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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:
10-12-2019 to 07-01-2020
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Definitive test: 0 (control), 10, 18, 32, 56 and 100 mg/L prepared as WSF (water soluble fraction ; the result of a WAF (water accommodated fractions) subject to a separation stage).
These were, respectively: 0 (control), 9.3, 16, 26, 47 and 69 mg/L based on geometric mean measured average concentrations, respectively; controls: Test medium without test item or other additives. Spacing factor determined from range-finding test and physico-chemical properties of the substance.
- Sampling method: Singular samples for possible analysis were taken from all test concentrations and the control according to the schedule:
1. At the start of the test 0-hours and 24-hours from the freshly prepared solutions.
2. At the first renewal (t=24 hours)
3. The end of the test from the 24-hour old solutions, volume: 1 mL from the approximate centre of the test vessels.
- Sample storage conditions before analysis: Samples were used on day of sampling; reserve samples were stored in a freezer until analysis (≤ -15°C). Reserve samples of 1 mL were taken for possible analysis if needed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The preparation procedure for test solutions was based on a non-GLP saturation test and range-finder (cited in the full study report). Preparation of test solutions started with loading rates individually prepared at 10, 18, 32, 56 and 100 mg/L. The test item was added to stirring test medium on a volume basis by means of an automated displacement pipette. The required volume of test item for each treatment group was determined based on the test item’s density of 0.978 g/cm3. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. The obtained mixtures were allowed to settle for a period of approximately two hours. Thereafter, the aqueous Water Soluble Fractions (WSFs) were collected by means of siphoning through glass wool and used as test concentrations. All test solutions were clear and colourless at the end of the preparation procedure. The test solutions preparation were performed under dimmed lighting and aluminium wrapped glassware with minimal headspace. Five test item concentrations in a geometric series with a separation factor of 1.8, were prepared as follows: 0 (control), 10, 18, 32, 56 and 100 mg/L loading rates as WSF. For the definitive test: equivalent geometric mean measured concentrations were: 0 (control), 9.3, 16, 26, 47 and 69 mg/L which were based on analysis during the definitive test period.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: For positive control - reference item: potassium dichromate were prepared in a separately conducted reference test (documented in the full study report). 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. Undissolved test item was excluded on the basis of application of loading rates as WSFs.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphinds
- Strain/clone: Strain: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
- Justification for species other than prescribed by test guideline: Not applicable.
- Age at study initiation (mean and range, SD): < 24 hours old daphnids from a healthy stock were used for the study
- Weight at study initiation (mean and range, SD): Not applicable.
- Length at study initiation (length definition, mean, range and SD): Not applicable.
- Stage and instar at study initiation: Juvenile ; < 24 hours
- Valve height at study initiation, for shell deposition study (mean and range, SD): Not applicable.
- Peripheral shell growth removed prior to test initiation: Not applicable.
- Method of breeding: Parthenogenesis
- Source: in-house laboratory cultures
- Age of parental stock (mean and range, SD):
- Feeding during test: No. The daphnids were not fed during the study. During culture: Daily, a suspension of fresh water algae. The algae are cultured at the test facility
- Food type: Not applicable.
- Amount: Not applicable.
- Frequency: Not applicable.

ACCLIMATION
- Acclimation period: None.
- Acclimation conditions (same as test or not): Not applicable.
- Type and amount of food: Not applicable.
- Feeding frequency: Not applicable.
- Health during acclimation (any mortality observed): Not applicable.

