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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:
1 May 2015 to 8 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to EU, OECD guidelines to a GLP recognised standard
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
Principles of method if other than guideline:
Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples for analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter used to prepare the WSF was retained for analysis of the residue.
Frequency at t=0 h and t=48 h
Volume 2.0 mL from the approximate centre of the test vessels
Storage Samples were stored in a freezer until analysis.
At the end of the exposure period, the replicates were pooled at each concentration before sampling. Additionally, reserve samples of 2.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
yes
Details on test solutions:
Stock and spiking solutions
Stock solutions of the test substance were prepared in acetonitrile at concentrations not corrected for purity of 1000 mg/l.
For spiking, dilutions of a stock solution were used. The solvent of the spiking solutions was acetonitrile.

Calibration solutions
From two stock solutions, five solutions with the test substance in the concentration range not corrected for purity of 10 - 1000 μg/l were prepared in acetonitrile. The solutions were diluted by a factor of 100 with 50/50 (v/v) acetonitrile/ISO-medium to obtain calibration solutions in the concentration range not corrected for purity of 0.1 - 10 μg/l.
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
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.
Characteristics: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.

Breeding
Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres 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).
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
180 mg/l expressed as CaCO3 and the pH: 7.7 ± 0.3.
Test temperature:
Temperature of medium: 18-22°C
pH:
pH: 6.0-8.5
Dissolved oxygen:
≥3 mg/L
Salinity:
Not applicable.
Nominal and measured concentrations:
loading rate of 100 mg/L
Details on test conditions:
1,2-Dioxoethylen)bis(iminoethylen) bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat]: 1.0, 10 and 100% of the WSF prepared at a loading rate of 100 mg/L.
Control Test: medium without test substance or other additives
Test duration: 48 hours
Test type: Static
Test vessels: 100 mL, all-glass
Medium: Adjusted ISO medium
Number of daphnids: 20 each for the control and the highest test concentration 10 for each lower test concentration
Loading: 5 per vessel containing 80 mL of test solution
Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions
Introduction of daphnids: Within 35 minutes after preparation of the test solutions
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7, art. 1.04864, batch no. K44879664) was obtained from Merck, Darmstadt, Germany.
Duration:
48 h
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 48h-EC50 was above the maximum solubility of the test substance in the test medium
Details on results:
The 24 and 48h-EC50 values could not be determined because the observed effects were below 50%.
Results with reference substance (positive control):
The actual responses in this reference test with K2Cr2O7are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50was between 0.3 and 1.0 mg/L. Hence, the sensitivity of the daphnia was within the range determined with the historical data collected at WIL Research Europe
The 24h-EC50was 0.72 mg/L with a 95% confidence interval between 0.64 and 0.82 mg/L.
The 48h-EC50was estimated to correspond with 0.36 mg/L with 10% effect at 0.32 mg/L and 100% effect at 0.56 mg/L.

Number of introduced daphnids and incidence of immobility

Time (H)

Replicate

(1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-

hydroxyphenyl)propionat], % WSF prepared at 100 mg/L

Control

1.0

10

100

0

A

5

5

5

5

 

B

5

5

5

5

 

C

5

 

 

5

 

D

5

 

 

5

 

TOTAL INTRODUCED

20

10

10

20

24         

A

0

0

 

1

 

B

0

0

 

 

 

C

0

 

 

2

 

D

0

 

 

1

 

TOTAL IMMOBILISED 

0

0

0

4

 

Effect%

0

0

0

20

48         

A

0

0

0

2

 

B

0

0

0

0

 

C

0

 

 

2

 

D

0

 

 

1

 

TOTAL IMMOBILISED 

0

0

0

 

5

 

Effect%

0

0

0

25

 

pH and oxygen concentrations

(1,2-Dioxoethylen)bis(iminoethylen)bis[3-

(3,5-di-tert-butyl-4-hydroxyphenyl)propionat]

% WSF prepared at 100 mg//L

Start (t=0 h)

End (t=48 h)

pH

O2

pH

O2

Control

8.0

9.0

8.1

9.1

100

8.0

8.7

8.2

9.1

Acute immobilization of daphnia after 24 and 48 hours in the reference test with potassium

dichromate:

Concentration

K2Cr2O7

(mg/L)

Number

Exposed

% immobile

Expected response (%)

After 48 hours1

24h

48h

Minimal

Maximal

Control

20

0

0

0

102

0.10

20

0

0

0

10

0.18

20

0

0

0

10

0.32

20

0

10

0

30

0.56

20

10

100

0

100

1.0

20

95

100

40

100

1.8

20

100

100

100

100

1Based on historical data of the previous years (n>60).

2A maximum response of 10% does not invalidate the results of the test.

 

The actual responses in this reference test with K2Cr2O7 are within the ranges of the expectedresponses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of the daphnia was within the range determined with the historical data collected at WILResearch Europe.

The 24h-EC50 was 0.72 mg/L with a 95% confidence interval between 0.64 and 0.82 mg/L.

The 48h-EC50 was estimated to correspond with 0.36 mg/L with 10% effect at 0.32 mg/L and 100% effect at 0.56 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
Due to the very low solubility of (1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4- hydroxyphenyl)propionat] in water, concentration levels that might induce ≥50% effects on the mobility of daphnids could not be reached.
The measured concentration in the WSF prepared at a loading rate of 100 mg/L was below the lowest calibration standard of 0.2 μg/L. 25% of the daphnids exposed to this WSF were immobilised. Hence, the 48h-EC50 was above the maximum solubility of the test substance in the test medium.
Executive summary:

The purpose of the study was to evaluate the test substance for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC50 at 24 and 48 hours of exposure. Study was conducted in accordance to:

-OECD guidelines for Testing of Chemicals, guideline No. 202: "Daphnia sp., Acute Immobilisation Test", Adopted April 13, 2004. In addition, the procedures were designed to meet the test methods of the following guideline and guidance document:

-Commission Regulation (EC) No 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C.2. "Daphnia Sp. Acute Immobilisation Test".

-Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000. 

 

In conclusion Due to the very low solubility of (1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4 -hydroxyphenyl)propionat] in water, concentration levels that might induce≥50% effects on the mobility of daphnids could not be reached.

The measured concentration in the WSF prepared at a loading rate of 100 mg/L was below the lowest calibration standard of 0.2 μg/L. 25% of the daphnids exposed to this WSF were immobilised. Hence, the 48h-EC50 was above the maximum solubility of the test substance in the test medium. No classification is applicable.

Description of key information

The 24 and 48h-EC50 values could not be determined because the observed effects were below 50%.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

The purpose of the study was to evaluate the test substance for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC50 at 24 and 48 hours of exposure. Study was conducted in accordance to:

-OECD guidelines for Testing of Chemicals, guideline No. 202: "Daphnia sp., Acute Immobilisation Test", Adopted April 13, 2004. In addition, the procedures were designed to meet the test methods of the following guideline and guidance document:

-Commission Regulation (EC) No 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C.2. "Daphnia Sp. Acute Immobilisation Test".

-Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000. 

 

In conclusion Due to the very low solubility of (1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4 -hydroxyphenyl)propionat] in water, concentration levels that might induce ≥50% effects on the mobility of daphnids could not be reached.

The measured concentration in the WSF prepared at a loading rate of 100 mg/L was below the lowest calibration standard of 0.2 μg/L. 25% of the daphnids exposed to this WSF were immobilised. Hence, the 48h-EC50 was above the maximum solubility of the test substance in the test medium. No classification is applicable.