring time (30 minutes), while it did at a rate of 100% as a solution exposed to EA-3098 prepared at 100 mg/l for a longer period (72 -hours).

The analytical programme did not detect concentrations of polymeric compounds related to the test material, while the original monomers were not measured by the analytical method used. The method of separation of the water phase (filtration or centrifugation) did not influence this. Hence, no pronouncement can be given on the possible relationship between the observed toxicity and concentrations of either of the monomers. In addition, the difference in effect between solutions incubated for a short or prolonged period indicated the presence of a possible toxic degradation product that could also not be identified with the analytical method available.

Validity criteria fulfilled:
yes
Conclusions:
EA -3098 induced immbolisation of Daphnia magna at the maximum solubility of EA-3098 in water prepared at a nominal loading of 100 mg/l. The 48-hr EC50 was between 56 and 100% of the maximum solubility of EA-3098 in water, i.e. at a highest level of 1.0 x 10-4 g/l.
Executive summary:

The study procedures were designed to meet the test methods and validity criteria of the EEC Directive 92/69, Part C.2 and the OECD Guideline No. 202.

The water solubility of EA-3098 was at the highest < 1.0 x 10-4g/l (determined in a water solubility study).

The project commenced with a range-finding test. Aqueous mixtures with test material loading rates of 100 mg/l were treated with ultra-sonic waves for 10 minutes and stirred for 30 minutes. Subsequently they were filtered through a 0.45 µm filter. The filtrate was used for further dilution to test 0.1 to 100%. No immobility was observed at any of the test solutions during the test period. Chemical analysis revealed the concentrations of all three major components were below their limits of detection. Since no detectable concentrations were found it was decided to prolong the period of stirring and to separate the soluble fraction by centrigugation.

In a limit study, an aqueous mixture of test material in de-ionised water (100 mg/l) was stirred for up to 72 hours. Subsequently it was ultra-centrifuged and the supernatent tested after addition of the necessary medium salts. all daphnids exposed to this solution became immobilised after 48 hours. Samples were taken for chemical analysis during the limit test. However, the levels of three major components remained below the limit of detection (<0.15 mg/l).

A final study was performed starting with a supernatent prepared at 100 mg/l, prepared in the same way as the limit test. The supernatent was used for further dilution to test 10 to 100%. Twenty organisms (in quadruple, five per vessel) were exposed per dilution and a blank-control. No analytical support was considered relevant because levels of test material constituents, measureable with the analytical method available, were expected to be below the level of detection.

The study met the acceptability criteria prescribed by the protocol and was considered valid. The total effect rate was 100% at the maximum solubility level of the test material in water. No effects occurred up to an including 56% of the maximum solubility level of the test material in water.

The analytical support did not detect concentrations of the major compounds related to the test material, while the original monomers were not measureable by the analytical method used. Hence, any presence of a possible toxic component or degradation product could not be identified with the analytical method available.

In conclusion, the test material induced acute immbolisation of Daphnia magna at the maximum solubility of the test material in water prepared at a nominal loading rate of 100 mg/l. The 48 -hr EC50 was between 56 and 100% of the maximum solubility of the test material in water, i.e. at a highest level of 0.0001 g/l.

Description of key information

The test material induced acute immbolisation of Daphnia magna at the maximum solubility of the test material in water prepared at a nominal loading rate of 100 mg/l. The 48 -hr EC50 was between 56 and 100% of the maximum solubility of the test material in water, i.e. at a highest level of 0.0001 g/l.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.1 mg/L

Additional information

The study procedures were designed to meet the test methods and validity criteria of the EEC Directive 92/69, Part C.2 and the OECD Guideline No. 202.

The water solubility of EA-3098 was at the highest < 1.0 x 10-4g/l (determined in a water solubility study).

The project commenced with a range-finding test. Aqueous mixtures with test material loading rates of 100 mg/l were treated with ultra-sonic waves for 10 minutes and stirred for 30 minutes. Subsequently they were filtered through a 0.45 µm filter. The filtrate was used for further dilution to test 0.1 to 100%. No immobility was observed at any of the test solutions during the test period. Chemical analysis revealed the concentrations of all three major components were below their limits of detection. Since no detectable concentrations were found it was decided to prolong the period of stirring and to separate the soluble fraction by centrigugation.

In a limit study, an aqueous mixture of test material in de-ionised water (100 mg/l) was stirred for up to 72 hours. Subsequently it was ultra-centrifuged and the supernatent tested after addition of the necessary medium salts. all daphnids exposed to this solution became immobilised after 48 hours. Samples were taken for chemical analysis during the limit test. However, the levels of three major components remained below the limit of detection (<0.15 mg/l).

A final study was performed starting with a supernatent prepared at 100 mg/l, prepared in the same way as the limit test. The supernatent was used for further dilution to test 10 to 100%. Twenty organisms (in quadruple, five per vessel) were exposed per dilution and a blank-control. No analytical support was considered relevant because levels of test material constituents, measureable with the analytical method available, were expected to be below the level of detection.

The study met the acceptability criteria prescribed by the protocol and was considered valid. The total effect rate was 100% at the maximum solubility level of the test material in water. No effects occurred up to an including 56% of the maximum solubility level of the test material in water.

The analytical support did not detect concentrations of the major polymeric compounds related to the test material, while the original monomers were not measureable by the analytical method used. Hence, any presence of a possible toxic component or degradation product could not be identified with the analytical method available.

In conclusion, the test material induced acute immbolisation of Daphnia magna at the maximum solubility of the test material in water prepared at a nominal loading rate of 100 mg/l. The 48 -hr EC50 was between 56 and 100% of the maximum solubility of the test material in water, i.e. at a highest level of 0.0001 g/l.