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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:
26 May 2019 to 12 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
13 April 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals, OECD series on testing and assessment number 23.
Version / remarks:
2019
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.
- Sampling method: Frequency: at t = 0 and 48 h; volume: 6.0 mL from the approximate centre of the test vessels.
- Sample storage conditions before analysis: During the final test, 100 μL acetone was added to the taken samples before storage in a freezer, in accordance with the analytical method. Storage Samples were stored in a freezer (≤-15 °C) until analysis.
Additionally, reserve samples of 6.0 mL were taken for possible analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The batch tested was not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition. All glassware used during the preparation of test solutions was airtight closed with minimal headspace to minimise vaporisation of test material.
Preparation of test solutions started with a loading rate of 100 mg/L applying a one-day period of magnetic stirring to ensure maximum dissolution of the test material in test medium. The obtained mixture was allowed to settle for a period of 30 minutes. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colourless at the end of the preparation procedure. Furthermore, tyndall effect was checked with the use of a laser pen, no tyndall effect was observed.
Any residual volumes were discarded.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain/clone: Crustacea, Cladocera (Straus, 1820)
- Source: In-house laboratory culture with a known history at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Age of parental stock: Daphnia used in the test were less than 24 hours old, from parental daphnids of more than two weeks old.
- Feeding during test: No

BREEDING
For the start of each batch, approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of medium in an all glass culture vessel. The maximum age of the cultures was 4 weeks and after 7 days of cultivation, half of the medium was renewed twice a week.
The temperature of the medium 18-22 °C. Culture medium was M7. Salts and vitamins were added to freshly prepared test medium to reach the following concentrations:
Salts (concentrations in mg/L): H3BO3 0.71, FeSO4.7H2O 0.25, MnCl2.4H2O 0.090, LiCl 0.076, RbCl 0.018, SrCl2.6H2O 0.038, Na2MoO4.2H2O 0.015, NaBr 0.0040, CuCl2.2H2O 0.0042, ZnCl2 0.013, CoCl2.6H2O 0.010, KI 0.0032, Na2SeO3 0.0022, NH4VO3 0.00057, Na2EDTA.2H2O 0.62, Na2SiO3.5H2O 7.5, NaNO3 0.27, KH2PO4 0.14 and K2HPO4 0.18 mg/L.
Vitamins (concentrations in µg/L): Thiamine hydrochloride 75.0, B12 1.0 and Biotin 0.75 μg/L
- Type and amount of food: A suspension of fresh water algae
- Feeding frequency: Feeding occurred daily
- Health: Daphnids originated from a healthy stock, 2nd to 5th brood, showing 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:
The hardness of test medium expressed as CaCO3: 180 mg/L
Test temperature:
20 - 22 °C
pH:
8.0 - 8.2
Dissolved oxygen:
8.1 - 8.9 mg/L
Nominal and measured concentrations:
- Nominal concentrations: Solutions containing 10, 18, 32, 56 and 100 % of the SS, prepared at a loading rate of 100 mg/L.
- Measured concentrations at the start of the test: 1.5, 2.8, 4.5, 10 and 19 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 mL, all-glass
- Type: Airtight
- Material, size, headspace, fill volume: Closed without headspace
- Aeration: No
- No. of organisms per vessel: 5 per vessel
- No. of vessels per concentration (replicates): 4 (20 daphnids per concentration)
- No. of vessels per control (replicates): 4 (20 daphnids)
- Introduction of daphnids: Within 40 minutes after preparation of the test solutions

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands): CaCl2.2H2O 211.5 mg/L, MgSO4.7H2O 88.8 mg/L, NaHCO3 46.7 mg/L and KCl 4.2 mg/L
- Intervals of water quality measurement: pH and dissolved oxygen were measured at the beginning and at the end of the test, for all concentrations and the control. 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: A daily photoperiod of 16 hours.

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

VEHICLE CONTROL PERFORMED: Not required

RANGE-FINDING STUDY
- Test concentrations: Control and a SS prepared at a loading rate of 100 mg/L (20 daphnids) and 1.0 and 10 % of the SS (10 daphnids)
- Results used to determine the conditions for the definitive study: Yes. No immobility was observed in the control and at the two lowest test concentrations throughout the test. At the end of the test, complete immobility was observed at 100 % of the SS prepared at a loading rate of 100 mg/L. Therefore, the expected EC50 was between 10 and 100 % of the SS.

INTERPRETATION
- Acceptability of the Test
1. In the control, no daphnids became immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium.
2. The dissolved oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (K2Cr2O7)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
16 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % CL 15 - 18 mg/L
Details on results:
Effect parameters were expressed as initially measured concentrations.

IMMOBILITY
Table 1 shows the responses recorded during the final test. No immobility was observed in the control and at the four lowest test concentrations throughout the exposure period. At the end of the test, 95 % immobility was observed at the highest test material concentration.
The responses recorded in this test allowed for reliable determination of an EC50 and were in agreement with what was expected based on the results of the combined limit/range-finding test.
The study met the acceptability criteria and was considered valid.

