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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:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study performed under GLP. All relevant validity criteria were met.
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
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23 (2000)
Deviations:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: June 2013; signature: February 2016
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The saturated solution and 5 test item concentrations in a geometric series with a separation factor of 2, prepared by dilution of the stock solution 1 mL/L with dilution water were tested as follows: 0 (control), 6.25, 12.5, 25.0, 50.0, 100% of the saturated solution, which corresponds to nominal test item concentrations (based on mean relative density of the mixture of 1.01) of: 0 (control), 63.2, 126, 253, 506, 1010 mg/L. The test concentrations are based on the results of a non-GLP preliminary range finding test. All concentration levels and the control were analytically verified via GC-MS in all tested concentration levels and the control at the start of the exposure intervals (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours). This resulted in analytically measured geometric mean measured test item concentrations of the sum of two components of 0.156, 0.295, 0.594, 1.200and 2.29 mg/L, respectively.- Sampling method: At the start of the exposure intervals (0 and 24 hours), sampling was carried out after preparation of the concentration levels. At the end of the exposure (48 hours), samples for the analysis of the aged media were taken from additionally prepared replicates with test media, but without daphnids, since the volumes of the test replicates were insufficient for the analytical determination of the test concentrations. These replicates were incubated under test conditions until sampling. 1 mL cyclohexane was added and the samples were shaken properly for 1 min. The cyclohexane phase was removed with a pipette and transferred into a 4 mL vial containing a hint of sodium sulphate for drying the samples. For measurement 475 µl sample were mixed with 25 µl of a 20 mg/L gamma-decalactone solution as internals standard. The full dilution steps for samples is documented in the full study report.- Sample storage conditions before analysis: All original samples were stored at 6 ± 2 °C before preparation, if necessary. Prepared samples were stored in an autosampler at room temperature until analysis.
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM- Common name: Daphinds- Strain: Daphnia magna STRAUS (Clone 5) - Source: in-house laboratory cultures; origin documented in full study report.- Age at study initiation (mean and range, SD): Less than 24 hours old daphnids from a healthy stock were used for the study- Method of breeding: Not reported.- Feeding during test: No. The daphnids were not fed during the study. During culture: 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 > 10^6 cells/mL. The algae are cultured at the test facility- Food type: Not applicable. - Amount: Not applicable.- Frequency: Not applicable.ACCLIMATION- Acclimation period: Acclimatization was not necessary, because the dilution water was equivalent to the culture medium
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
Start of the Exposure Intervals for the Control Replicates (0 and 24 hours): 0 hours: Total Hardness: 172 mg CaCO3/L ; 24 hours Total Hardness: 174 mg CaCO3/L
Test temperature:
During the test period, the temperature in the incubator was 19 – 20 °C.
pH:
Measured in one additional replicate (without daphnids) per concentration level and control: Test replicates: 0 hours: pH 8.38 - 8.88 ; 24 hours: pH 8.14 - 8.81; mean pH at 48 hours all replicates: did not differ by more than 1.5 unitsControls: 0 hours: pH 8.54 ; 24 hours: pH 8.44; mean pH at 48 hours all replicates: did not differ by more than 1.5 units
Dissolved oxygen:
Measured in one additional replicate (without daphnids) per concentration level and control: Test replicates: 0 hours: O2 dissolved: 8.61 - 8.99 mg/L ; 24 hours: O2 dissolved: 8.49 - 8.77mg/L; mean dissolved oxygen at 48 hours all replicates: did not decrease to less than 8.49 mg/L.Controls: 0 hours: O2 dissolved: 9.32 mg/L ; 24 hours: O2 dissolved: 8.92 mg/L; mean dissolved oxygen at 48 hours to less than 8.92 mg/L.
Nominal and measured concentrations:
Range finding tests were performed (non-GLP) prior to the definitive test: Information is provided in table 1. Measured concentrations were determined and are available in the full study report.Definitive test: nominal concentrations: 5 test item concentrations in a geometric series with a separation factor of 2, prepared by dilution of the stock solution 1 mL/L with dilution water with nominal loading levels of the test item of: 6.25 – 12.5 – 25.0 – 50.0 - 100 % saturated solution. This corresponds to nominal test item concentrations (based on mean relative density of the mixture of 1.011) of: 63.2 - 126 - 253 - 506 - 1010 mg/L.
Details on test conditions:
TEST SYSTEM- Test vessel: Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses- Type: Glass, semi-static- Material, size, headspace, fill volume: 20 mL fill volume- Aeration: No aeration of the test solutions.- No. of organisms per vessel: Control and test item: 20 per concentration, 5 per vessel (divided into 4 replicates)- No. of vessels per concentration (replicates): 4 (four replicates, 5 daphnia per vessel).- No. of vessels per control (replicates): 4 (four replicates, 5 daphnia per vessel).- No. of vessels per vehicle control (replicates): Not applicable.TEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: Same as culture medium (Elendt M4 according to OECD TG 202 annex 3 (2004)).- Culture medium different from test medium: No. Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.20 g test solution per replicate were weighed out into the 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. 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.OTHER TEST CONDITIONS- Adjustment of pH: None.- Photoperiod: 16/8 h light/dark cycle- Light intensity: Diffuse light, light intensity of max. 1500 lxEFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility (including mortality), 24 hours and at 48 hours. An organism was considered to be 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.TEST CONCENTRATIONS- Spacing factor for test concentrations: Two (2)- Justification for using less concentrations than requested by guideline: Not applicable- Range finding study: Yes.- Test concentrations: 0 (control), 1 , 10 and 100 % of saturated solution; measured concentrations (sum of two components) reported within the full study report.- Results used to determine the conditions for the definitive study: Yes.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
1.69 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 1.57 - 1.92 mg/L; GMM based on sum of two constituents
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.27 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 1.14 - 1.34 mg/L; GMM based on sum of two constituents
Details on results:
- Behavioural abnormalities: None- Observations on body length and weight: Not applicable.- Other biological observations: None.- Mortality of control: No mortalities.- Other adverse effects control: None.- Abnormal responses: None.- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None. All tested concentration levels were visually clear throughout the exposure. No Tyndall-effect was observed in the saturated solution.- Effect concentrations exceeding solubility of substance in test medium: Not reported. Effect levels were based on geometric mean measured (GMM) of two measured components.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes.- Mortality: None. Acute immobilisation observed only.- EC50/LC50: 24h-EC50 was 1.91 mg/L (C.I. 1.09 – 3.29 mg/L).- Other: The EC50-value of the reference item potassium dichromate after 24 hours is within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) listed in DIN 38412 - L 30 for Daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD Guideline TG 202 (Clone A).

Table 1. Immobilisation Rates in the non GLP Preliminary Range Finding Test

(n = 20, divided into 2 replicates with 10 daphnids each)

Dilution level of the
saturated solution

[%]

IMMOBILIZATION [%]

24 hours

48 hours

Replicates

Replicates

1

2

MV

1

2

MV

100*

30

30

30

100

100

100

10

0

0

0

0

0

0

1

0

0

0

0

0

0

Control

0

0

0

0

0

0

* = saturated solution

 

Table 2. Immobilisation Rates after 24 and 48 h of Exposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

Dilution level of the
saturated solution

[%]

 

Geometric mean measured concentration of the sum of two constituents [mg/L]

IMMOBILIZATION [%]

24 hours

48 hours

Replicates

Replicates

1

2

3

4

MV

1

2

3

4

MV

100*

2.52

100

100

100

100

100

100% mortality after 24 hours

50.0

1.46

40

40

20

40

35

80

80

60

60

70

25.0

0.763

20

0

0

0

5

20

0

0

0

5

12.5

0.363

0

0

0

0

0

0

0

0

0

0

6.25

0.200

0

0

0

0

0

0

0

0

0

0

Control

< LOQ

0

0

0

0

0

0

0

0

0

0

Meas. conc. = measured concentration of the test item, enrichment and dilution factors taken into account

