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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 April 6 to 21, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 4.6, 10, 22, 46 and 100 (% WSF of 100 mg/L loading rate)
- Sampling method: During the definitive test, singular samples for TOC-analysis were taken from all test groups (incubated without algae) according to the schedule presented below:
- Sampling frequency: 0 and 48 h
- Sampling volume: 40 mL
- Sample storage: Samples were stored in a refrigerator until analyses
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Preparation of test solutions started with an aqueous mixture at a loading rate of 100 mg/L. This mixture was magnetically stirred for 48 h and then left to settle overnight. This resulted in a hazy dispersion with a floating layer and precipitate. Subsequently, the hazy water phase was collected by siphoning it through glass wool. This resulted in a slightly hazy solution that was used for testing. The lower test concentrations were prepared by subsequent dilution of the Water Soluble Fraction (WSF). The final test solutions were all clear and colourless except for the undiluted WSF that was slightly hazy.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: Notox in-house culture
- Age at study initiation: <24 h
- Method of breeding: Using daphnids less than 3 d old, by placing about 250 of them into 5L of M7 medium in an all-glass culture vessel.
- Renewal of culture: After 7 d of cultivation half of the medium twice a week
- 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.
- Feeding during test: No feeding


ACCLIMATION
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: Suspension of fresh water algae.
- Feeding frequency: Daily
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
180 mg/L (as CaCO3)
Test temperature:
19.1-19.6 °C (Measured electronically using REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA))
pH:
7.6-7.9
Dissolved oxygen:
8.5-9.3 mg/L
Salinity:
Not reported
Nominal and measured concentrations:
Measured concentration (Time weighted average): 0, 2.0, 4.8, 10 and 21 mg/L containing 0, 10, 22, 46 and 100% WSF (Water Soluble Fraction) , respectively. For 4.6% WSF, TWA concentration was not measured.
For details on measured concentrations versus nominal concentrations, please refer to 'table 1' in the 'Over all remarks' section
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass
- Type (delete if not applicable): Open
- Material, size, headspace, fill volume: 80 mL
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: There were 5 animals/vessel containing 80mL test solution. Loading rate was therefore 1 animal/16 mL test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water purified by Reverse Osmosis
- Metals: M7 medium contained the following elements:
Fe: 0.05 mg/L
Mn: 0.025 mg/L
Cu: 0.0016 mg/L
Zn: 0.0063 mg/L
Co: 0.0025 mg/L

OTHER TEST CONDITIONS
- Photoperiod: 16 hours photoperiod daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobility and mortality were recorded at 24 and 48 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approx. 2
- Range finding study: Yes. Prior to the initiation of definitive exposure, preliminary range-finding test was conducted and data obtained from preliminary exposure was used to select the definitive test concentration. Test procedure and conditions were similar to those applied in the final test with the following exceptions: (1)Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to 0.1, 1.0, and 10% of the WSF in the combined range-finding test. (2) Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration. (3)Samples for determination of actual exposure concentrations were taken from the control and the highest test concentration at the start and the end of the test.
- Test concentrations: 0, 0.1, 1.0, 10 and 100% of the WSF
- Results used to determine the conditions for the definitive study: All daphnids exposed to the undiluted WSF were immobile within 24 h, whereas no significant immobility was observed in any the WSF dilutions during the 48 h test period (See 'Table 1' in the section 'Any other information on results'). Thus, the concentrations of definitive study were selected based on the fact that EC50 should be between 10 and 100% of a WSF prepared at 100 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (20 Daphnia/conc. were exposed for a period of 48 h to K2Cr2O7 at the concentrations of 0, 0.1, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
3.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: (3.3-4.4)
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Following 48 h of exposure, immobility of 100, 100 and 80% was observed in the treatment levels 22, 46 and 100 % WSF of 100 mg/L loading rate respectively. For details please refer to 'Table 2' in the section 'Any other information on results'.
Results with reference substance (positive control):
- Results with reference substance valid: Yes
- EC50:
The 24h-EC50 was 0.75 mg/L with a 95% confidence interval between 0.56 and 1.0 mg/L.
The 48h-EC50 was 0.36 mg/L with a 95% confidence interval between 0.28 and 0.49 mg/L.
These are the results of the most recent reference test conducted with potassium dichromate at the test facility.
Reported statistics and error estimates:
The EC50-value was calculated at 24 and 48 h of exposure from the probits of the percentages of affected daphnids and the logarithms of the corresponding test material concentrations using the maximum likelihood estimation method.

