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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:
March 25 - June 13, 2019
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: OECD Series on Testing and Assessment No. 23 (Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals)
Version / remarks:
2nd edition; 2019
GLP compliance:
yes (incl. certificate)
Remarks:
Swiss Federal Office of Public Health, Consumer protection directorate, Notification authority for chemicals, Bern, Switzerland
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The control and loading rates of 12.5, 25, 50 and 100 mg/L were analysed analytically. The samples of the lowest loading rate (6.25 mg/L) were not analyzed since this loading rate was below the 48-hour NOEL determined in this test and were therefore not relevant for the interpretation of the biological results.
- Sampling method: Duplicate samples were taken from each loading rate and the control at the start and end of the test. For sampling from the aged test media, the contents of the respective replicates were combined prior to sampling.
- Sample storage conditions before analysis: All samples were stored frozen (at -20 ± 5 °C) immediately after sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Water Accommodated Fractions (WAFs) of the test item with the loading rates of 6.25, 12.5, 25, 50 and 100 mg/L were prepared. The five individual emulsions were intensively stirred for 3 hours to dissolve the different components of the test item as far as possible within a reasonable time period. After stirring the emulsions were let to settle for 30 minutes and thereafter the lower phases were filtered through a 0.45 µm membrane filter. The filter was saturated by at least 200 mL test medium and the negative pressure of the filtration unit was reduced to a minimum to avoid losses of the volatile test item. These undiluted filtrates were tested as WAFs.
- Eluate: no
- Differential loading: yes
- Controls: yes, test water without test item
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: in-house culture (originally obtained from the Daphnia Collection of the University of Basel/Switzerland in 2015)
- Feeding during test: no
- Age: 6-24 h

BREEDING
- Breeding conditions (same as test or not): same as test
- Type and amount of food: algal suspension of the green algae Desmodesmus subspicatus supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Germany) and cultivated at the laboratory under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, Melle, Germany)
- Feeding frequency: three times a week

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg CaCO3/L
Test temperature:
22 °C
pH:
7.7 - 8.1
Dissolved oxygen:
8.0 - 8.2 mg/L (0 h)
8.0 - 8.6 mg/L (48 h)
Nominal and measured concentrations:
nominal loading rates: control, 6.25, 12.5, 25, 50, 100 mg/L
measured (0 h): < LOQ, not measured, 3.0, 9.1, 15, 19 mg/L
measured (48 h): < LOQ, not measured, 2.6, 8.0, 13, 16 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: beakers
- Type: sealed with glass stoppers
- Material, size, headspace, fill volume: glass, 100 mL, without headspace, fill volume: 60 mL
- Aeration: no
- 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: reconstituted test water according to guideline

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light to 8-hour dark cycle with a 30-minute transition period
- Light intensity: 16 - 17 µmol m-2 s-1

EFFECT PARAMETERS MEASURED
- Mobility after 24 h and 48 h

RANGE-FINDING STUDY
- Test concentrations: control, 1.0, 10, 100 mg/L loading rate
- Results used to determine the conditions for the definitive study: 100 % immobilisation at WAF loading rate of 100 mg/L
Reference substance (positive control):
yes
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % CI: 26-34 mg/L
Duration:
48 h
Dose descriptor:
EL0
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EL100
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Immobilisation of control: 0 %
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50 (24 h): 1.5 mg/L (tested April 2019)
- Other: sensitivity of the test organisms was within the internal historical range (24-hour EC50 from 2015 to 2019: 0.78-1.60 mg/L)
Reported statistics and error estimates:
The 24- and 48-hour EL50 and the 95 % confidence limits were calculated by Trimmed Spearman-Karber method for estimating median lethal concentrations, since the estimation based on the Probit or logit models was not be valid (p(F) > 0.05).
Statistical analysis was performed using ToxRat Professional®. The 24-hour and 48-hour NOEL, EL0 and EL100 were determined directly from the raw data.

Table 1: Mobility of Daphnia magna after 24 and 48 h

Treatment

 

Loading Rate

No. of
Daphnids
Tested

Immobilized
Daphnids after
24 Hours

Immobilized
Daphnids after
48 Hours

[mg/L]

 

No.

[%]

No.

[%]

Control

20

0

0

0

0

6.25

20

0

0

0

0

12.5

20

0

0

0

0

25

20

0

0

5

25

50

20

4

20

20

100

100

20

20

100

20

100

Table 2: Measured concentrations of WAF loading rates

Sampling Day/ Sample Age

Water Accomodated Fraction Loading Rate mg Test Item/L

Measured Concentration
 of Test Item
x

Sample Preparation Factor
F

Determined Concentration of Test Item
c

% of Initially Measured

[d/h]

 

 

[mg/L]

[%]

0/0

Control

n.d.

0.1

<LOQ

-

(fresh)

12.5

30.2

0.1

3.02

-

 

25

90.6

0.1

9.06

-

 

50

147

0.1

14.7

-

 

100

190

0.1

19.0

-

2/48

Control

n.d.

0.1

<LOQ

n.a.

(aged)

12.5

26.3

0.1

2.63

87

 

25

79.7

0.1

7.97

88

 

50

128

0.1

12.8

87

 

100

155

0.1

15.5

82

Validity criteria fulfilled:
yes
Conclusions:
The test item had acute toxic effects on Daphnia magna in a 48-hour static test. The 48-hour EL50 was calculated to be 30 mg/L (calculation based on loading rates), with 95% confidence limits of 26 and 34 mg/L.

Description of key information

EL50 (48 h): 30 mg/L (nominal, OECD 202)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
30 mg/L

Additional information

One experimental study is available investigating the short-term toxicity of the test item to aquatic invertebrates. The acute toxicity of the test item to Daphnia magna was determined in a 48-hour static test according to OECD 202 (2004) under GLP conditions. Since the test item is a complex mixture consisting of several constituents with differently low solubility, Water Accommodated Fractions (WAFs) with the loading rates of 6.25, 12.5, 25, 50 and 100 mg/L were tested to assess the toxicity of the test item to daphnids. Additionally, a control group (test water without test item) was tested in parallel. For preparation of the test media, five individual emulsions with the corresponding test item loading rate were intensively stirred for 3 hours to dissolve the different components of the test item as far as possible within a reasonable time period. After stirring the emulsions were let to settle for 30 minutes and thereafter the lower phase was filtered through a 0.45 µm membrane filter. These undiluted filtrates were tested as WAFs. The test item gave a chromatographic profile consisting of several peaks. The analytical measurements of the soluble components were based on the sum of these three peaks. At the start of the test, the measured concentrations of the analyzed components of the test item in the test media with loading rates of 12.5, 25, 50 and 100 mg/L were 3.0, 9.1, 15and 19 mg/L, respectively. The measured concentrations at the end of the test were in the range of 82 and 88 % of the initially measured values. The 48-hour EL50 was calculated to be 30 mg/L with 95% confidence limits of 26 and 34 mg/L. Since WAFs were tested, all effect values were based on the nominal loading rates of the test item. The preparation of the WAFs and procedure of evaluation of data of WAFs is based on recommendations of the OECD Guidance Document No. 23 on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, 2019.