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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:
06 January 2017 to 09 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples for possible analysis were taken from all test concentrations and the control. In addition, the filter used to prepare the saturated solution (SS) was retained for possible analysis of the residue.
- Sampling method: At t = 0 and t = 48 h, 2.4 mL was sampled from the approximate centre of the test vessels. At the end of the exposure period, the replicates were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤ -15 °C) until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Preparation of test solutions started with a loading rate of 100 mg/L applying three days of magnetic stirring to reach the maximum dissolution of the test material in medium. The obtained aqueous mixture was filtered through a 0.45 μm membrane filter (RC55, Whatman) and the resulting saturated solution (SS) was used as the highest test concentration for the combined limit/range-finding test and as a stock solution for the final test. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and increasingly yellow with increasing concentration at the end of the preparation procedure.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain/clone: Straus, 1820, at least third generation,
- Source: In-house laboratory culture with a known history; obtained by a cyclical parthenogenesis under specified breeding conditions.
- Age of parental stock (mean and range, SD): Daphnids originated from a healthy stock, 2nd to 5th brood. For the test, young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old were selected.
- Feeding during test: No

ACCLIMATION
- Acclimation period: Each batch is started with newborn daphnids, i.e. less than 3 days old, by placing about 250 into 5 litres of medium in an all-glass culture vessel. The maximum age of the cultures is 4 weeks. After 7 days of cultivation, half of the medium is renewed twice a week.
- Acclimation conditions (same as test or not): Yes. Daphnids were kept in medium M7 at 18 to 22 °C.
- Type and amount of food: A suspension of fresh water algae
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): Daphnia showed no signs of stress such as mortality >20 %, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
180 mg/L expressed as CaCO3
Test temperature:
19 to 20 °C
pH:
7.9 to 8.1
Dissolved oxygen:
8.5 to 9.9 mg O2/L
Nominal and measured concentrations:
- Nominal concentrations: 1.0, 1.8, 3.2, 5.6 and 10 % of a SS prepared at 100 mg/L
- Measured concentrations: 1.1, 1.9, 3.3, 5.6 and 10 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL test vessels
- Material, size, headspace, fill volume: All-glass vessels containing 80 mL of solution
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The following chemicals are dissolved in tap water purified by Reverse Osmosis (RO-water):
ISO medium: Macro salts: CaCl2.2H2O 211.5 mg/L, MgSO4.7H2O 88.8 mg/L, NaHCO3 46.7 mg/L and KCl 4.2 mg/L.
Medium M7: trace elements, macronutrients and vitamins are added to freshly prepared ISO medium to reach the following concentrations:
Trace elements: B 0.125 mg/L, Fe 0.05 mg/L, Mn 0.025 mg/L, Li, Rb and Sr 0.0125 mg/L, Mo 0.0063 mg/L, Br 0.0025 mg/L, Cu 0.0016 mg/L, Zn 0.0063 mg/L, Co and I 0.0025 mg/L, Se 0.0010 mg/L, V 0.0003 mg/L and Na2EDTA.2H2O 2.5 mg/L.
Macro nutrients: Na2SiO3.9H2O 10.0 mg/L, NaNO3 0.27 mg/L, KH2PO4 0.14 mg/L and K2HPO4 0.18 mg/L.
Vitamins: Thiamine 75.0 μg/L, B12 1.0 μg/L and Biotin 0.75 μg/L.
- Culture medium different from test medium: Daphnids were cultured in medium M7 and the test carried out in adjusted ISO medium.
- Intervals of water quality measurement: At the beginning and at the end of the test, pH and dissolved oxygen were determined. Additionally, after 24 hours the oxygen level was measured in the highest test concentration. Temperature of medium was monitored continuously in a temperature control vessel, beginning at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: None, there was no illumination

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

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 % of a SS at 100 mg/L.
- Results used to determine the conditions for the definitive study: Yes. All daphnids exposed to the two highest test concentrations were immobilised from 24 hours onwards. No immobility was observed at the lowest test concentration and the control during the test period. The expected EC50 was between concentrations obtained in 1.0 and 10 % of a SS prepared at 100 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
2.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % confidence interval between 1.9 and 2.6 mg/L
Details on results:
After 48 hours of exposure all daphnids exposed to measured initial concentrations of 3.3 mg/L and higher were immobilised. In addition, 30 % of the daphnids exposed to 1.9 mg/L were immobilised while no immobility was observed at the lowest test concentration and the control. The responses recorded in this test allowed for reliable determination of an EC50.
The 48h-EC50 was 2.1 mg/L based on measured initial concentrations (95 % confidence interval between 1.9 and 2.6 mg/L).

