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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:
27-07-2015 to 07-12-2015
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)
Version / remarks:
see above
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 SPME GC-MS 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 room temperature. The test item samples were diluted with methanol and demin prior to analysis. The dilution steps and indicated dilution factors are indicated in the full study report. Prepared samples were stored in an autosampler at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test item exposures was prepared from stock solution. The stock solution (1.20 mg/L of the test item were weighed out) was prepared with dilution water (ISO test water as specified below) two days before the start of the exposure intervals (at -48 and -24 hours). The stock solution was prepared under light exclusion with dilution water and pre-incubated at 30 ± 2 °C with constant stirring with approximately 1100 rpm for 24 hours. Thereafter, the stock solution was stirred also under light exclusion at 20 ± 1 °C (test temperature) for further 24 hours with approximately 1100 rpm and used for the test. Prior to start of the exposure, the test solutions were checked for undissolved test item. Presence of undissolved test item during preparation and during the test was not observed. 5 test item concentrations in a geometric series with a separation factor of 2, were prepared by diluting the stock solution of 1.20 mg/L with dilution water. Nominal concentrations for definitive test: 0 (control), 0.0750, 0.150, 0.300, 0.600, 1.200 mg/L with equivalent geometric mean measured concentrations: 0 (control), 0.0583, 0.117, 0.242, 0.469 and 0.903 mg/L. Which were based on analysis.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: For positive control - reference item: 1.0, 2.0 and 4.0 mg/L were prepared. A negative/blank control without test item or reference item was also included.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Prior to start of the exposure, the test solutions were checked for undissolved test item. Presence of undissolved test item during preparation and during the test was not observed.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphinds
- Strain: Daphnia magna STRAUS (Clone 5)
- Justification for species other than prescribed by test guideline: Not applicable.
- Source: in-house laboratory cultures (origin of stock reported in full study report).
- Age of parental stock (mean and range, SD): 2 to 24 hours old daphnids from a healthy stock were used for the study
- 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
Remarks on exposure duration:
In accordance with the OECD TG 202 guideline.
Hardness:
Dilution water quality parameters: 0 hours: Total Hardness: 260 mg CaCO3/L ; 24 hours Total Hardness: 262 mg CaCO3/L
Test temperature:
Dilution water quality parameters: 0 hours: Temperature 21.0 °C ; 24 hours: Temperature 21 °C
pH:
Dilution water quality parameters: 0 hours: pH 7.66 ; 24 hours: pH 7.83; mean pH at 48 hours all replicates: did not differ by more than 0.1 units
Dissolved oxygen:
Dilution water quality parameters: 0 hours: O2 dissolved: 8.79 mg/L ; 24 hours: O2 dissolved: 8.90 mg/L; mean dissolved oxygen at 48 hours all replicates: did not decrease to less than 8.33 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), 0.0750, 0.150, 0.300, 0.600, 1.200 mg/L
with equivalent geometric mean measured concentrations: 0 (control), 0.0583, 0.117, 0.242, 0.469 and 0.903 mg/L.
Measured concentrations were determined and are available in the full study report.
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.
- 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), 0.120, 1.20 and 12.00 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.:
> 0.903 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: - mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.799 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 0.642 - > 0.903 mg/L
Details on results:
- Behavioural abnormalities: None reported.
- Observations on body length and weight: Not applicable.
- Other biological observations: None reported.
- 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 1.88 mg/L (C.I. 1.62 - 2.19).
- 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 - L30 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

12.00

100

100

100

100

100

100

1.20

10

20

15

100

100

100

0.12

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)

Geometric mean measured test item concentration

[mg/L]

Immobilisation [%]

 

24 hours

48 hours

 

Replicates

Replicates

 

 

 

 

 

1

2

3

4

MV

1

2

3

4

MV

0.903

0

0

0

0

0

60

40

60

80

60

0.469

0

0

0

20

5

0

20

0

40

15

0.242

0

20

0

0

5

0

20

0

0

5

0.117

0

0

0

0

0

0

0

0

0

0

0.0583

0

0

0

0

0

0

0

0

0

0

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]

Meas.

conc.

[mg/L] *

%

Meas.

conc.

[mg/L]

%

Meas.

conc.

[mg/L]

%

Meas.

conc.

[mg/L]

%

 

1.200

0.943

79

0.815

68

0.964

80

0.897

75

0.903

0.600

0.517

86

0.436

73

0.493

82

0.435

73

0.469

0.300

0.263

88

0.212

71

0.272

91

0.225

75

0.242

0.150

0.132

88

0.100

67

0.120

80

0.117

78

0.117

0.075

0.0610

81

0.0525

70

0.0602

80

0.060

80

0.0583

Control

< LOQ

 

< LOQ

 

< LOQ

 

< LOQ

 

< LOQ

 

 

 

 

 

 

 

 

 

 

Meas. Conc. = measured concentration of the test item, single injections, dilution factors taken into account

% = percent of the nominal concentration on the test item

LOQ = limit of quantification of the analytical method (0.005 mg/L of the test item)

* = reanalysed, mean value of two replicates, dilution factors taken into account

Validity criteria fulfilled:
yes
Conclusions:
The test item 48h-EC50 was 0.799 (95% CL: 0.642 – > 0.903) 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 range of 0.0750 to 1.204 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 1.20 mg/L was prepared with ISO test water (according to OECD 202, Annex 3) which was pre-incubated under light exclusion at 30 ± 2°C with constant stirring at approximately 1100 rpm for 24 hours and stirred for further 24 hours with a magnetic stirrer at approximately 1100 rpm at 20 ± 1°Cunder light exclusion and used as highest concentration level and for preparation of further test concentrations. Presence of undissolved test item was not observed and the stock solution and tested solutions were visually clear throughout. 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 SPME-GC-ME 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 79 to 91% 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 67 to 80% of the nominal concentrations. Since the measured test item concentrations were not all within ± 20% of the nominal concentrations, effect levels were based on geometric mean measured concentrations of the test item which were: 0 (control), 0.0583, 0.117, 0.242, 0.469 and 0.903 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 0.799 mg/L (C.I: 0.642 – > 0.903 mg/L) based on geometric mean measured concentrations of the test item in the test system.

Description of key information

EC50 (invertebrates) = 0.799 (C.I. 0.642 - > 0.903) mg/L ; geometric mean measured concentrations, 48hour-freshwater, OECD TG 202, 2015

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.799 mg/L

Additional information

Key Study : OECD TG 202, 2015 : 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 range of 0.0750 to 1.204 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 1.20 mg/L was prepared with ISO test water (according to OECD 202, Annex 3) which was pre-incubated under light exclusion at 30 ± 2°C with constant stirring at approximately 1100 rpm for 24 hours and stirred for further 24 hours with a magnetic stirrer at approximately 1100 rpm at 20 ± 1°Cunder light exclusion and used as highest concentration level and for preparation of further test concentrations. Presence of undissolved test item was not observed and the stock solution and tested solutions were visually clear throughout. 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 SPME-GC-ME 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 79 to 91% 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 67 to 80% of the nominal concentrations. Since the measured test item concentrations were not all within ± 20% of the nominal concentrations, effect levels were based on geometric mean measured concentrations of the test item which were: 0 (control), 0.0583, 0.117, 0.242, 0.469 and 0.903 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 0.799 mg/L (C.I: 0.642 – > 0.903 mg/L) based on geometric mean measured concentrations of the test item in the test system.