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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:
5 August 2013 - 7 August 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to EC method C2 and OECD 202. GLP study.
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100, 31.3, 9.8, 3.1 and 1.0 mg/L
- Sampling method: Analytical measurements were performed at the applied test concentration levels daily in the case of fresh media and at the end of representative semi-static periods as appropriate, and on control at the start and at the end of the test.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Test concentrations were prepared with test item and test medium (ISO medium) according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23. prior to treatment. The test item is a UVCB material, a complex multi-component substance, which is only partially soluble in water. According to OECD No. 23., the toxicity of such substances were determined by preparing water-accommodated fractions (WAFs). Saturated test item solutions (nominal loading rate of 100, 31.3 and 9.8 mg/L) were prepared by dispersing/dissolving the appropriate amount of test item into the test medium (ISO medium) two days before treatment. These solutions were shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. A short settling time was considered to be inappropriate as the liquid phases did not separate visibly in the period after mixing. Hence a period of approximately 24 hour time was considered to be the most practicable settling time. After settling, centrifugation was found not to work for aiding separation (density is near that of water); filtration through a fine (0.22 μm) filter was found to be useful to prepare a suitable media for the study. Nominal test concentrations of 3.1 and 1.0 mg/L were prepared by appropriate dilution of nominal test concentrations of 9.8 mg/L and 3.1 mg/L respectively, since production of such low concentrations is impractical (as described in OECD 23).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: National Institute of Public Health 1097 Budapest Gyáli u. 2-6. Hungary
- Age at study initiation (mean and range, SD): Less than 24 h old at the beginning of the test
- Method of breeding: The Daphnia are bred in Ecotoxicological Laboratory of CiToxLAB Hungary Ltd. The health of the stock animals is continuously monitored by visual daily checking. Abnormal behaviour or significant decrease of population is recorded.
- Feeding during test: No

ACCLIMATION
- Acclimation period: There was no acclimatization because the water used was similar to the culture water
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
249 mg/L (as CaCO3)
Test temperature:
19.7 – 20.1 °C
pH:
7.51 – 7.71
Dissolved oxygen:
7.7 – 8.6 mg/L
Nominal and measured concentrations:
Nominal concentrations: 1.0, 3.1, 9.8, 31.3 and 100 mg/L.
Measured concentrations: See "Any other information on results incl. tables".
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beaker
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume:
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): 24h
- No. of organisms per vessel: 5 animals/replicate
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO medium in accordance with guidelines.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16-hour light and 8-hour dark cycle

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The immobility or mortality of the daphnids was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile. The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours. The water temperature, oxygen concentrations and pH of the control and the test solutions were measured at the start and at the end of each renewal period where it was possible.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2. Because significant immobility was observed at the highest examined concentration levels during the preliminary range-finding test and taking into account that the lowest concentration tested should preferably give no effect in the definitive test, therefore a wide concentration range was used and five test concentrations in a geometric series (using factor 3.2, which does not affect the quality of the study) and one control was used in the main test in a semi-static system.
Range finding study 1:
- Test concentrations: 0 (control), 0.1, 10 and 100 mg/L loading rates (5 daphnids per replicate, 2 replicates per concentration).
- Results used to determine the conditions for the definitive study: Immobilisations: 10/0; 10/0; 10/0, 10/1 and 10/10 at 0, 0.1, 10 and 100 mg/L.
- Observations: A residue of the test item was observed on the test beaker and a small amount on the surface as well, after media preparation according to WAF method.
Range finding study 2:
- Test concentrations: 0 (control), 0.1, 10 and 100 mg/L loading rates (5 daphnids per replicate, 2 replicates per concentration).
- Results used to determine the conditions for the definitive study: 10/0; 10/0; 10/0, 10/5 and 10/10 at 0, 0.1, 10 and 100 mg/L.
- Observations: No test material or droplets were observed on the beaker and on the surface. Therefore it was determined that the effects were related to chemical toxicity rather than physical effects.
Reference substance (positive control):
not required
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
40.46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 33.40 – 49.03 mg/L
Details on results:
- Mobililty of control: There were no immobilized animal in the control group.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: “Oil-like” droplets were observed on the surface of the test solution of 100 mg/L nominal loading rate prepared by WAF method according to OECD No. 23.
Results with reference substance (positive control):
For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year by the laboratory to demonstrate satisfactory test conditions. The date of the last study (Study Code: 13/223-023DA) with reference item Potassium dichromate (batch no.: 0769128) is: 16-17 July 2013. The 24h EC50 results were considered to be fully satisfactory: 0.82 mg/L (95 % confidence limits: 0.76 – 0.87 mg/L).
Reported statistics and error estimates:
The 24 hours EL50 was calculated by Probit analysis using SPSS software.The 48 hours EL50 was calculated by Probit analysis using TOXSTAT software. The 48 hours EL100 were determined directly from the raw data. For the determination of the LOELR and NOELR, the immobilization at the nominal test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.

Number and percentage of immobilised animals:

Nominal concentration (mg/L)

Number of treated animals

Immobilised animals

24 hours

48 hours

Number

Percent

Number

Percent

Control

20

0

0

0

0

1.0

20

0

0

0

0

3.1

20

0

0

2

10

9.8

20

0

0

1

5

31.3

20

0

0

2

10

100

20

20

100

20

100*

*: statistically significantly different compared to the control values (Dunnett’s Test; α = 0.05)

The 24h EL50 value: 54.43 mg/L nominal loading rate (with 95 % conf. limits: 42.70 – 71.24 mg/L)

The 48h EL50 value: 40.46 mg/L nominal loading rate (with 95 % conf. limits: 33.40 – 49.03 mg/L)

The 48h EL100 value: 100 mg/L nominal loading rate

The statistical result indicates a LOELR of 100 mg/L nominal loading rate and NOELR of 31.3 mg/L nominal loading rate. LOELR and NOELR can be determined on the basis of the raw data as 3.1 mg/L nominal loading rate and 1.0 mg/L nominal loading rate respectively. Taking into account the biological and statistical results at the same time, LOELR and NOELR can be expressed as > 1 but < 100 mg/L nominal loading rate.

Analytical results:

At the nominal concentration of 100 mg/L the calculated geometric mean test item concentration of the main components was 0.762 mg/L. At lower concentrations the results were below the LOQ (< 0.02 mg/L). The analytical results indicate that the saturation concentration of the measured material after settling and filtration was low but detectable, and that about 24 hours later it was generally below the LOQ. It was found not to be possible to measure concentrations below 0.02 mg/L, even taking into account the concentration step. It was concluded that the WAF method used was the most appropriate for ensuring exposure of organisms to water-soluble fractions of the UVCB test item. This procedure, performed under semi-static conditions maximised exposure and is suitable for risk assessment. The analytical method was designed to measure the presence of the main components of the UVCB test item; however the WAF method is designed to test any extractable components that may enter the aquatic environment. Hence the results of the study are expressed in units of nominal test item, the analytical data provide evidence of exposure and supplementary information about the main components of the test item.

Validity criteria fulfilled:
yes
Remarks:
(immobilisation in the control <10%, dissolved oxygen concentration at the end of the test >= 3 mg/l of the air saturation)
Conclusions:
The 48h-EL50 of the substance was determined to be 40.46 mg/L nominal loading rate in Daphnia magna.
Executive summary:

An acute toxicity of the substance on Daphnia magna was assessed in an acute immobilisation test according to EC method C2 and OECD 202. Based on preliminary range-finding results, 20 daphnids per dose (5 daphnids per replicate) were exposed to 0 (control) 1.0, 3.1, 9.8, 31.3 and 100 mg/L over an exposure period of 48 hours in a semi-static test system. The test item is a UVCB material, a complex multi-component substance, which is only partially soluble in water. According to OECD No. 23., the toxicity of such substances can be determined by preparing water-accommodated fractions (WAFs). Test concentrations were analytically determined at the beginning of the test and 24-hour intervals thereafter during the experiment. All validity criteria were met during this study. The 48h-EL50 value of the substance was determined to be 40.46 mg/L nominal loading rate. The statistical result indicates a LOELR of 100 mg/L nominal loading rate and NOELR of 31.3 mg/L nominal loading rate. LOELR and NOELR can be determined on the basis of the raw data as 3.1 mg/L nominal loading rate and 1.0 mg/L nominal loading rate respectively. Taking into account the biological and statistical results at the same time, LOELR and NOELR can be expressed as > 1 but < 100 mg/L nominal loading rate.

Description of key information

Key study: Test method EC C2, OECD 202. GLP study. The 48h-EL50 of CNLS was determined to be 40.46 mg/L nominal loading rate in Daphnia magna.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
40.46 mg/L

Additional information

Key study: An acute toxicity of the substance on Daphnia magna was assessed in an acute immobilisation test according to EC method C2 and OECD 202. Based on preliminary range-finding results, 20 daphnids per dose (5 daphnids per replicate) were exposed to 0 (control) 1.0, 3.1, 9.8, 31.3 and 100 mg/L (WAFs) over an exposure period of 48 hours in a semi-static test system. The 48h-EL50 of the substance was determined to be 40.46 mg/L nominal loading rate in Daphnia magna.