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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:
29 Aug -20 Oct 2016
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/Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods
pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the
Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals
(REACH), C.2, Acute Toxicity for Daphnia
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Swiss federal office of public health; consumer protection directorate; notification authority for chemicals; CH-3003 Bern
Analytical monitoring:
yes
Details on sampling:
- Sampling method: samples were taken at the start and at the end of the two 24-hour test medium renewal periods
- Sample storage conditions before analysis: deep-frozen (at about -20 °C)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Differential loading: highest test concentration (with a loading rate of 100 mg/L) intensive stirring for three hours in the dark in a closed stirring vessel; after stirring the solution was filtered through a 0.45 μm membrane filter (Whatman, NC45 , pre-conditioned with 200 mL filtrate). The undiluted filtrate was used as highest test concentration. It was further diluted with test water to prepare the test media with the lower test concentrations of the dilutions 1:2, 1:4, 1:8 and 1:16.
- Controls: yes
- Others:
At the start of the test (Day 0) and at the test medium renewal (Day 1) the highest test concentration (undiluted filtrate) was prepared with a loading rate of 100 mg/L considering the relative density of the test item of 0.90. The test item was mixed into test water by intensive stirring for three hours in the dark in a closed stirring vessel to dissolve a maximum of test item in test water. The stirring time was based on the stirring pre-experiment which showed, that the maximum amount of dissolved test item was reached after this stirring time.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna Straus
- Age of organisms at test start: 6-24 hours
- Feeding during test: no
- Food type: stock culture was fed with algal suspension of the green algae Desmodesmus subspicatus or a mixture of this algal suspension and a commercial fish diet
- Frequency: stock culture was fed three times a week

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg/L CaCO3; 2.5 mmol/L
Test temperature:
20 and 21 °C.
pH:
7.7 to 7.9 (control)
7.5 to 7.9 (test concentrations)
Dissolved oxygen:
8.5 to 8.8 (control)
8.3 to 8.8 (test concentrations)
Nominal and measured concentrations:
control; 1:16 (not analysed since below the NOEC); 1:8 (not analysed since below the NOEC); 1:4 (0.051 mg/L); 1:2 (0.11 mg/L) and undiluted filtrate (0.24 mg/L) (mean measured concentration of the test item)
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: closed
- Material, size, headspace, fill volume: glass vessels completely filled (without headspace) with 60 mL, tightly sealed with glass stoppers
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Other: for each daphnid 12 mL (60 mL per replicate)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water according to ISO was used (test water was aerated prior to test start until oxygen saturation was achieved)
- Ca/mg ratio: 4:1
- Na/K ratio: 10:1
- Alkalinity: 0.8 mmol/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: the pH values, dissolved oxygen concentrations and water temperature were determined in each treatment at test start and end of the test medium renewal periods

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light to 8-hour dark cycle with a 30-minute transition period
- Light intensity: between 15 and 18 μmol m-2 s-1

EFFECT PARAMETERS MEASURED: immobility of the daphnids was determined by visual inspection after 24 and 48 hours of exposure

RANGE-FINDING STUDY
- Test concentrations: control; 1:20; 1:5 and undiluted filtrate (100 mg/L)
- Results used to determine the conditions for the definitive study: Based on the results of the pre-experiments the following test concentrations for the main test were chosen
Reference substance (positive control):
yes
Remarks:
Potassium chlorid
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits
Remarks:
0.085 - 0.12 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.051 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
0.24 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
0.051 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Mortality of control: no effect was oberved
Results with reference substance (positive control):
- Dose-response test: yes
- ECx: 24-hour EC50 1.0 mg/L
- Other: For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control twice a year. The result of the latest positive control test in October 2016 (IES Laboratories Study 20160278) showed that the sensitivity of the test organisms was within the range given by the guideline (24-hour EC50: 0.60-2.1 mg/L).
Reported statistics and error estimates:
The 48-hour EC50 was calculated by Probit Analysis using linear maximum likelihood regression. Statistical analysis was performed using ToxRat Professional®.

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

Treatment /Dilution

Mean measured concentration

No. of daphnids tested

Immobilized daphnids after 24 hours

Immobilized daphnids after 48 hours

 

[mg/L]

 

No.

[%]

No.

[%]

Control

---

20

0

0

0

0

Dilution 1:16

n.a.

20

0

0

0

0

Dilution 1:8

n.a.

20

0

0

0

0

Dilution 1:4

0.051

20

0

0

0

0

Dilution 1:2

0.11

20

0

0

13

65

Undilutedfiltrate*

0.24

20

14

70

20

100

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

*: Undiluted filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L

Validity criteria

The test is considered to be valid, as in the control no daphnids showed immobilization or other signs of disease or stress. Furthermore, the dissolved oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.

Analytical results:

At the end of the two renewal periods, the test item concentrations were in the range of 70 to 85% of the initially measured values, demonstrating a slightly decrease of the test item concentrations during the 24-hour renewal periods. The mean measured test item concentrations over the test period of 48 hours were calculated as the arithmetic mean of the two geometric means which were determined from the test item concentrations measured at the start and end of each of the two test medium renewal periods.

Table 3: 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.

1.67

<LOQ

-

(fresh)

Dilution 1:4

0.0335

1.67

0.0559

-

 

Dilution 1:2

0.0731

1.67

0.122

-

 

Undiluted Filtrate

0.152

1.67

0.254

-

1/24

Control

n.d.

1.67

<LOQ

n.a.

(aged)

Dilution 1:4

0.0257

1.67

0.0428

77

 

Dilution 1:2

0.0541

1.67

0.0901

74

 

Undiluted Filtrate

0.120

1.67

0.200

79

1/0

Control

n.d.

1.67

<LOQ

-

(fresh)

Dilution 1:4

0.0383

1.67

0.0639

-

 

Dilution 1:2

0.0823

1.67

0.137

-

 

Undiluted Filtrate

0.168

1.67

0.279

-

2/24

Control

n.d.

1.67

<LOQ

n.a.

(aged)

Dilution 1:4

0.0269

1.67

0.0449

70

 

Dilution 1:2

0.0620

1.67

0.103

75

 

Undiluted Filtrate

0.142

1.67

0.237

85

LOQ: 0.017 mg/L

n.d. = not detected

n.a. = not applicable

Validity criteria fulfilled:
yes
Conclusions:
The test item had acute toxic effects on Daphnia magna in a 48-hour semi-static test. The 48-hour EC50 was calculated to be 0.10 mg/L (arithm. mean measured concentration).

Description of key information

EC50 (48h) = 0.10 mg/L (Daphnia magna; OECD 202; arithmetic mean)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.1 mg/L

Additional information

There is one GLP guideline study available, which assessed the effects of the 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 as saturated solution with a loading rate of 100 mg/L. Therefore 100 mg/L test item was mixed in test water, 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, 1:4, 1:8, and 1:16 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 70 to 80% of the initially measured values, demonstrating a slightly decrease of the test item concentrations during the 24 -hour renewal periods. The measured test item concentrations were calculated as the arithmetic mean of the two geometric means. The EC50 (48 h) value was 0.10 mg/L (based on the arithmetic mean).