Registration Dossier

Diss Factsheets

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

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:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
An Acute Immobilisation Test to Daphnia magna STRAUS was carried out to determine the EC50-values of the test item after 24 and 48 hours of exposure under semi-static conditions in a closed system and in darkness.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected June 2013; signature: November 2013
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All relevant concentration levels and the control were analytically verified via HPLC-DAD at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours).
- Sampling method: At the start of the exposure intervals (0 and 24 hours), sampling was carried out after preparation of the test concentrations. At the end of the exposure intervals (24 and 48 hours), samples were taken directly from the test vessels. The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).
- Sample storage conditions before analysis: All original samples were stored at 6 ± 2 °C 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
- Age at study initiation (mean and range, SD): 2 to 24 hours old daphnids from a healthy stock were used for the study. Juveniles born within this period of max. 22 hours preceding the exposure were used for the test after an acclimatisation phase of at least 2 hours in the dilution water. No brood progeny were used for the test.
- 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: At least 2 hours in dilution water.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
Dilution water quality parameters: 0 hours: Total Hardness: 263 mg CaCO3/L ; 24 hours Total Hardness: 265 mg CaCO3/L
Test temperature:
Dilution water quality parameters: 0 hours: Temperature 20.4 °C ; 24 hours: Temperature 20.3 °C
pH:
Dilution water quality parameters: The dilution water was adjusted to pH 6 ±0.2 by addition of 1 M HCI one day prior to the start of the exposure intervals (at -24 and 0 hours). Pre adjustment: 0 hours: pH 7.74 ; 24 hours: pH 7.69; Post adjustment: 0 hours: pH 6.17 ; 24 hours: pH 6.01.
The mean pH at 48 hours all replicates: did not differ by more than 0.3 units: Range in treatment group and control replicates: pH: 6.14 - 6.29
Dissolved oxygen:
Dilution water quality parameters: 0 hours: O2 dissolved: 8.66 mg/L ; 24 hours: O2 dissolved: 8.71 mg/L; dissolved oxygen at 48 hours all replicates: did not decrease to less than 6.88 mg/L.
Nominal and measured concentrations:
Range finding tests were performed (non-GLP) prior to the definitive test: Information is provided in table 1.
Definitive test: nominal test item concentrations: 0 (control), 0.625, 1.25, 2.50, 5.00, 10.00 mg/L.
Geometric mean measured concentrations were determined and are provided in table 3.
Corresponding to geometric mean measured concentrations of the test item of: 0.400, 0.494, 1.02, 1.80, 4.62 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 4.5 (ID) x 9.5 (H) cm, ca. 130 mL
- Type: A semi-static test design with a daily renewal of the test solutions in glass flasks sealed with screw caps (made from polypropylene) and in darkness was chosen to reduce contact with air and losses of the test item by evaporation.
- Material, size, headspace, fill volume: Sealed glass flasks with screw caps (made from polypropylene).
- 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: ISO Test water, according to OECD 202, Annex 3: composition (mg/L): KCl 5.75; NaHCO3: 64.8; CaCl2.2H2O: 294 ; MgSO4.7H2O: 123.
- Culture medium different from test medium: Yes. Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L

The test vessels were filled up with the test solutions. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by pipette. Thereafter, the test vessels were closed immediately with screw caps. The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels were filled with the freshly prepared test solutions and the daphnids were transferred by pipette.

OTHER TEST CONDITIONS
- Adjustment of pH: No. Prior to test item exposure the pH of dilution water was pH adjusted. The dilution water was adjusted to pH 6 ±0.2 by addition of 1 M HCI one day prior to the start of the exposure intervals (at -24 and 0 hours). The test vessels were not pH adjusted during or after test item exposure.
- Photoperiod: 16/8 h light/dark cycle during culture conditions. During testing: Light exclusion.
- Light intensity: Diffuse light, light intensity of max. 20 µE /m2 s (1340 lx) during culture conditions. During testing: light exclusion.