% = percent of the initially meas. conc. of the test item at start of the corresponding exposure interval

LOQ = limit of quantification of the single components

* = saturated solution

 

Measured concentrations (of two constituents) at the test initiation, 24 hours (end of exposure interval), 24 hours (start of the exposure interval) and 48 hours end of the exposure interval are reported in the full study report. This data was used to calculate the Geometric mean measured concentrations as reported in table 2.

Validity criteria fulfilled:
yes
Conclusions:
The test substance 48h-EC50 was 1.27 mg/L (95% CL: 1.14 – 1.34 mg/L) based geometric mean measured concentrations of the sum of two constituents.
Executive summary:

The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines. The study was conducted under semi-static conditions over a period of 48 hours with a saturated solution of the test item and a further four dilution levels in a geometric series with a separation factor of 2 (nominal concentrations of 6.25 to 100% of the saturated solution; with control). A saturated solution of the test item was prepared as specified in section 4.2 with demineralized water four days before the start of each exposure intervals. After addition of mineral components of the test medium (Elendt M4 medium) and the saturated solution was used as the highest concentration level and as the stock solution for the preparation of all further tested dilution levels. The tested concentration levels were visually clear throughout the exposure period. No Tyndall effect was observed in the saturated solution. Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the untreated control. The measured constituents were analytically verified via GC-MS in all tested concentration levels and the control at the start of the exposure intervals (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours). The measured concentrations of the constituent 1 were in the range of 102 to 116% of the 0 hour measured concentrations at 24 hours and 64 to 85% of the 24 hour fresh test solution measured concentrations at 48 hours. The measured concentrations of the constituent 2 were in the range of 72 to 99% of the 0 hour measured concentrations at 24 hours and 37 to 74% of the 24 hour fresh test solution measured concentrations at 48 hours. Effect values are given based on the geometric mean measured concentrations of the sum of the two components. The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled. The 48h-EC50 was 1.27 mg/L (C.I: 1.14 – 1.34 mg/L) based on geometric mean measured concentrations of the sum of two constituents, within the test system.

Description of key information

EC50 (invertebrates) = 1.27 (C.I. 1.14 - 1.34) mg/L geometric mean measured concentrations; 48hour-freshwater, OECD TG 202, 2016

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1.27 mg/L

Additional information

The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines. The study was conducted under semi-static conditions over a period of 48 hours with a saturated solution of the test item and a further four dilution levels in a geometric series with a separation factor of 2 (nominal concentrations of 6.25 to 100% of the saturated solution; with control). A saturated solution of the test item was prepared as specified in section 4.2 with demineralized water four days before the start of each exposure intervals. After addition of mineral components of the test medium (Elendt M4 medium) and the saturated solution was used as the highest concentration level and as the stock solution for the preparation of all further tested dilution levels. The tested concentration levels were visually clear throughout the exposure period. No Tyndall effect was observed in the saturated solution. Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the untreated control. The measured constituents were analytically verified via GC-MS in all tested concentration levels and the control at the start of the exposure intervals (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours). The measured concentrations of the constituent 1 were in the range of 102 to 116% of the 0 hour measured concentrations at 24 hours and 64 to 85% of the 24 hour fresh test solution measured concentrations at 48 hours. The measured concentrations of the constituent 2 were in the range of 72 to 99% of the 0 hour measured concentrations at 24 hours and 37 to 74% of the 24 hour fresh test solution measured concentrations at 48 hours. Effect values are given based on the geometric mean measured concentrations of the sum of the two components. The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled. The 48h-EC50 was 1.27 mg/L (C.I: 1.14 – 1.34 mg/L) based on geometric mean measured concentrations of the sum of two constituents, within the test system.