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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:
10-07-2014 to 03-02-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met with acceptable minor deviations (lack of pre-test acclimation period).
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
Acclimatisation for Daphnids was 2 hours rather than guideline specified 'good practice' of 48 hours. The control groups did not indicate significant signs of stress and this is not considered a significant deviation.
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
see above.
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: June 2013; signature: November 2013
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Definitive test: 0 (control), 6.25, 12.5, 25.0, 50.0 and 100 mg/L (nominal). All concentrations levels and the control were analytically verified via GC-MS at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours).
- Sampling method: The analysis described within this report was the analysis of three of the constituents measured peak area and mass spectrum scan for identification. At the start of the exposure intervals (0 and 24 h), sampling was carried out after preparation of the test concentrations. At the end of the exposure intervals (24 and 48 h), samples were taken directly from the test vessels.
- Sample storage conditions before analysis: All original samples were stored refrigerated (6 +/-2 °C) before preparation if necessary. All prepared samples were stored at room temperature (in auto sampler) before start of analysis if necessary.
Vehicle:
no
Details on test solutions:
PPREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution (100 mg/L of the test item were weighed out) was freshly prepared with dilution water before the start of both exposure intervals (at 0 and 24 h). The stock solution was stirred with a magnetic stirrer at approximately 1100 rpm for 1 h at room temperature. 5 test item concentrations in a geometric series with a separation factor of 2 were tested as follows: 0 (control) - 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L. The definitive test concentrations are based on the results of a non GLP preliminary range finding test under static conditions.
- Eluate: No
- Controls: Dilution water without test item tested under the same conditions as the test groups.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphinds
- Strain: Daphnia magna (Straus)
- Source: Bred at test facility - Origin: Institut fur Wasser-, Boden- und Lufthygiene (WaBoLu)
- Age at study initiation (mean and range, SD): 2 to 24 hours. They were obtained by removing the juvenile daphnids from the culture vessels 22 h before the start of the exposure. The juveniles born within the 22 h preceding the exposure were used for the test after an acclimatisation phase of 2 hours in the dilution water. No first brood progeny was used for the test.
- Method of breeding: Not reported (expected to be: Parthenogenesis)
- Feeding during test: No.
- Food type: Cultures were 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
- Amount: See above.
- Frequency: See above. No feeding during test conditions.

ACCLIMATION
- Acclimation period: At least 2 hours in dilution water
- Acclimation conditions (same as test or not): Yes.
- Type and amount of food: Not reported
- Feeding frequency: Not reported
- Health during acclimation (any mortality observed): Not reported
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
The adjusted-ISO medium hardness: 263 (start) to 260 (24h) mg/L expressed as CaCO3 and the pH: 7.7 ± 0.1 (pH 7.79 start ; pH 7.66 end)
Test temperature:
The temperature continuously measured in a temperature control vessel was 19 to 20°C°C during the test, and complied with the requirements as laid down in the guideline (18-22°C, constant within 2°C).
pH:
Test conditions remained within the limits prescribed by the guideline; pH: 7.4-8.1 not varying by 1.5 units the end of the test. Test medium actual pH: 7.7 ± 0.7
Dissolved oxygen:
Test conditions remained within the limits prescribed by the guideline; oxygen: > 3 mg/L at the end of the test. Actual oxygen: Dilution water: start: 8.42 mg/L and end: 8.45 mg/L and Test medium: start: 8.0 ± 0.1 mg/L and end: 8.0 ± 0.3 mg/L
Nominal and measured concentrations:
Range finding tests were performed prior to the definitive test: Information is provided in table 1. at concentrations 0 (control), 1, 10 and 100 mg/L.
Definitive test: nominal concentrations:
Nominal: 0 (control), 6.25, 12.5, 25, 50 and 100 mg/L
Measured: The measured concentrations of the test item at the start of the exposure intervals (0 and 24 h) were in the range of 90 to 111 % of the nominal values. At the end of the exposure intervals (24 and 48 h), the measured concentrations of the test item were in the range of 91 to 109 % of the nominal values. See Table 1.
Details on test conditions:
TEST SYSTEM
- Test vessel: Flask, 120 mL
- Type (delete if not applicable): closed, Glass flasks sealed with screw caps (made from polypropylene)
- Material, size, headspace, fill volume: Glass, (4.5 (ID) x 9.5 (H) cm, ca. 130 ml), full - no headspace
- Aeration: No aeration of the test solutions.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): Every 24 hours
- No. of organisms per vessel: Control and test item: 20 per concentration, 5 per vessel containing ca. 100 ml of test solution (definitive test)
- 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: not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water, according to OECD 202
- Culture medium different from test medium: Culture medium = Elendt M4, modified to a total hardness of 160 to 180 mg CaCO3/L; Dilution water for test: ISO Test water, according to OECD 202
- Intervals of water quality measurement: Prior to the start of the exposure (0 h) and the water renewal (24 h), the water quality parameters (i.e. pH value, dissolved oxygen concentration, temperature, conductivity and total hardness) of the dilution water were measured. At the start of the exposure intervals (0 and 24 h), the water quality parameters of the fresh media (i.e. pH value, dissolved oxygen concentration) were measured in one additional replicate per concentration level and control. At the end of the exposure intervals (24 and 48 h), the water parameters of the old media were measured in all replicates per concentration level and control. The incubator temperature was recorded throughout the period of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light, 8 h dark
- Light intensity: Diffuse light; light intensity of max. 20 µE m-2 S-1

