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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:
17 August 2015 to 19 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
In the vessel A of the control treatment, seven, instead of five, daphnids were exposed. