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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:
2003-04-23 to 2003-09-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
December 1992
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
adopted April 4, 1984
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Frequency: One day before the start from the highest and lowest test group (check test system), at the start and the end of the exposure period from all test concentrations.
Volume: 10 ml from the approximate centre of the test vessels.
Storage: Not applicable, samples were analysed on the day of sampling.

Vehicle:
yes
Remarks:
DMSO
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions were prepared in DMSO at a factor of 10,000 higher than the target concentrations and dosed via a computer-controlled system consisting of 5 dispensers. Via this system the dosed volumes entered mixing flasks separately from the tap-water supply. The tap-water was supplied via a flow-meter with a flow rate of ca. 6 L/h.
- Controls: Tap-water without test substance but with the additive used in the treatment of the stocks, i.e. DMSO.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylsulfoxide (DMSO)
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
- Age of parental stock (mean and range, SD): Daphnids originated from a healthy stock, 2nd to 5th brood
- Feeding during test: No
- Food type: During breeding daily, a suspension of fresh water algae.

ACCLIMATION
- Acclimation period: With new-born animals, i.e. less than 3 days old, by placing about 250 of them into 10 litres of medium in an all-glass culture vessel.
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: fresh water algae
- Feeding frequency: daily
- Health during acclimation (any mortality observed): no mortality > 20% observed


Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg/L CaCO3
Test temperature:
18-22°C, constant within 2°C.
pH:
Between 6.0 and 8.5. Should not vary by more than 1 unit at the end of the test in any test solution.
Dissolved oxygen:
> 7 mg/l at the start of the test,
>= 5 mg/l at the end of the test.
Nominal and measured concentrations:
nominal concentrations:
0, 0.1, 1, 10, 100 mg/L for the combined limit/range-finding test (static)
0, 0.1, 1.0, 10 mg/L for the additional range-finding test (flow-through)
0, 0.10, 0.22, 0.46, 1.0, 2.2 mg/L for the final flow-through study
Measured conentrations:
0, 0.035, 0.4, 5.0 mg/L for the additional range-finding test (flow-through)
0, 0.059, 0.085, 0.19, 0.31, 0.82 mg/L for the final flow-through study (mean measured)
Details on test conditions:
TEST SYSTEM
- Test vessel:Stock solutions were prepared in DMSO at a factor of 10,000 higher than the target concentrations. The stocks were dosed via a computer-controlled system consisting of 5 dispensers (Gilson). Via this system the dosed volume entered a mixing flask separately from the tap-water supply. The tap-water was supplied via a flow meter with a flow rate of ca. 6 L/h. In the mixing flask the dosed volume and the tap-water were mixed under continuous stirring. The test system was checked daily.
- Material, size, headspace, fill volume: Stainless steel vessels with a volume of 1.5 litres containing 4 mesh containers of stainless steel.
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): The tap-water was supplied via a flow meter with a flow rate of ca. 6 I/h.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap Water
- Metals:
Selenium < 0.1 µg/L
Arsenic< 5.0 µg/L
Cadmium< 0.4 µg/L
Chromium <1.0 µg/L
Copper 65/32 µg/L
Mercury < 0.05 µg/L
Lead < 5.0 µg/L
Zinc 24/62 mg/L
Manganese < 0.010 mg/L
Calcium 12 mg/L
Magnesium 1.4 mg/L
Iron 0.061 mg/L
- Culture medium different from test medium: Medium used for culturing: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

OTHER TEST CONDITIONS
- Photoperiod: 16 h daily
- Light intensity: 671 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mobility

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: nominal:
0, 0.1, 1, 10, 100 mg/L for the combined limit/range-finding test (static)
0, 0.1, 1.0, 10 mg/L for the additional range-finding test (flow-through)

- Results used to determine the conditions for the definitive study:
All daphnids exposed to the target concentration 10 mg/L became immobilised at the end of the test period, while 70% immobility was observed in the 1.0 mg/L target concentration. No immobility was observed in the 0.10 mg/L target concentration and the control. Analytical results showed that measured concentrations were at 30 to 60% of the target concentrations. The relatively low recoveries were not expected as the test solutions appeared to be completely dissolved and continuous renewal should avoid decrease of test concentrations by degradation. Analyses of a three-day old stock solution of 10 g/L used to dose the 1.0 mg/L target concentration showed that the stock was prepared correctly and was stable for at least three days. Consequently, it was decided to prepare stock solutions only once during the final test and to reduce the flow of the dilution water from 12 L/h to 6 L/h to prepare test solutions with actual exposure concentrations that were closer to the target concentrations.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
712.5 µg/L
Nominal / measured:
meas. (arithm. mean)
Remarks on result:
other: 603.6 - 938.6 µg/l
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
591.4 µg/L
Nominal / measured:
meas. (arithm. mean)
Remarks on result:
other: 524.5 - 715.8 µg/l
Results with reference substance (positive control):
- Results with reference substance valid?
- Relevant effect levels:
The 24h-EC50 was 1.2 mg/L with a 95% confidence interval between 1.0 and 1.5 mg/L.
The 48h-EC50 was 0.60 mg/L with a 95% confidence interval between 0.52 and 0.76 mg/L.
Reported statistics and error estimates:
The EC50 at the various observation times was determined using the maximum likelihood estimation method with the probits of the percentages of immobilised daphnids as function of the logarithms of the corresponding concentrations (Finney, D.J., 1971: Probit analysis, Cambridge University Press, Cambridge, U.K., 3rd edition)