EFFECT 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: 2.0
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study
- Test concentrations: 0 (control), 0.50, 5.00 and 50.00 mg/L nominal test item concentration. See table 1.
- 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.:
> 4.62 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: not applicable ; geometric mean measured concentration
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.23 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 1.01 - 1.55 mg/L ; geometric mean measured concentration
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: not applicable ; nominal concentration
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
3.05 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 2.33 - 4.12 mg/L ; nominal concentration
Details on results:
- Behavioural abnormalities: None reported.
- Observations on body length and weight: Not applicable.
- Other biological observations:
- Mortality of control: No mortalities in control.
- Other adverse effects control: None reported.
- Abnormal responses: None reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None. Prior to start of the exposure intervals, the test solutions were checked for undissolved test item via laser beam (Tyndall effect). Presence of undissolved test item during preparation and during the test was not observed.
- Effect concentrations exceeding solubility of substance in test medium: No.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes.
- Mortality: None. Acute immobilisation observed only.
- EC50/LC50: 24h-EC50 was 1.89 mg/L (C.I. 1.75 - 2.04).
- 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
Reported statistics and error estimates:
The EC50-value after 24 and 48 hours of exposure were calculated by sigmoidal dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t-distribution.

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

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

Nominal

test item

concentration

[mg/L]

Immobilisation [%]

24 hours

48 hours

Replicates

Replicates

1

2

MV

1

2

MV

50.0

90

50

70

100

100

100

 5.00

30

20

25

 70

 80

 75

 0.500

 0

 0

 0

   0

   0

   0

Control

 0

 0

 0

   0

   0

   0

* measured concentrations presented in the full study report.

 

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)

Nominal

test item

concentrations

 

[mg/L]

Geometric mean

measured

test item

concentrations

[mg/L]

Immobilisation [%]

 

24 hours

48 hours

Replicates

Replicates

1

2

3

4

MV

1

2

3

4

MV

10.0

4.62

20

40

40

60

40

80

80

100

80

85

5.00

1.80

20

40

60

20

35

60

80

 80

80

75

2.50

1.02

 0

 0

 0

 0

 0

20

60

 40

20

35

1.25

0.494

 0

 0

 0

 0

 0

40

20

 20

20

25

0.625

0.400

 0

 0

 0

 0

 0

 0

20

   0

 0

 5

Control

 0

 0

 0

 0

 0

 0

 0

   0

 0

 0

  

Table 3. Measured Concentrations and Percent of the Nominal and the Initially Measured Concentrations of the Test Item during the Definitive Test

Sampling

0 hours

Start of the

exposure interval

24 hours

End of the

exposure interval

24 hours

Start of the

exposure interval

48 hours

End of the

exposure interval

 

Nominal

test item

concentration

 

[mg/L]

Test item concentration

Geometric mean*

measured

test item

concentration

[mg/L]

Meas.

conc.

[mg/L]

%

Meas.

conc.

[mg/L]

%

Meas.

conc.

[mg/L]

%

Meas.

conc.

[mg/L]

%

10.0

7.56

76

5.64

56

5.19

52

2.06

21

4.62

 5.00

3.81

76

2.67

53

2.59

52

< LOQ

1.80

 2.50

1.90

76

1.12

45

1.26

50

< LOQ

1.02

 1.25

0.931

74

< LOQ

< LOQ

< LOQ

0.494

 0.625

< LOQ

< LOQ

< LOQ

< LOQ

0.400

Control

< LOQ

< LOQ

< LOQ

< LOQ

 

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

% = percent of the nominal concentration of the test item

LOQ = limit of quantification of the analytical method (0.8 mg/L of the test item)*

