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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:
20 Apr 2016 to 22 Apr 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
May, 2008
Principles of method if other than guideline:
Organisms were exposed to a Water Accommodated Fraction (WAF) / Water Soluble Fraction (WSF), because the test item is a UVCB and poorly soluble in water.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report): Fatty acids, C12-14, a-sulfo, disodium salts
- Batch No.: Ra-He 2014-054
- Purity test date: 100 % UVCB
- Appearance: Beige powder
- Expiry date: Oct 2016
- Retention: at least 1 g has been retained on 2016-02-24 and stored at 6 ± 2 °C
- Storage condition of test material: Room temperature, protected from light, in the tightly closed original container
Analytical monitoring:
yes
Details on sampling:
TYPE OF ANALYSIS
The carbon content of the test item was determined. For this purpose, three samples of the test item of 30 mg were analysed via solid TOC measurement. All loading levels of the test item and the control were analytically verified via analysis of the total organic carbon (TOC, according to DIN EN 1484) at the start of the exposure intervals (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours). The control replicates were measured in triplicates to determine the variability or reproducibility. The limit of quantification (LOQ) was calculated for each sampling date as follows: LOQ = mean value (control) + 9 standard deviation. For the evaluation, the background (i.e. carbon content of the control) was subtracted from the carbon content of each loading level in order to calculate the net carbon content. The percentage of TOC was calculated in relation to the nominal TOC concentrations.

SAMPLING FOR ANALYTICAL MONITORING
At the start of the exposure intervals (0 and 24 hours), sampling was carried out after preparation of the loading levels. At the end of the exposure intervals (24 and 48 hours), samples were taken from additional replicates with test media, but without daphnids, which were incubated under test conditions until sampling
Vehicle:
no
Details on test solutions:
PREPARATION OF WATER ACCOMMODATED FRACTION
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000) is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture, an UVCB substance and is poorly soluble in water and in the permitted solvents. Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.

PREPARATION OF THE WATER SOLUBLE FRACTIONS
Five Water Soluble Fraction (WSF) were prepared with nominal loadings as specified below. For each loading level, an appropriate amount of the test item was weighed out and transferred into a brown glass flask with an appropriate amount of dilution water. The test item dispersions were shaken for 24 hours with 20 rpm at room temperature. Then undissolved test item was removed by membrane filtration (membrane filter 0.45 pm, RC, MACHEREY„ NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of filtrate were discarded. The filtration was interrupted for ca. 15 minutes to allow for adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the WSF, was used in the test. During filtration, the filter was always kept covered, This procedure was carried out one day before the start of each exposure interval (at -24 and 0 hours).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain/clone: STRAUS (clone 5)
- Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
- Breeder: Noack Laboratorien GmbH, Käthe-PaulusStr. 1, 31157 Sarstedt, Germany
- Feeding: The daphnids were not fed during the study.

ACCLIMATION
Acclimatisation was not necessary, because the dilution water was equivalent to the culture medium.

STOCK CULTURE MAINTENANCE CONDITIONS
- Culture conditions: in glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2 °C, in an incubator, 16 hours illumination, light intensity of max. 20 µE·m-2·s-1 (max. 1340 lx)
- Culture medium: Elendt M4, according to OECD 202, Annex 3 (2004) is used. The composition is modified to a total hardness of approximately 160 to 180 mg CaCO3/L.
- Feeding: The culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 1E+06 cells/mL. The algae are cultured at the test facility.
- Origin food: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
168 - 171 mg/L (as CaCO3/L)
Test temperature:
20.4 - 20.3
pH:
7.35 - 8.23
Dissolved oxygen:
5.82 - 8.57 mg/L
Conductivity:
459 - 487 µS/cm
Nominal and measured concentrations:
- Nominal loading levels: 0 (control), 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L (based on a range-finding test)
- Nominal TOC concentrations: 2.61, 5.23, 10.5, 20.9 and 41.8 mg C/L
- Meausred concentrations (t=0h; new): 3.02 (control), 7.05, 9.49, 12.74, 21.1 and 39.2 mg C/L
- Meausred concentrations (t=24h; old): 2.95 (control), 8.19, 10.3, 13.2, 21.7 and 39.7 mg C/L
- Measured concentrations (t=24h; new): 3.02 (control), 5.76, 8.35, 13.0, 22.1 and 39.0 mg C/L
- Measured concentrations (t=48h; old): 3.32 (control), 7.33, 8.70, 13.4, 22.3 and 39.0 mg C/L. For more information, please see 'Any other information on materials and methods incl. tables'
Details on test conditions:
TEST SYSTEM
- Age of the daphnids: Less than 24 hours old daphnids from a healthy stock were used for at the start of the exposure the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.
- Test vessel: Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses
- Test volume: 20 mL
- Renewal rate of test solution: The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Application: 20 g test solution per replicate were weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by pipette.