Table 1: Incidence of immobility in the additional range-finding test (flow-through):

Concentration Test substance; Target/Acutal (mg/L)

Vessel Number

Number Daphnia exposed

Response at 24 h

Response at 48 h

number

%

number

%

DMSO-control

A

5

0

0

0

0

B

5

0

 

0

 

0.1/0.035

A

5

0

0

0

0

B

5

0

 

0

 

1.0/0.40

A

5

2

20

3

70

B

5

0

 

4

 

10/5.0

A

5

4

70

5

100

B

5

3

 

5

 

Table 2: Measured and average measured concentrations per test group (final test)

Concentration Target (mg/L)

Measured T=0 (mg/L)

Measured T=48 h (mg/L)

Mean Measured (mg/L)

0.10

0.054

0.064

0.059

0.22

0.074

0.096

0.085

0.46

0.188

0.193

0.19

1.0

0.277

0.343

0.31

2.2

0.755

0.883

0.82

Table 3:Acute immobilisation of Daphnia after 24 and 48 hours in the final EC50 study.

Concentration Test substance; Target/Mean (mg/L)

Vessel Number

Number Daphnia exposed

Response at 24 h

Response at 48 h

number

%

number

%

DMSO-control

A

5

0

0

0

0

B

5

0

0

C

5

0

0

D

5

0

0

0.10/0.059

A

5

0

0

0

0

B

5

0

0

C

5

0

0

D

5

0

0

0.22/0.085

A

5

0

0

0

0

B

5

0

0

C

5

0

0

D

5

0

0

0.46/0.19

A

5

0

0

0

0

B

5

0

0

C

5

0

0

D

5

0

0

1.0/0.31

A

5

0

0

0

0

B

5

0

0

C

5

0

0

D

5

0

0

2.2/0.82

A

5

4

63

5

84

B

4*

3

3

C

5

4

5

D

5

1

3

*Inadvertently 4 instead of 5 Daphnids were exposed

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study 2,4-Bismaleimidotoluene did not induce acute immobilisation of Daphnia magna at or below target concentrations of 1.0 mg/L, corresponding to a mean exposure concentration of 0.31 mg/L under flow-through test conditions (NOEC).
The 24h-EC50 for Daphnia was 0.71 mg/L with a 95% confidence interval between 0.60 and 0.94 mg/L (based on mean exposure concentrations).
The 48h-EC50 for Daphnia was 0.59 mg/L with a 95% confidence interval between 0.52 and 0.72 mg/L (based on mean exposure concentrations).

Description of key information

- short-term toxicity to aquatic invertebrates: according to OECD TG 202 (adopted April 4th, 1984); RL 1; GLP; test with Daphnia magna 48 h EC50 = 591.4 µg/L

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
591.4 µg/L

Additional information

The 48-hr-acute toxicity of 2,4-Bismaleimidotoluene to Daphnia magna was studied under flow-through conditions  according  to  OECD  Guideline  202  (2004).  Daphnids  were  exposed  to  control  and  test chemical at nominal concentrations of 0.10, 0.22, 0.46, 1.0 and 2.2 mg a.i./L (corresponding to 0.059, 0.085, 0.19, 0.31 and 0.82 mg/L mean measured concentrations) in tap water for 48 h. Mortality/immobilization was observed daily, pH was recorded at the beginning and at the end of the test and temperature was measured continuously. 2,4-Bismaleimidotoluene  did  not  induce  acute  lmmobilisation  of  Daphnia  magna  at  or  below  nominal concentrations  of  1.0  mg/l,  corresponding  to  a  mean exposure  concentration  of  0.31  mg/l  under flow-through test conditions (NOEC). The 48-hour EC50 was determined to be 0.59 mg a.i./L (95% confidence limits: 0.52 and 0.72 mg/L).