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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:
19 May - 26 July 2016
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:
Adopted 13 April, 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Swiss Federal Office for the Environment
Analytical monitoring:
yes
Details on sampling:
- Concentrations: each test concentration and the control
- Sampling method: duplicate samples at the start and end of the two 24-hour test medium renewal periods
- Sample storage conditions before analysis: Immediately after sampling, acetonitrile (6 mL acetonitrile per 9 mL sample volume) was added to each sample for stabilization; storage at -20°C
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: stirring/filtration: The test item was mixed in test water at a loading rate of 100 mg/L, and was intensively stirred for 3 hours. After stirring the emulsion was filtered through a 0.45 μm membrane filter. The undiluted filtrate and the dilutions 1:2.2, 1:4.6, 1:10, and 1:22 of the filtrate were used as test media.
- Controls: blank water control
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain/clone: Daphnia magna Straus
- Feeding during test: no feeding
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg CaCO3/L
Test temperature:
21 °C
pH:
7.7 - 7.8 (control)
7.6 - 8.0 (test concentrations)
Dissolved oxygen:
8.2 - 8.5 mg/L (control)
7.9 - 8.5 mg/L (test concentrations)
Nominal and measured concentrations:
nominal: control, 1:22 dilution, 1:10 dilution, 1:4.6 dilution, 1:2.2 dilution, undiluted filtrate (loading rate of 100 mg/L)
measured (arithmetic mean): < 0.069 mg/L, n/a, n/a, 0.12 mg/L, 0.32 mg/L, 0.74 mg/L
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: closed
- Material, size, headspace, fill volume: glass vessels completely filled (without headspace) with 60 mL test medium and tightly sealed with glass stoppers
- Renewal rate of test solution: test medium renewal after 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 20
- No. of vessels per control (replicates): 20

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: test item was diluted with test water which was prepared according to OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000
- Ca/Mg ratio: 4:1
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: 16 h light / 8 h dark with 30 min transition period
- Light intensity: 15 - 18 μmol m-2 s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobility at 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: control, dilution 1:20, dilution 1:5, undiluted filtrate (undiluted filtrate with loading rate of 100 mg/L)
- Results used to determine the conditions for the definitive study: test concentrations and test media preparation in definitive test based on range-finding study
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.41 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CI: 0.32 - 0.53 mg/L
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
0.12 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 0.74 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.12 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none
- Other: Biological and analytical results are summarized within the section "Any other information on results incl.tables"
Results with reference substance (positive control):
- Results with reference substance valid? yes; potassium dichromate is tested as a positive control twice a year
- EC50 (24h): 1.6 mg/L (range given by test guideline: 0.60-2.1 mg/L)
Reported statistics and error estimates:
The 24-hour EC50 could not be calculated due to the low toxicity of the test item (25% immobility at the highest test concentration). The 48-hour EC50 was calculated by Probit Analysis using linear maximum likelihood regression.Statistical analysis was performed using ToxRat Professional®. The 24-hour and 48-hour NOEC, EC0 and EC100 were determined directly from the raw data.

Table 1: Effect of the test material on the mobility of Daphnia magna

Treatment /

Dilution

 

Mean

measured

concentration

[mg/L]

No. of

daphnids

tested

 

Immobilized

daphnids after

24 hours

Immobilized

daphnids after

48 hours

No.

[%]

No.

[%]

Control

--

20

0

0

0

0

Dilution 1:22

n.a

20

0

0

0

0

Dilution 1:10

n.a

20

0

0

0

0

Dilution 1:4.6

0.12

20

0

0

0

0

Dilution 1:2.2

0.32

20

0

0

7

35

Undiluted Filtrate*

0.74

20

5

25

17

85

n.a.: Not analyzed since below the NOEC determined in this test

*: Undiluted filtrate with a loading rate of 100 mg/L

Table 2: ECx values based on mean measured test item concentrations

ECxValues

Immobility

Mean measured concentration [mg/L]

24-hour EC50

>0.74

24-hour EC0

0.32

24-hour EC100

>0.74

48-hour EC50

0.41

(95%-confidence limits)

(0.32 – 0.53)

48-hour EC0and 48-hour NOEC

0.12

48-hour EC100

>0.74*

*: At the undiluted filtrate, 85% of the daphnids were found to be immobile.

