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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 February 06, 2012 to April 05, 2012
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 (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all concentrations.
- Sampling method: On the day of analysis, the samples were defrosted at room temperature. The test samples were diluted in a 1:3 (v:v) ratio with acetonitrile and analysed. If necessary, the samples were further diluted with 75/25 (v/v) acetonitrile/ISO-medium to obtain concentrations within the calibration range.
- Sample storage conditions before analysis: The samples were stored in the freezer (≤ -15°C).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test substance was not completely soluble in test medium at the initial loading rates prepared. Preparation of test solutions started with individually prepared loading rates between 1.0 and 100 mg/L that were magnetically stirred for a period of ~22 hours. The resulting aqueous mixtures were then left to settle for 2 hours. The resulting Water Accommodated Fractions (WAFs) were then collected by means of siphoning the middle fractions. The final test solutions were in general slightly hazy for the WAFs of 22 mg/L and higher and appeared clear and colourless at WAFs prepared up to 10 mg/L.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnids.
- Source: In-house laboratory culture with a known history.
- Age at study initiation (mean and range, SD): young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.
- Method of breeding:
Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 L 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, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).
- Feeding during test: no feeding.

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
180 mg CaCO3/L
Test temperature:
19.5 - 20.4ºC
pH:
7.6 - 8.0
Dissolved oxygen:
8.5 - 9.3 mg O2/L
Nominal and measured concentrations:
WAFs prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL
- Material, size, headspace, fill volume: all-glass; 80 mL
- Aeration: No aeration of the test solutions.
- Renewal rate of test solution (frequency/flow rate): renewal of test solutions after 24 hours.
- No. of organisms per vessel: 5 daphnids.
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 5 per vessel containing 80 mL of test solution

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement:
pH and dissolved oxygen: At the beginning, after 24 hours of exposure 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.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours photoperiod daily.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (including mortality): At 24 hours and at 48 hours.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: The expected EC50 was between concentrations present in WAFs prepared at 10 and 100 mg/L.
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
>= 10 - <= 22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
13 mg/L
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: (95% confidence interval 12 - 15 mg/L). The average exposure concentrations were calculated as mathematical means of the concentrations of test item measured during the first and the last refreshment period.
Details on results:
- Immobility: Significant immobility was observed at loading rates of 22 mg/L and higher.

Table1. Acute immobilisation of daphnids after 26 and 48 hours in the final test.

Loading rate

WAF*

(mg/l)

Average measured

(mg/l)

Vessel

number

 

Number

Daphnia

exposed

Response at 26 h

Response at 48 h

 

number

Total

%

 

number

Total

%

Control

Control

A

B

C

D

5

5

5

5

0

1

0

0

5

0

1

1

0

10

4.6

3.5

A

B

C

D

5

5

5

5

0

0

0

0

0

0

0

0

0

0

10

9.2

A

B

C

D

5

5

5

5

0

0

0

0

0

1

0

0

0

5

22

17

A

B

C

D

5

5

5

5

0

0

2

1

15

5

4

5

3

85

46

42

A

B

C

D

5

5

5

5

3

3

3

3

60

5

5

5

5

100

100

98

A

B

C

D

5

5

5

5

5

5

4

5

95

5

5

5

5

100

* WAF prepared at the given Glycerides, coco mono- and di-, ethoxylated loading rate.

Table 2. Effect parameters.

Parameter

 

Loading rate

WAF* (mg/l)

 

Average measured conc. (mg/l)

95%-confidence interval (mg/l)

NOEC

10

9.2

-

26h-EC50

>22, <46

35

26-45

48h-EC50

>10, <22

13

12-15

* WAF prepared at the given Glycerides, coco mono- and di-, ethoxylated loading rate.

Validity criteria fulfilled:
yes
Remarks:
(In the control, no more than 10% of the daphnids became immobilised; the oxygen concentration at the end of the test was ≥ 3 mg/L in control and test vessels).
Conclusions:
The 48h-EC50 was between WAFs prepared at loading rates of 10 and 22 mg/L. The 48h-EC50 based on average exposure concentrations corresponded to 13 mg/L (95% confidence interval between 12 and 15 mg/L).
Executive summary:

Assessment of acute toxic effects on the mobility of Daphnia magna with Glycerides, coco mono- and di-, ethoxylated (Semi-static Method) was determinated according to the OECD Guideline 202, with GLP.

A final test was performed with daily renewal of test solutions based on the result of a preceding static combined limit/range-finding test. Twenty daphnia per test group (5 per vessel, 4 vessels) were exposed to a control and WAFs prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L. Samples for analytical confirmation of actual exposure concentrations were taken from freshly prepared solutions at the start and after 24 hours of exposure and from 24-hour old solutions at 24 and 48 hours of exposure.

 

Analysis of the samples taken during the two renewal periods showed that measured concentrations in freshly prepared solutions were close to the expected concentrations based on the loading rates and that concentrations generally decreased slightly over the two 24-hour test periods.Based on these results, the average exposure concentrations were calculated. The range tested based on average exposure concentrations was 3.5, 9.2, 17, 42 and 98 mg/L.

 

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

The test substance not induce significant acute immobilisation of Daphnia magna at or below a WAF prepared at a loading rate of 10 mg/L. This corresponded to an average measured exposure concentration of 9.2 mg/L (NOEC).

 

The 48h-EC50was between WAFs prepared at loading rates of 10 and 22 mg/L. The 48h-EC50based on average exposure concentrations corresponded to 13 mg/L (95% confidence interval between 12 and 15 mg/L).

Description of key information

Key study: Experimental results: OECD Guideline 202 and EU method C.2. GLP study.
The 48h-EC50 was between WAFs prepared at loading rates of 10 and 22 mg/L. The 48h-EC50 based on average exposure concentrations corresponded to 13 mg/L (95% confidence interval between 12 and 15 mg/L).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
13 mg/L

Additional information

Key study: Experimental results: OECD Guideline 202 and EU method C.2. GLP study.

The 48h-EC50 was between WAFs prepared at loading rates of 10 and 22 mg/L. The 48h-EC50 based on average exposure concentrations corresponded to 13 mg/L (95% confidence interval between 12 and 15 mg/L).