* = measured concentrations below the LOQ taken into account with ½ LOQ

Validity criteria fulfilled:
yes
Conclusions:
The test substance 48h-EC50 was 3.05 mg/L (C.I: 2.33 – 4.12 mg/L) based on nominal concentrations and 48h-EC50 was 1.23 mg/L (C.I: 1.01 – 1.55 mg/L) based on geometric mean measured concentrations.
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 under GLP. The study was conducted in a closed system (sealed glass flasks) without headspace under semi-static conditions and under light exclusion over a period of 48 hours with five concentration levels of the test item in the nominal concentration range of 0.625 to 10.0 mg/L prepared in a geometric series with a separation factor of 2. To reduce losses of the test item, the study was conducted under semi-static conditions in darkness and in a closed system. A stock solution of 10.0 mg/L was prepared with ISO test water (according to OECD 202, Annex 3) which was adjusted to a pH of 6 ± 0.2 by addition of 1 M HCI prior to testing. Stirred with a magnetic stirrer at approximately 1100 rpm and without headspace and light exclusion and was used as highest concentration level and for preparation of further test concentrations. At the end of the stirring phase, Tyndall effect was observed in the stock solutions of 10 mg/L. However, all test concentrations were visually clear throughout the exposure intervals. Twenty daphnids were exposed to each concentration level and the control. 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. The concentrations of the test item were analytically verified via HPLC-DAD at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours) in all relevant concentration levels and the control. The measured concentrations of the test item at the start of the exposure intervals (0 and 24 hours) were in the range of< LOQ to 76% of the nominal concentrations. At the end of the exposure intervals (24 and 48 hours), the measured concentrations of the test item were in the range of< LOQ to 56% of the nominal concentrations. Based on the decline of the analytical recoveries and the nature of the test item (chemical class ester), the observed effects may be related to the test item or possible degradation products. Since the measured test item concentrations did not remain within ± 20% of the nominal concentrations, the EC50-values are based upon the nominal and geometric mean measured concentrations. The GMM were 0.400, 0.494, 1.02, 1.80, 4.62 mg/L. 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 3.05 mg/L (C.I: 2.33 – 4.12 mg/L) based on nominal concentrations and 48h-EC50 was 1.23 mg/L (C.I: 1.01 – 1.55 mg/L) based on geometric mean measured concentrations of the test item in the test system.

Description of key information

EC50 (invertebrates) = 1.23 (C.I. 1.01 - 1.55) mg/L based on geometric mean measured concentrations, 48-hour, freshwater, OECD TG 202, 2017

EC50 (invertebrates) = 3.05 mg/L (C.I: 2.33 – 4.12 mg/L) mg/L based on nominal concentrations, 48-hour, freshwater, OECD TG 202, 2017

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
1.23 mg/L

Additional information

Key study: OECD TG 202, 2017 : 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 under GLP. The study was conducted in a closed system (sealed glass flasks) without headspace under semi-static conditions and under light exclusion over a period of 48 hours with five concentration levels of the test item in the nominal concentration range of 0.625 to 10.0 mg/L prepared in a geometric series with a separation factor of 2. To reduce losses of the test item, the study was conducted under semi-static conditions in darkness and in a closed system. Twenty daphnids were exposed to each concentration level and the control. 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. The concentrations of the test item were analytically verified via HPLC-DAD at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours) in all relevant concentration levels and the control. The measured concentrations of the test item at the start of the exposure intervals (0 and 24 hours) were in the range of< LOQ to 76% of the nominal concentrations. At the end of the exposure intervals (24 and 48 hours), the measured concentrations of the test item were in the range of< LOQ to 56% of the nominal concentrations. Based on the decline of the analytical recoveries and the nature of the test item (chemical class ester), the observed effects may be related to the test item or possible degradation products. Since the measured test item concentrations did not remain within ± 20% of the nominal concentrations, the EC50-values are based upon the nominal and geometric mean measured concentrations. The GMM were 0.400, 0.494, 1.02, 1.80, 4.62 mg/L. 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 3.05 mg/L (C.I: 2.33 – 4.12 mg/L) based on nominal concentrations and 48h-EC50 was 1.23 mg/L (C.I: 1.01 – 1.55 mg/L) based on geometric mean measured concentrations of the test item in the test system.

Categories Display