TEST MEDIUM
- Source/preparation of dilution water: Composition of the Culture Medium Elendt M4 according to OECD 202, Annex 3 (2004)
- Culture medium different from test medium: No
- Intervals of water quality measurement: 24 h

WATER QUALITY PARAMETERS
Prior to the start of the exposure (0 hours) and the water renewal (24 hours), the water quality parameters (i.e. pH-value, dissolved oxygen concentration, temperature and conductivity) of the dilution water were measured. Acid capacity, acidity and total hardness are determined at least quarterly. At the start of the exposure intervals (0 and 24 hours), the water quality parameters of the fresh media (i.e. pH-value, dissolved oxygen concentration) were measured in one additional replicate per loading level and control. At the end of the exposure intervals (24 and 48 hours), the water quality parameters of the old media were measured in all replicates per loading level and control. The incubator temperature was recorded throughout the period of the test.

OTHER TEST CONDITIONS
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 20 µE·m-2·s-1 (max. 1340 Ix)

EFFECT PARAMETERS MEASURED
Immobilisation was determined in all groups after 24 and 48 hours. An animal was considered immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Other observations were not made.

RANGE-FINDING STUDY
- Nominal test concentrations: 0 (control), 1.0, 10.0 and 100,0 mg/L
- Results used to determine the conditions for the definitive study: At 100 mg/L, 70% of the daphnids were immobile after 48 hours exposure. No immobility was observed at the other concentrations.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
93 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% C.I.: 82.2 - > 100 mg/L
Details on results:
- Mobility: No immobility was observed in the negative control group and up to a concentration of 25.0 mg/L throughout the test. At 50 and 100 mg/L, 15 and 55% daphnia were immobile after 48 h, respectively. See 'Any other information on results incl. tables'.
- Test item solubiltiy: All loading levels were visually clear throughout the exposure in both exposure intervals.
Results with reference substance (positive control):
The 24-h EC50 value of the reference substance was determined to be 1.95 mg/L (0: 1.14 - 3.30 mg/L). (a reference test is conducted once per month at the test facility).
Reported statistics and error estimates:
The EL10-value after 24 hours and the EL0- and EL50-values (after 48 hours) were calculated by sigmoidal dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism5. The EC50-vaIue for the reference item and its 95% confidence limits were calculated accordingly. All data were computer-processed and rounded. Calculations were made using the following software: GraphPad Prism5 (GRAPHPAD SOFTWARE, INC.) and Excel (MICROSOFT CORPORATION).

Table: Immobilisation rates after 24 and 48 hours of exposure

Nominal loading level of the test item [mg/L]

Immobilisation [%]

24 hours

48 hours

Replicates

Replicates

1

2

3

4

Mean

1

2

3

4

mean

100 (WSF)

20

0

40

40

25

60

40

60

60

55

50.0  (WSF)

0

0

20

20

10

0

0

20

40

15

25.0  (WSF)

0

0

0

0

0

0

0

0

0

0

12.5  (WSF)

0

0

0

0

0

0

0

0

0

0

6.25  (WSF)

0

0

0

0

0

0

0

0

0

0

Control

0

0

0

0

0

0

0

0

0

0

WSF = Water soluble fraction

USE OF NOMINAL CONCENTRATIONS

The carbon content of the test item was determined via solid TOC measurement to be 41.8%. The percentage related to the nominal TOC concentrations attributable to the test item were calculated after subtraction of the background value from the control. The TOC in the three highest loadings was in the range of 85 - 98% of the nominal values throughout the exposure period. With decreasing loadings the TOC increased markedly in comparison to the nominal values and values of 124 — 201% of the nominal value were determined. Generally, the measured values were constant during the exposure period with exception of a marked increase in the test solutions 6.25 and 12.5 mg test item/L. The reason for the high TOC values could not be identified. The TOC values attributable to the test item were close to or partly below the LOQ. Assuming that the additional C-content was caused by the test item, the TOC measurement confirmed that the loadings were close to or above the nominal loadings during the test period.

Validity criteria fulfilled:
yes

Description of key information

The 48-h EC50 value is 93 mg/L in aquatic invertebrates (Daphnia magna).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
93 mg/L

Additional information

The acute toxicity to aquatic invertebrates was determined in a study according to OECD TG 202 and in compliance with GLP criteria. In this study daphnids (D. magna, 20 per concentration) were exposed to nominal loading levels of 0 (control), 6.25, 12.5, 25.0, 50.0 and 100 mg/L for 48 hours under semi-static conditions. The TOC in the three highest loadings was in the range of 85 - 98% of the nominal values throughout the exposure period. In the two lower loadings, the TOC increased markedly in comparison to the nominal values and values of 124 - 201% of the nominal value were determined. The TOC measurement confirmed that the loadings were close to or above the nominal loadings during the test period (assuming that the additional C-content was caused by the test item). Therefore, the effect values are based on nominal concentrations. Immobilization was recorded after 24 and 48 hours exposure. The water quality parameters measured at 24 and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guidelines were fulfilled. After 48 hours, immobility was determined to be 15 and 55% in the two highest test concentrations, whereas at the other (lower) test concentrations no effects were observed. Based on these findings the 48-h EC50 value was estimated to be 93.0 mg/L (95% C.L.: 82.2 - 100 mg/L).