Table 3: Analytical Results for Test Samples

Sampling Day / Age of Sample

Dilution Factor of Filtrate of Loading Rate 100 mg Test Item /L

Measured Concentration of Test Item x

Sample Preparation Factor

 

F

Determined Concentration of Test Item

c

% of Initially Measured Concentration

[d/h]

 

[mg/L]

 

[mg/L]

 

0/0

Control

n.d.

1.667

<LOQ

 

(fresh)

1:4.6-Dilution

0.116

1.667

0.193

 

 

1:2.2-Dilution

0.247

1.667

0.412

 

 

Undiluted Filtrate

0.572

1.667

0.954

 

0/24

Control

n.d.

1.667

<LOQ

n.a.

(aged)

1:4.6-Dilution

0.051

1.667

0.084

44%

 

1:2.2-Dilution

0.147

1.667

0.246

60%

 

Undiluted Filtrate

0.410

1.667

0.683

72%

1/0

Control

n.d.

1.667

<LOQ

 

(fresh)

1:4.6-Dilution

0.093

1.667

0.156

 

 

1:2.2-Dilution

0.243

1.667

0.405

 

 

Undiluted Filtrate

0.563

1.667

0.939

 

2/24

Control

n.d.

1.667

<LOQ

n.a.

(aged)

1:4.6-Dilution

0.051

1.667

0.085

55%

 

1:2.2-Dilution

0.152

1.667

0.253

63%

 

Undiluted Filtrate

0.288

1.667

0.480

51%

LOQ: 0.069 mg/L

n.d. = not detected

n.a. = not applicable

Table 4: Mean measured Concentrations based on Analytical Results

Treatment/ Dilution

Measured Test Item Concentations at the Start of the Renewal Periods (Day 0/Day 1)

Measured Test Item Concentations at the End of the Renewal Periods (Day 1/Day 2)

Mean Measured Concentration of the Test Item

[mg/L]

[mg/L]

[mg/L]

Dilution 1:22

n.a.

n.a.

n.a.

Dilution 1:10

n.a.

n.a.

n.a.

Dilution 1:4.6

0.19 / 0.16

0.084 / 0.085

0.12

Dilution 1:2.2

0.41 / 0.41

0.25 / 0.25

0.32

Undiluted Filtrate*

0.95 / 0.94

0.68 / 0.48

0.74

*: Undiluted filtrate with a loading rate of 100 mg/L

n.a.: Not analyzed since below the NOEC determined in this test

Validity criteria fulfilled:
yes
Conclusions:
The test item had acute toxic effects in a 48-hour semi-static test based on immobility of Daphnia magna with an EC50 (48 h) value of 0.41 mg/L (arithmetic mean measured concentration) (OECD 202, GLP).

Description of key information

EC50 (48 h) = 0.41 mg/L (arithmetic mean measured concentration, OECD 202, Daphnia magna)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.41 mg/L

Additional information

There is one GLP guideline study available, which assessed the effects of substance towards aquatic invertebrates (Daphnia magna) according to the OECD guideline 202. As the test item is a volatile substance, the test was performed using glass tubes completely filled with test medium that were tightly sealed with glass stoppers to avoid losses of test item by evaporation. A semi-static test design with a test medium renewal after 24 hours was used. Due to the low water solubility of the test item, the test media were prepared following the filtration method. Therefore the test item was mixed in test water at a loading rate of 100 mg/L, and was intensively stirred for 3 hours. After stirring the emulsion was filtered through a 0.45 μm membrane filter. The undiluted filtrate and the dilutions 1:2.2, 1:4.6, 1:10, and 1:22 of the filtrate were used as test media. The concentration range was previously established in a preliminary range-finding test. The loading rates of the test item were analytically verified by HPLC-UV. At the end of the two renewal periods, the test item concentration were in a range of 44 to 72 % of the initially measured values, demonstrating a decrease of the test item concentrations during the 24-hour renewal periods. The mean measured test item concentrations were calculated as the arithmetic mean of the two geometric means. The EC50 (48 h) value was 0.41 mg/L.

 

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