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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 March 2012 - 24 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Guideline:
other: European Commission Regulation (EC), EC No. 440/2008, Part C, PubNo. L142, C.2.
Guideline:
other: ISO International Standard 6341.
Deviations:
no
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report: MTDID 7831
- Physical state: Waxy solid
- Analytical purity: 93.96 w%
- Storage condition of test material: At room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: During the final test, single 2 mL samples were taken from all test concentrations and control at time 0h and 48h. At both time points, the replicates were pooled at each concentration before sampling. Samples were taken from near the center of the water column.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤ -15 °C) until analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Loading rates were individually prepared by applying a 30min treatment period of ultrasonic waves followed by 1 day of magnetic stirring. The resulting dispersions were left to settle for a maximum of 30min, after which the clear WAFs were collected by siphoning and used for testing.
- Controls: Test medium without test substance or other additives.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no visible undissolved test substance was observed at the test initiation.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
- Source: In-house laboratory culture with a known history.
- Age at study initiation (mean and range, SD): < 24 hours, from parental daphnids of more than two weeks old
- Method of breeding: Breeding batch started by placing approximately 250 daphnids less than 3 days old into 5 liters of M7 medium (adjusted ISO medium) in an all-glass culture vessel. Cultures were maintained for a maximum age of 4 weeks. After the first 7 days, cultures are renewed by replacement of half of the medium twice weekly. Cultures were maintained at 18 - 22 °C and fed daily with a suspension of freshwater algae.
- Feeding during test: None

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h

Test conditions

Hardness:
180 mg/L (CaCO3)
Test temperature:
19.7 - 20.5 °C
pH:
7.7 - 8.0
Dissolved oxygen:
8.7 - 9.1 mg/L
Nominal and measured concentrations:
Nominal concentrations of WAFs: 0.5, 0.9, 1.6, 2.9, and 5.0 mg/L
Mean measured concentrations of WAFs: 0.31, 0.46, 0.91, 0.14, and 1.4 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass containers covered with plastic lid; test volume was 80 mL
- Aeration: No
- 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 purified by reverse-osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands)
- Culture medium different from test medium: no, standard M7 medium used throughout. M7 is based on standard ISO medium supplemented with additional minerals.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h:8 h light:dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: yes
Daphnia magna exposed to a range of 0.1 to 100 mg/l (nominal concentration of MTDID 20422) increasing by a factor of 10 and to a control.
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% C.I. 1.0 to 1.4 mg/L
Details on results:
Measured concentrations at beginning and conclusion of test are shown in Table 1. Mean measured concentrations were used to calculate EC50 (except for the measured concentration of 0.14 mg/L/nominal 2.9 mg/L. The laboratory does not have an explanation for the resulting WAF prepared at 2.9 mg/L being much lower than expected. It is suspected that this concentration was prepared incorrectly in some way, therefore the results from this concentration were not used to determine the EC50). Immobilisation data in Table 2.

- Mortality of control: 5% immobilized at 48 hours. Number of daphnids that became immobilized was under 10%
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Chemical has low water solubility.
- Effect concentrations exceeding solubility of substance in test medium: Possible. WAFs were also prepared at a loading rate of 5 mg/L in the study with zebrafish. Resulting concentrations were considerably lower (0.40 and 0.42 mg/L at the start of the first and last renewal, respectively) in the fish WAFs despite the fact that the same medium was used as the Daphnia test. This difference was likely caused by the difference in the volume of the medium used for the preparation of test solutions (1 vs 25 liters) and the lower stirring force used to prepare the fish test solutions. It is believed by the testing laboratory that the maximum solubility in this medium was reached in the zebra-fish study and that likely the higher concentrations in the Daphnia study were above the medium solubility although no undissolved test substance was visible.
Results with reference substance (positive control):
The 48-hr EC50 was 0.38 mg/L with a 95% confidence interval of 0.34 - 0.45 mg/L for the positive control, potassium dichromate. This result was in the lower range of the expected historical response (a 48h EC50 between 0.3 and 1.0 mg/L) for this laboratory. Hence, the sensitivity of this batch of D. magna was in agreement when compared to the historical data collected at this laboratory.
Reported statistics and error estimates:
The 48 EC50 value was calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding test substance concentrations using the maximum likelihood estimation method (Finney, D.J., 1971: Probit analysis, Cambridge University Press, Cambridge, U.K., 3rd edition).

The result for the WAF of 2.9 mg/L (measured 0.14 mg/L) was lower than expected based on results obtained from the remaining WAFs. It is not known why but the results were not used in the calculation of the EC50 value.

Any other information on results incl. tables

Table 1. Concentrations of test substance in the test medium – final test

Time of sampling

                              Concentration

Nominal WAF

C4 Acrylate

(mg/I)

Measured

C4 Acrylate

(mg/l)

Percent of initial concentration

0 hours

Control

n.d.

0.5

0.282

0.9

0.523

1.6

0.971

2.9

0.148

5.0

1.59

 

48 hours

Control

n.d.

0.5

0.330

117

0.9

0.397

76

1.6

0.847

87

2.9

0.128

87

5.0

1.28

80

n.d. Not detected

Table 2. Acute immobilization of daphnia after 24 and 48 hours in the final test

Measured Concentration

C4 Acrylate

(mg/L)

Vessel replicate

Number Daphnia exposed

Response at 24 h

Response at 48 h

Number

% of Total

Number

% of Total

Control

A

5

0

0

0

5

B

5

0

0 [1]

C

5

0

0

D

5

0

1

0.31

A

5

0 [1]

0

0

0

B

5

0

0

C

5

0 [1]

0

D

5

0 [1]

0 [1]

0.46

A

5

0 [1]

0

0

0

B

5

0 [2]

0

C

5

0

0

D

5

0

0

0.91

A

5

1

10

0

15

B

5

1

2

C

5

0 [1]

1

D

5

0

0

0.14

A

5

0

0

0

0

B

5

0 [1]

0

C

5

0 [3]

0

D

5

0 [4]

0 [3]

1.4

A

5

0

0

5

75

B

5

0

4

C

5

0

2

D

5

0

4

[ ] between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
In the control, less than 10% of daphnids became immobilised. The oxygen concentration at the end of the test was ≥ 3 mg/l in control and test vessels.
Conclusions:
The 48-hour EC50 (immobilisation) of C4 acrylate to Daphnia magna was 1.2 mg/l, 95% C.I. 1.0 -1.4 mg/l (OECD 202).
Executive summary:

The toxicity of C4 acrylate to the water flea, Daphnia magna, was assessed in a 48-hour toxicity test conducted according to the OECD 202 (2004) method. The average exposure concentrations were calculated by geometric means of the concentrations of C4 acrylate measured in the samples taken at the start (0h) and the end of the test (48h).

The 48-hour EC50 of C4 acrylate to Daphna magna for immobilisation was 1.2 mg/L from the probits of the percentages of affected daphnids and the logarithms of the corresponding test substance concentrations using the maximum likelihood estimation method.

The study was performed in accordance with internationally-accepted test guidelines and Good Laboratory Practice (GLP) standards. Therefore, this study is reliable without restrictions and the results are suitable for purposes of Risk Assessment, Classification & Labeling, and PBT Analysis.

Results Synopsis

Test type: static (based on data obtained using OECD 202 methodology) 48-hr EC50 = 1.2 mg/L