Table 1: Acute immobilisation of daphnids esposed to amides, C8-C18, C18 unsatd.,N-(2-hydroxypropyl) (range finding study)

Test group

 (% WSF of 100 mg/L loading rate)

Vessel number

 

Number

Daphnia

exposed

Response at 24 h

Response at 48 h

 

number

Total

%

 

number

Total

%

Control

A

B

C

D

5

5

5

5

0

0

0

0

0

0

0

0

0

0

       0.1

A

B

5

5

0

0

0

0

0

0

       1.0

A

B

5

5

0

0

0

0

1

10

     10

A

B

5

5

0

0

0

0

1

10

   100

A

B

C

D

5

5

5

5

5

5

5

5

100

5

5

5

5

100

Table 2: Acute immobilisation of daphnids esposed to amides, C8-C18, C18 unsatd.,N-(2-hydroxypropyl) (definite test)

Test group

 (% WSF of 100 mg/L loading rate)

Test group

Average meas. conc.

(mg/L)

Vessel

number

 

Number

Daphnia

exposed

Response at 24 h

Response at 48 h

 

number

Total

%

 

number

Total

%

Control

-

A

B

C

D

5

5

5

5

0 (3)

0

0

0

0

0

0

0

0 (1)

0

4.6

n.m.

A

B

C

D

5

5

5

5

0

0

0

0

0

0

0

0

0

0

10

2.0

A

B

C

D

5

5

5

5

0

0

0

0

0

0

0

0

0

0

22

4.8

A

B

C

D

5

5

5

5

0

1

1

2

20

4

4

4

4

80

46

10

A

B

C

D

5

5

5

5

4

3

2

3

60

5

5

5

5

100

100

21

A

B

C

D

5

5

5

5

4

5

5

4

90

5

5

5

5

100

n.m. Not measured

 ( ) 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.

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the 48 h EC50 was determined to be 3.7 mg/L (3.3-4.4 mg/L). The 48 h NOEC was 2.0 mg/L.
Executive summary:

A study was conducted to evaluate the acute toxicity of the test substance to Daphnia magna under static conditions according to OECD Guideline 202, in compliance with GL¨P. The substance was not completely soluble in test medium at the concentrations tested and hence the preparation included a 2 d magnetic stirring period followed by overnight settlement and subsequent collection of the Water Soluble Fraction (WSF). Twenty daphnia per test group (4 vessels, 5 Daphnia/vessel) were exposed at concentrations of 4.6, 10, 22, 46 and 100% of the WSF prepared at a loading rate of 100 mg/L. The total test period was 48 h. Samples for measurement of Total Organic Carbon (TOC) in solution were taken at the start and the end of the test period. The average exposure concentrations were calculated to correspond to 2.0, 4.8, 10 and 21 mg/L for the test groups containing 10, 22, 46 and 100% WSF, respectively. Under the study conditions, the 48 h EC50 was determined to be 3.7 mg/L (3.3-4.4 mg/L). The 48 h NOEC was 2.0 mg/L (Migchielsen, 2010).

Description of key information

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
3.7 mg/L

Additional information

A study was performed to evaluate the acute toxicity of the test substance to Daphnia magna under static conditions according to OECD Guideline 202. The substance was not completely soluble in test medium at the concentrations tested and hence the preparation included a 2 d magnetic stirring period followed by overnight settlement and subsequent collection of the Water Soluble Fraction (WSF). Twenty daphnia per test group (4 vessels, 5 Daphnia/vessel) were exposed at concentrations of 4.6, 10, 22, 46 and 100% of the WSF prepared at a loading rate of 100 mg/L. The total test period was 48 h. Samples for measurement of Total Organic Carbon (TOC) in solution were taken at the start and the end of the test period. The average exposure concentrations were calculated to correspond to 2.0, 4.8, 10 and 21 mg/L for the test groups containing 10, 22, 46 and 100% WSF, respectively. Under the test conditions, the 48 h EC50 was determined to be 3.7 mg/L (3.3-4.4 mg/L). The 48 h NOEC was 2.0 mg/L (Migchielsen, 2010).