QUARANTINE (wild caught)
- Duration: Not applicable.
- Health/mortality: Not applicable.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: Not applicable.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
In accordance with the OECD TG 202 guideline.
Hardness:
The ISO medium hardness: 180 mg/L expressed as CaCO3 and the pH: 7.7 ± 0.3
Test temperature:
The temperature continuously measured in a temperature control vessel varied between 19 and 21°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).
pH:
Test conditions remained within the limits prescribed by the guideline; pH: 7.5-9.0, not varying by more than 1.5 unit at the end of the test (actual pH range = 7.9 – 8.1).
Dissolved oxygen:
Test conditions remained within the limits prescribed by the guideline; oxygen: >= 3 mgO2/L at the end of the test (actual O2 = 8.1 to 9.2 mgO2/L).
Nominal and measured concentrations:
Range-finding test: 0 (control), 1.0, 10 and 100 mg/L prepared as WSF
Definitive test: 0 (control), 10, 18, 32, 56 and 100 mg/L prepared as WSF (water soluble fraction ; the result of a WAF (water accommodated fractions) subject to a separation stage) with equivalent geometric mean measured concentrations were: 0 (control), 9.3, 16, 26, 47 and 69 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL, all glass, closed airtight without headspace.
- Type (delete if not applicable): Closed
- Material, size, headspace, fill volume: 50 mL, all glass, closed airtight without headspace.
- Volume of solution: ca. 50 mL
- Aeration: No.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): renwal after 24 hours intervals
- 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.
- Biomass loading rate: See above.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse Osmosis (RO-water, GEON Waterbehandeling, The Netherlands); used to prepare ISO Medium M7 (culture medium) and/or test medium
- Total organic carbon: Not applicable.
- Particulate matter: Not applicable.
- Metals: Not applicable.
- Pesticides: Not applicable.
- Chlorine: Not applicable.
- Alkalinity: Not applicable.
- Ca/mg ratio: See below.
- Conductivity: Not applicable.
- Salinity: Not applicable.
- Culture medium different from test medium: Yes. The following were added to Reverse Osmosis (RO-water, GEON), dilution water: CaCl2.2H2O 211.5 mg/L ; MgSO4.7H2O 88.8 mg/L ; NaHCO3 46.7 mg/L ; KCl 4.2 mg/L and the hardness of test medium expressed as CaCO3: 180 mg/L
- Intervals of water quality measurement: pH and dissolved oxygen: at the beginning, after 24 hours of exposure (fresh and spent solutions) and at the end of the test, for all concentrations and the control ; temperature : continuously.

OTHER TEST CONDITIONS
- Adjustment of pH: None.
- Photoperiod: The study was performed in the dark
- Light intensity: Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility (including mortality), 24 hours and at 48 hours.

VEHICLE CONTROL PERFORMED: Not applicable.

RANGE-FINDING STUDY
- Test concentrations: 0 (control), 1.0, 10 and 100 mg/L prepared as WSF
- 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.:
19 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 17 to 21 mg/L
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
16 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 14 - 19 mg/L
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.
- Effect concentrations exceeding solubility of substance in test medium: No.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes.
- Mortality: See below.
- EC50/LC50: 24h- and/or 48h- EC50 was within the expected range. The reference test was conducted in a separate study (cited in the full study report) under GLP.
- Other: The sensitivity of the test system was in agreement with the historical data.
Reported statistics and error estimates:
The 24 and 48h-EC50-values were calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding test item concentrations (geometric mean) using the maximum likelihood estimation method.
ToxRat Professional v 3.2.1 was the software package utilised.

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

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

Test item, WSF loading rate concentration (mg/L)

Immobilisation [%]

24 hours

 

48 hours

Replicates

 

Replicates

1

2

3

4

MV (%)

1

2

3

4

MV (%)

0 (control)

0

0

0

0

0

0

0

0

0

0

1.0

0

0

-

-

0

0

0

-

-

0

10

0

0

-

-

0

0

0

-

-

0

100

5 #

5

5

5

100

5

5

5

5

100

* measured concentrations presented in the full study report.

# Microscopic observation revealed no test item was attached to the daphnids.

 

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)

Test item, WSF loading rate concentration (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 (%)

0 (control)

0

0

0

0

0

0

0

0

2

0

10

10

9.3

0

0

0

0

0

0

1

0

0

5

18

16

1

1

2

1

25

2

1

2

2

35

32

26

5 #

4 (3)

5 (1)

4

90

5

5

5

5

100

56

47

5

5

5

5

100

5

5

5

5

100

100

69

5

5

5

5

100

5

5

5

5

100

( )= number of daphnids observed to be trapped at the surface of the test solutions. These organisms were re-immersed into the respective solutions before recording of mobility

# = Microscopic observation revealed no test item was attached to the daphnids.

 

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

Test item, WSF loading rate concentration (mg/L)

Measured concentrations (mg/L)

Geometric mean

measured

test item

concentration

[mg/L]

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

 

 

 

 

 

 

10

8.61

8.42

10.1

10.3

9.3

18

16.5

16.2

14.4

15.5

16

32

26.4

26.7

26.3

25.9

26

56

48.4

46.2

47.8

46.2

47

100

62.5

60.8

75.7

76.5

69

Validity criteria fulfilled:
yes
Conclusions:
The test item 48h-EC50 was 16 (C.I: 14 – 19) mg/L based on geometric mean measured concentrations.
Executive summary:

