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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:
30 October 2017 - 08 December 2017
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:
2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Solubility in water: 6300.4 mg/L at 24°C
- Stability in water: Not indicated
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations and control.
- Frequency: At the start of the test and after 24 hours from the freshly prepared solutions. At the first renewal (t=24h) and the end of the test from the 24-hour old solutions.
- Sampling method: 15 mL from the approximate center of the test vessels. At the end of each refreshment period, the replicates were not pooled at each concentration before sampling. Instead, samples were taken from replicate 1 of each test concentration.
- Sample storage conditions before analysis: samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
Details on test solutions:
PREPARATION OF TEST SOLUTION
Preparation of test solutions started with loading rates individually prepared at 1.0 to 100 mg/L. A 57- to 74-minutes 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 60 to 63 minutes. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain/clone: Daphnia magna (Straus, 1820)
- Source: In-house laboratory culture with a known history.
- Age of parental stock: >2 weeks old
- Validity of batch: 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.
- Age of test organisms at start of test: <24 h
- Feeding during the test: no

BREEDING:
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
- Temperature of the medium: 18-22 °C
- Feeding: Daily, a suspension of fresh water algae
- Medium: M7 medium
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
180 mg/L expressed as CaCO3
Test temperature:
19 - 21 °C
pH:
7.9 - 8.1
Dissolved oxygen:
8.6 - 11.0 mg/L
Nominal and measured concentrations:
- Nominal concentrations: WAFs individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L and a blank control.
- Measured concentrations (average*): 9.0, 17, 29, 49 and 76 mg/L.

* For more detail on nominal versus measured concentrations, see 'Any other information on materials and methods'.
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 mL, all-glass, air-tight closed vessels with headspace reduced to minimum.
- Aeration: no aeration
- No. of organisms per vessel: 5 daphnids per vessel
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Introduction of daphnids: within 5 minutes after preparation of the test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO Medium, made by dissolving in Reverse-Osmosis purified tap water the following 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).
- Culture medium different from test medium: Yes.
- Intervals of water quality measurement: pH and dissolved oxygen were measured at the beginning, after 24 hours of exposure and at the end of the test, for all concentrations and the control. Temperature of the medium was continuously monitored in a temperature control vessel, beginning at the start of the test. In addition, Temperature of of fresh and old medium was measured upon refreshment.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16h light : 8h dark

EFFECT PARAMETERS MEASURED
- Immobility at 24 h and at 48 h.

RANGE-FINDING STUDY
- Test concentrations: WAFs prepared at loading rates of 1, 10 and 100 mg/L and a blank control.
Reference substance (positive control):
yes
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
21 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: 95% C.I.: 18 - 24 mg/L
Details on results:
- Observations: After 24 hours of exposure, no immobility was observed in the two lowest test concentrations and complete immobilisation in the two highest test concentrations. After 48 hours of exposure, immobilisation increased with increasing concentration at 9.0 to 49 mg/L. Complete immobilisation was observed at 49 and 76 mg/L.
- Mortality of control: None
- Other adverse effects control: At the end of the test, a single daphnid showed signs of stress, i.e. was found trapped at the surface of the test solution. This was accepted since the observation was within the limits of the acceptability criteria.
- Any indication that effect concentrations exceeded the solubility of substance in test medium: No.
Results with reference substance (positive control):
- Results with reference substance valid? Yes.
- Dose-response test: Yes
- Relevant effect levels: 48h-EC50 = 0.41 mg/L (95% C.I.: 0.34 - 0.46 mg/L).
- Other: The actual response was within the ranges of the expected responses (48h-EC50 between 0.3 and 1.0 mg/L). The sensitivity of this batch of D. magna was in agreement with the historical data collected at the Test Facility.
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 item concentrations (average) using the maximum likelihood estimation method. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

DETAIL ON RESULTS

Table: Incidence of Immobility in the final test:

Time (h)

Replicate

Average test concentration (mg/L)

Control

9

17

29

49

76

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

5

5

B

0

0

0 (3)

1

5

5

C

0

0

0

3#

5

5

D

0

0

0

3

5

5

Total immobilised

0

0

0

7

20

20

Effect %

0

0

0

35

100

100

48

A

0

0 (5)

0

4

5

5

B

0

1 (5)

2

5

5

5

C

0

0

0

5

5

5

D

0 (1)

0

1 (1)

5

5

5

Total immobilised

0

1

3

19

20

20

Effect %

0

5

15

95

100

100

( ) between brackets: number of daphnia 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 item attached to the daphnids.

Validity criteria fulfilled:
yes
Remarks:
, see 'Overall remarks'.
Conclusions:
The 48h-EC50 for Daphnia magna was 21 mg/L based on average exposure concentrations (95% confidence interval between 18 and 24 mg/L).
Executive summary:

The short-term toxicity to Dapnids was examined in a study according to OECD TG 202 and in compliance with GLP criteria. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

Twenty daphnids per group (D. magna, 5 per replicate, quadruplicate) were exposed to an untreated control and toWAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L. The total exposure period was 48 hours and test solutions were renewed after 24 hours of exposure. The test was performed in closed vessels without headspace. Samples for analytical confirmation of actual exposure concentrations were taken from freshly prepared and spent solutions in both renewals. Analysis of the samples taken at the start of the test and at the start of the second renewal phase showed concentrations close to the applied loading rates. Theseconcentrations remained stable during the following 24-hour period, i.e. were at 93-103% of the respective initial concentration.Based on these results, theaverage exposure concentrations were calculated to be 9.0, 17, 29, 49 and 76 mg/L in WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L. Dose-related effects were observed after 24 and 48 hours of exposure with complete immobilisation at the two highest test concentrations. The study met the acceptability criteria prescribed by the study plan and was considered valid.

After 24 hours of exposure, no immobility was observed in the two lowest test concentrations and complete immobilisation in the two highest test concentrations. After 48 hours of exposure, immobilisation increased with increasing concentration at 9.0 to 49 mg/L. Complete immobilisation was observed at 49 and 76 mg/L. In conclusion, the 48h-EC50 for daphnids was 21 mg/L based on average exposure concentrations (95% confidence interval between 18 and 24 mg/L).

Description of key information

The short-term toxicity to Dapnids was examined in a study according to OECD TG 202 and in compliance with GLP criteria. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

Twenty daphnids per group (D. magna, 5 per replicate, quadruplicate) were exposed to an untreated control and toWAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L. The total exposure period was 48 hours and test solutions were renewed after 24 hours of exposure. The test was performed in closed vessels without headspace. Samples for analytical confirmation of actual exposure concentrations were taken from freshly prepared and spent solutions in both renewals. Analysis of the samples taken at the start of the test and at the start of the second renewal phase showed concentrations close to the applied loading rates. These concentrations remained stable during the following 24-hour period, i.e. were at 93-103% of the respective initial concentration. Based on these results, the average exposure concentrations were calculated to be 9.0, 17, 29, 49 and 76 mg/L in WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L. Dose-related effects were observed after 24 and 48 hours of exposure with complete immobilisation at the two highest test concentrations. The study met the acceptability criteria prescribed by the study plan and was considered valid.

After 24 hours of exposure, no immobility was observed in the two lowest test concentrations and complete immobilisation in the two highest test concentrations. After 48 hours of exposure, immobilisation increased with increasing concentration at 9.0 to 49 mg/L. Complete immobilisation was observed at 49 and 76 mg/L. In conclusion, the 48h-EC50 for daphnids was 21 mg/L based on average exposure concentrations (95% confidence interval between 18 and 24 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
21 mg/L

Additional information