In conclusion, the 48h-EC50 for Daphnia magna exposed to the test material was 16 mg/L based on measured concentrations (95% confidence interval between 15 and 18 mg/L).
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. Daphnia 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 study met the acceptability criteria prescribed by the study plan and was considered valid.
The 24h-EC50 in this reference test was within the expected range of 0.6 and 2.1 mg/L and the 48h-EC50 was within the expected range of 0.28 and 0.75 mg/L, which is based on reference tests performed at the Test Facility during the last ten years.
The 24h-EC50 was 0.92 mg/L with a 95 % confidence interval between 0.78 and 1.1 mg/L.
The 48h-EC50 was 0.55 mg/L with a 95 % confidence interval between 0.47 and 0.64 mg/L.
Reported statistics and error estimates:
The 24h-EC50-value could not be determined because the observed effects were below 50 %. The 48h-EC50-value was calculated from the logits 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 analysis.

Table 1: Number of Introduced Daphnids and Incidence of Immobility in the Final Test

Time (h)

Replicate

Measured concentration (mg/L)

Control

1.5

2.8

4.5

10

19

0

A

5

5

5

5

5

5

B

5

5

5

5

5

5

C

5

5

5

5

5

5

D

5

5

5

5

5

5

Total introduced

20

20

20

20

20

20

24

A

0

0

0

0

0

2 (5)

B

0

0

0

0

0

1 (2)

C

0

0

0

0

0

2 (5)

D

0

0

0

0

0

3 (5)

Total immobilised

0

0

0

0

0

8

Effect %

0

0

0

0

0

40

48

A

0

0

0

0

0

5 (3)

B

0

0

0

0

0

4 (1)

C

0

0

0

0

0

5 (3)#

D

0

0

0

0

0

5 (2)

Total immobilised

0

0

0

0

0

19

Effect %

0

0

0

0

0

95

 ( ) between brackets: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

# Microscopic observation revealed no test material attached to the daphnids.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the 48h-EC50 was 16 mg/L based on measured concentrations (95 % confidence interval between 15 and 18 mg/L).
Executive summary:

An acute toxicity test with daphnia magna (static) was carried out in accordance with the standardised guidelines OECD 202 and OECD series on testing and assessment number 23 under GLP conditions.

The batch of material tested was a clear colourless liquid and was not completely soluble in test medium at the loading rate initially prepared. All glassware used during the preparation of test solutions was airtight closed with minimal headspace to minimise vaporisation of test material.

A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium. A final test was performed based on the results of a combined limit/range-finding test.

Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to solutions containing 10, 18, 32, 56 and 100 % of the SS prepared at a loading rate of 100 mg/L. 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.

Samples taken from all test concentrations and the control were analysed. No test material was detected in the control throughout the test. The measured concentrations at the start of the test were 1.5, 2.8, 4.5, 10 and 19 mg/L, respectively. During the exposure period, the concentrations remained stable, i.e. were at 90 - 106 % relative to the initial concentrations at the end of the test. Based on these results, effect parameters were expressed as initially measured concentrations.

No immobility was observed in the control or the four lowest test concentrations throughout the exposure period. At the end of the test, 95 % immobility was observed at the highest test material concentration. The study met the acceptability criteria and was considered valid.

Under the conditions of this study, the 48h-EC50 was 16 mg/L based on measured concentrations (95 % confidence interval between 15 and 18 mg/L).

Description of key information

Under the conditions of this study, the 48h-EC50 was 16 mg/L based on measured concentrations (95 % confidence interval between 15 and 18 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
16 mg/L

Additional information

An acute toxicity test with daphnia magna (static) was carried out in accordance with the standardised guidelines OECD 202 and 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).

The batch of material tested was a clear colourless liquid and was not completely soluble in test medium at the loading rate initially prepared. All glassware used during the preparation of test solutions was airtight closed with minimal headspace to minimise vaporisation of test material.

A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium. A final test was performed based on the results of a combined limit/range-finding test.

Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to solutions containing 10, 18, 32, 56 and 100 % of the SS prepared at a loading rate of 100 mg/L. 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.

Samples taken from all test concentrations and the control were analysed. No test material was detected in the control throughout the test. The measured concentrations at the start of the test were 1.5, 2.8, 4.5, 10 and 19 mg/L, respectively. During the exposure period, the concentrations remained stable, i.e. were at 90 - 106 % relative to the initial concentrations at the end of the test. Based on these results, effect parameters were expressed as initially measured concentrations.

No immobility was observed in the control or the four lowest test concentrations throughout the exposure period. At the end of the test, 95 % immobility was observed at the highest test material concentration. The study met the acceptability criteria and was considered valid.

Under the conditions of this study, the 48h-EC50 was 16 mg/L based on measured concentrations (95 % confidence interval between 15 and 18 mg/L).