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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study
- Test concentrations: 1.00, 10.0 and 100 mg/L.
- Results used to determine the conditions for the definitive study: The percentage immobility was determined in all concentrations and control groups after 24 and 48 h of exposure. 48 h immobility at 1.0 mg/L = 0%; 48 h immobility at 10 mg/L = 20%; 48 h immobility at 100 mg/L= 100%.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
22.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Based on analytically confirmed nominal concentrations of the test item. 95 % C.I. 19.7 - 25.4 mg/L
Details on results:
- Behavioural abnormalities: None
- Observations on body length and weight: Not applicable.
- Other biological observations:
- 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
- EC50: 1.56 mg/L
- Other: C.I. 1.53 - 1.58 mg/L
Reported statistics and error estimates:
TThe EC50-values after 24 and 48 h were calculated by sigmoidal dose response regression. The 95 % confidence limits were calculated from the best-fit values, the standard error and the t-distribution with the software described in the full study report.

Table 1: Calculated Exposure Concentrations of the Test Item during the Definitive Test (based on the determination of three constituents)

Sampling date 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
Calc. conc. [mg/L] % Calc. conc. [mg/L] % Calc. conc. [mg/L] % Calc. conc. [mg/L] %
100 98.7 99 94.0 94 90.2 90 * not determined
50.0 48.2 96 47.0 94 47.8 96 46.7 93
25.0 23.1 92 24.0 96 23.7 95 25.7 103
12.5 12.4 99 11.4 91 12.5 100 12.7 102
6.25 5.98 96 5.98 95 6.91 111 6.84 109
Control < SYSQL < SYSQL < SYSQL < SYSQL

Calc. conc. = calculated concentration of the test item, based on the measurement (sum of peak area) of 3 constituent isomers, single determinations, dilution factor taken into account.

% = percent of the nominal concentration of the test item.

SYSQL = system quantification limit (5.00 ug test item/L).

* = not determined due to 100 % mortality after 24 h

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 concentration [mg/L] IMMOBILISATION [%]

24 h

48 h

Replicates

Replicates

1

2

3

4

MV

1

2

3

4

MV

100

100

100

100

100

100

100

100

100

100

100

50.0

80

80

80

100

85

80

80

100

100

90

25.0

20

60

40

60

45

40

80

60

60

60

12.5

0

0

0

0

0

20

0

0

0

5

6.25

0

0

0

0

0

0

0

0

0

0

Control

0

0

0

0

0

0

0

0

0

0

MV = mean value

Validity criteria fulfilled:
yes
Conclusions:
The test item 48h-EC50 was 22.8 mg/L (95% CL: 19.7 – 25.4 mg/L) based on analytically confirmed nominal 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. 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 6.25 to 100.0 mg/L prepared with dilution water 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 under diffuse light conditions and in a closed system. A stock solution of 100.0 mg/L was prepared with ISO test water (according to OECD 202, Annex 3), with a magnetic stirrer at approximately 1100 rpm for one hour and was freshly prepared at both exposure intervals (0 and 24 hours). Further test concentrations were prepared by diluting the stock solution of 100 mg/L with dilution water. The tested solutions were clear throughout the exposure period. 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 GC-MS 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 h) were in the range of 90 to 111 % of the nominal values. At the end of the exposure intervals (24 and 48 h), the measured concentrations of the test item were in the range of 91 to 109 % of the nominal values. The measured test item concentrations were all within ± 20% of the nominal concentrations. Indicating that the test item concentrations were successfully maintained for the duration of the exposure. Therefore, the EC50-values were based on analytically confirmed nominal concentrations of test item. 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 22.8 mg/L (C.I: 19.7 – 25.4 mg/L) based on analytically confirmed nominal concentrations of the test item in the test system.

 

Description of key information

EC50 (invertebrates) = 48h-EC50 was 22.8 mg/L (95% CL: 19.7 – 25.4 mg/L) ; analytically confirmed nominal concentrations, 48hour-freshwater, semi-static closed system, OECD TG 202, 2015

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
22.8 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 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 6.25 to 100.0 mg/L prepared with dilution water 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 under diffuse light conditions and in a closed system. A stock solution of 100.0 mg/L was prepared with ISO test water (according to OECD 202, Annex 3), with a magnetic stirrer at approximately 1100 rpm for one hour and was freshly prepared at both exposure intervals (0 and 24 hours). Further test concentrations were prepared by diluting the stock solution of 100 mg/L with dilution water. The tested solutions were clear throughout the exposure period. 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 GC-MS 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 h) were in the range of 90 to 111 % of the nominal values. At the end of the exposure intervals (24 and 48 h), the measured concentrations of the test item were in the range of 91 to 109 % of the nominal values. The measured test item concentrations were all within ± 20% of the nominal concentrations. Indicating that the test item concentrations were successfully maintained for the duration of the exposure. Therefore, the EC50-values were based on analytically confirmed nominal concentrations of test item. 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 22.8 mg/L (C.I: 19.7 – 25.4 mg/L) based on analytically confirmed nominal concentrations of the test item in the test system.

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