The acute toxicity to Daphnia magna was carried out according to OECD 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. Following preliminary range finding/limit test in the 1.0 to 100 mg/L range, a similarly conducted definitive study was completed in 50 mL, all glass, closed airtight without headspace under semi-static conditions. The preparation procedure for test solutions was based on a non-GLP saturation test and range-finder (cited in the full study report).Preparation of test solutions started with loading rates individually prepared at 10, 18, 32, 56 and 100 mg/L. The test item was added to stirring test medium on a volume basis by means of an automated displacement pipette. The required volume of test item for each treatment group was determined based on the test item’s density of 0.978 g/cm3. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. The obtained mixtures were allowed to settle for a period of approximately two hours. Thereafter, the aqueous Water Soluble Fractions (WSFs) were collected by means of siphoning through glass wool and used as test concentrations. All test solutions were clear and colourless at the end of the preparation procedure. The test solutions preparation were performed under dimmed lighting and aluminium wrapped glassware with minimal headspace. Five test item concentrations in a geometric series with a separation factor of 1.8, were prepared as follows: 0 (control), 10, 18, 32, 56 and 100 mg/L loading rates as WSF. Four replicates per test group containing five daphnids each were utilised. The total exposure period was 48 hours and samples for analytical confirmation of actual exposure concentrations were taken from freshly prepared solutions (t=0 and 24 h) and 24-hour old solutions (t=24 and 48 h). Samples taken from all test concentrations and the control were analysed. The concentrations measured at the start of the test were 8.6, 17, 26, 48 and 63 mg/L in the WSFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L, respectively. The concentrations in the freshly prepared solutions upon renewal after 24 hours were 10, 14, 26, 48 and 76 mg/L, respectively. The concentrations remained stable during both 24-hour renewal periods, i.e. were within 96 to 108% of the initial values in the spent solutions. Equivalent geometric mean measured concentrations were: 0 (control), 9.3, 16, 26, 47 and 69 mg/L. The study met all the relevant acceptability criteria and was considered valid. Concentration-related immobility was found at average exposure concentrations of 16 mg/L and higher, resulting in complete immobility at the three highest test concentrations after 48 hours of exposure. Under the conditions of this study, the 48h-EC50 was 16 (C.I: 14 – 19) mg/L based on geometric mean measured concentrations.

Description of key information

48h-EC50 (invertebrates) = 16 (C.I. 14 – 19) mg/L based on geometric mean measured exposure concentrations, 48-hour, freshwater, OECD 202, 2020

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
16 mg/L

Additional information

Key study : OECD TG 202, 2020 : The acute toxicity to Daphnia magna was carried out according to OECD 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. Following preliminary range finding/limit test in the 1.0 to 100 mg/L range, a similarly conducted definitive study was completed in 50 mL, all glass, closed airtight without headspace under semi-static conditions. The preparation procedure for test solutions was based on a non-GLP saturation test and range-finder (cited in the full study report).Preparation of test solutions started with loading rates individually prepared at 10, 18, 32, 56 and 100 mg/L. The test item was added to stirring test medium on a volume basis by means of an automated displacement pipette. The required volume of test item for each treatment group was determined based on the test item’s density of 0.978 g/cm3. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. The obtained mixtures were allowed to settle for a period of approximately two hours. Thereafter, the aqueous Water Soluble Fractions (WSFs) were collected by means of siphoning through glass wool and used as test concentrations. All test solutions were clear and colourless at the end of the preparation procedure. The test solutions preparation were performed under dimmed lighting and aluminium wrapped glassware with minimal headspace. Five test item concentrations in a geometric series with a separation factor of 1.8, were prepared as follows: 0 (control), 10, 18, 32, 56 and 100 mg/L loading rates as WSF. Four replicates per test group containing five daphnids each were utilised. The total exposure period was 48 hours and samples for analytical confirmation of actual exposure concentrations were taken from freshly prepared solutions (t=0 and 24 h) and 24-hour old solutions (t=24 and 48 h). Samples taken from all test concentrations and the control were analysed. The concentrations measured at the start of the test were 8.6, 17, 26, 48 and 63 mg/L in the WSFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L, respectively. The concentrations in the freshly prepared solutions upon renewal after 24 hours were 10, 14, 26, 48 and 76 mg/L, respectively. The concentrations remained stable during both 24-hour renewal periods, i.e. were within 96 to 108% of the initial values in the spent solutions. Equivalent geometric mean measured concentrations were: 0 (control), 9.3, 16, 26, 47 and 69 mg/L. The study met all the relevant acceptability criteria and was considered valid. Concentration-related immobility was found at average exposure concentrations of 16 mg/L and higher, resulting in complete immobility at the three highest test concentrations after 48 hours of exposure. Under the conditions of this study, the 48h-EC50 was 16 (C.I: 14 – 19) mg/L based on geometric mean measured concentrations.