Samples taken from 1.0, 1.8, 3.2, 5.6 and 10 % of the SS prepared at 100 mg/L were analysed. The measured concentrations were 1.1, 1.9, 3.3, 5.6 and 10 mg/L at the start of the test. During the exposure period the measured concentrations remained stable and were 92 to 94 % of initial at the end of the test. Given these results, the EC50 values were calculated using the measured initial concentrations.
Results with reference substance (positive control):
The reference test was carried out to check the sensitivity of the test system as used by the testing facility. Daphnids were exposed for a maximum of 48 hours to K2Cr2O7 concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L and to a control. Twenty daphnids were exposed per concentration.
The actual responses in this reference test were within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was within the expected range of 0.28 to 0.9 mg/L. Hence, the sensitivity of the daphnia was within the range determined with the historical data collected at the testing facility.
The 24 h EC50 was 0.91 mg/L with a 95 % confidence interval ranging from 0.77 to 1.1 mg/L.
The 48 h EC50 was 0.58 mg/L with a 95 % confidence interval ranging from 0.49 to 0.67 mg/L.
Reported statistics and error estimates:
The 24 and 48h-EC50-value was calculated from the weibits of the percentages of affected daphnids and the logarithms of the corresponding test material concentrations (initially measured) using the maximum likelihood estimation method.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Table 1: Incidence of Immobility

Time (h)

Replicate

Test Material Concentration (mg/L)

Control

1.1

1.9

3.3

5.6

10

24

A

0

0

0

0

3

5

B

0

0

0

0

5

5

C

0

0

0

0

4

5

D

0

0

0

0

4 [1]

5

Total immobilised

0

0

0

0

16

20

Effect %

0

0

0

0

80

100

48

A

0

0

1

5

5

5

B

0

0

2

5

5

5

C

0

0

1

5

5

5

D

0

0

2

5

5

5

Total immobilised

0

0

6

20

20

20

Effect %

0

0

30

100

100

100

5 Daphnids were introduced per replicate

[ ] Number of daphnids observed trapped at the surface of the test solutions. These organisms were re-immersed into the respective solutions before recording of mobility.

Validity criteria fulfilled:
yes
Conclusions:
The 48 h EC50 was 2.1 mg/L based on measured initial concentrations (95 % confidence interval between 1.9 and 2.6 mg/L).
Executive summary:

The potential for the test material to cause acute toxicity to Daphnia magna was investigated in accordance with the standardised guidelines OECD 202, EU Method C.2 and the OECD series on testing and assessment number 23 under GLP conditions.

A final test was performed based on the results of a preceding combined limit/range-finding test. The test material was not completely soluble in test medium at the loading rate initially prepared. Weighing and preparation of test solutions was performed under dimmed light. A saturated solution (SS) was prepared at a loading rate of 100 mg/L and used as a stock solution. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium.

Twenty daphnids per group (four replicates, five daphnids per replicate) were exposed to a control and to 1.0, 1.8, 3.2, 5.6 and 10 % of the SS prepared at 100 mg/L under static conditions. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Analysis of the samples taken from 1.0, 1.8, 3.2, 5.6 and 10 % of the SS prepared at 100 mg/L were analysed. The measured concentrations were 1.1, 1.9, 3.3, 5.6 and 10 mg/L at the start of the test. During the exposure period the measured concentrations remained stable and were 92 to 94 % of initial at the end of the test. Given these results, the EC50 values were calculated using the measured initial concentrations.

After 48 hours of exposure all daphnids exposed to measured initial concentrations of 3.3 mg/L and higher were immobilised. In addition, 30 % of the daphnids exposed to 1.9 mg/L were immobilised while no immobility was observed at the lowest test concentration and the control. The responses recorded in this test allowed for reliable determination of an EC50.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The 48 h EC50 was 2.1 mg/L based on measured initial concentrations (95 % confidence interval between 1.9 and 2.6 mg/L).

Description of key information

The 48 h EC50 was 2.1 mg/L based on measured initial concentrations (95 % confidence interval between 1.9 and 2.6 mg/L).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
2.1 mg/L

Additional information

The potential for the test material to cause acute toxicity to Daphnia magna was investigated in accordance with the standardised guidelines OECD 202, EU Method C.2 and the OECD series on testing and assessment number 23 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

A final test was performed based on the results of a preceding combined limit/range-finding test. The test material was not completely soluble in test medium at the loading rate initially prepared. Weighing and preparation of test solutions was performed under dimmed light. A saturated solution (SS) was prepared at a loading rate of 100 mg/L and used as a stock solution. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium.

Twenty daphnids per group (four replicates, five daphnids per replicate) were exposed to a control and to 1.0, 1.8, 3.2, 5.6 and 10 % of the SS prepared at 100 mg/L under static conditions. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Analysis of the samples taken from 1.0, 1.8, 3.2, 5.6 and 10 % of the SS prepared at 100 mg/L were analysed. The measured concentrations were 1.1, 1.9, 3.3, 5.6 and 10 mg/L at the start of the test. During the exposure period the measured concentrations remained stable and were 92 to 94 % of initial at the end of the test. Given these results, the EC50 values were calculated using the measured initial concentrations.

After 48 hours of exposure all daphnids exposed to measured initial concentrations of 3.3 mg/L and higher were immobilised. In addition, 30 % of the daphnids exposed to 1.9 mg/L were immobilised while no immobility was observed at the lowest test concentration and the control. The responses recorded in this test allowed for reliable determination of an EC50.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The 48 h EC50 was 2.1 mg/L based on measured initial concentrations (95 % confidence interval between 1.9 and 2.6 mg/L).