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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:
From August 2017 to May 2018
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:
April 13,2004.
Deviations:
yes
Remarks:
without any impact on the overall integrity of the study or the interpretation of the study results and conclusions
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Appearance: Pink liquid
- Test item storage: At room temperature protected from light
- Purity/Composition correction factor: No correction factor required
Analytical monitoring:
yes
Details on sampling:
Single samples for possible analysis were taken from all test concentrations, the stock solution and the control according to the schedule below:
- frequency: at t=0 h and t=48 h for all test concentrations and the control; at t=0 h for the undiluted Saturated Solution
- volume: 0.80 mL from the approximate centre of the test vessels;
- storage: samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.
At the end of the exposure, the replicates were not pooled at each concentrations before sampling. Instead, samples were taken from the same replicate at each test concentration.
Vehicle:
no
Details on test solutions:
Preparation of test solutions started with a loading rate of 100 mg/L applying a 3-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of 1 hour. 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 colorless at the end of the preparation procedure.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Species : Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Source: In-house laboratory culture with a known history
- 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

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 medium: 18-22°C
- Feeding: daily, a suspension of fresh water algae.
- Medium: M7
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
180 mg/L (expressed as CaCO3)
Test temperature:
19 - 21°C
pH:
8.0 - 8.3
Dissolved oxygen:
9.2 - 9.7 mg/L
Nominal and measured concentrations:
Nominal concentrations (final test): solutions containing 0 (control), 1, 2.2, 4.6, 10, 22 and 46% of the Saturated Solution prepared at a loading rate of 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 mL, all-glass, airtight closed vessels with no headspace.
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of organisms per vessel: 5 (20 per concentration)
- Feeding : No feeding

TEST MEDIUM / WATER PARAMETERS
- Medium: Adjusted ISO medium

OTHER TEST CONDITIONS
- Photoperiod: test was performed in the dark

EFFECT PARAMETERS MEASURED
- Immobility (including mortality): at 24 hours and at 48 hours

OTHER PARAMETERS MEASURED
- pH and dissolved oxygen: at the beginning and at the end of the test, for all concentrations and the control
- Temperature of medium: continuously in a temperature control vessel, beginning at the start of the test

RANGE-FINDING STUDY
- Range finding study: yes (solutions containing 0 (control), 1, 10 and 100% of a Saturated Solution prepared at a loading rate of 100 mg/L)
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
K2Cr2O7
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.75 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CI 0.60-0.94 mg/L
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CI 1.1-1.3 mg/L
Results with reference substance (positive control):
The responses in the reference test with K2Cr2O7 were within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.28 and 0.90 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at the CRO. The 24h-EC50 was 0.87 mg/L with a 95% confidence interval between 0.80 and 0.95 mg/L. The 48h-EC50 was 0.54 mg/L with a 95% confidence interval between 0.49 and 0.61 mg/L.
Reported statistics and error estimates:
The 24 and 48h-EC50-values were calculated using the Spearman-Karber method (without trim) on the percentages of affected daphnids and the logarithms of the corresponding test item concentrations. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Measured Concentrations

Analysis of the samples taken at the start (t=0) of the final test showed measured concentrations of 0.07, 0.20, 0.29, 0.81, 2.0 and 4.4 mg/L in solutions containing 1.0, 2.2, 4.6,

10, 22 and 46% of the SS, respectively. Additionally, analysis of the sample collected from the undiluted SS showed an initial concentration of 12 mg/L. During the exposure period, the

measured concentrations decreased to 59-87% of initially measured. A small response was observed in the control at the end of the test. However, since a similar response was observed

in the quality control samples, it was most probably due to carryover. It should be noted that the mean accuracies of the quality control samples at the target concentration of 0.01 mg/L

measured at the start of the test fell outside the criterion of 70-110%. Consequently, concentrations lower than 10 mg/L could have been slightly underestimated at the start of the test. Based on these results, the average exposure concentrations were calculated (see below table).

% SS prep. at loading rate of 100 mg/L

Measured concentrations (mg/L )

t=0h

Measured concentrations (mg/L)

t=48h

Average exp. conc.(mg/L)

1.0

0.0669

0.0454

0.055

2.2

0.200

0.118

0.15

4.6

0.292

0.220

0.25

10

0.807

0.549

0.67

22

2.03

1.76

1.9

46

4.41

3.50

3.9

Immobility

No immobility was recorded in the control and at the three lowest test concentrations throughout the test while a dose-related response was observed at the three highest test concentrations (see below table).

 

 

1,4-DIIODOPERFLUOROBUTANE

Average exposure concentration (mg/L)

Time (h)

Replicate

Control

0.055

0.15

0.25

0.67

1.9

3.9

0

A

5

5

5

5

5

5

5

B

4*

5

5

5

5

5

5

C

5

5

5

5

5

5

5

D

5

5

5

5

5

5

5

Total introduced

19

20

20

20

20

20

20

24

A

0

0

0(1)

0(1)

0

5

5

B

0

0

0

0

0

4

5

C

0(1)

0

0

0

0

5(2)

5

D

0

0

0

0

0

5(2)

5(2)

Total immobilised

0

0

0

0

0

19

20

%Immobility

0

0

0

0

0

95

100

48

A

0

0

0

0

1

5

5

B

0

0

0

0

1

5

5

C

0(2)

0

0

0

3

5

5

D

0

0

0

0    

3#

5

5

Total immobilised

0

0

0

0

8

20

20

% Immobility

0

0

0

0

40

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,

*Inadvertently, 4 instead of 5 daphnids were exposed

Acceptability of the Test

1. In the control, 10.5% of the 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. Although the validity criterion was only slightly exceeded, the test is considered valid.

2. The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, the 48h-EC50 for Daphnia magna exposed to the test item was 0.75 mg/L based on average exposure concentrations (95% confidence interval between 0.60 and 0.94 mg/L).
Executive summary:

The acute toxicity of the test item to the freshwater flea Daphnia magna was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 202. Under the conditions of the test, the 48h-EC50 for Daphnia magna exposed to the test item was 0.75 mg/L based on average exposure concentrations (95% confidence interval between 0.60 and 0.94 mg/L).

Description of key information

The acute toxicity of the test item to the freshwater flea Daphnia magna was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 202. Under the conditions of the test, the 48h-EC50 for Daphnia magna exposed to the test item was 0.75 mg/L based on average exposure concentrations (95% confidence interval between 0.60 and 0.94 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.75 mg/L

Additional information

The acute toxicity of the test item to the freshwater flea Daphnia magna was investigated in one GLP-compliant study performed in accordance with standard methods, with minor deviations. The study is considered as reliable (Klimisch 1) and is selected as a key study for the endpoint.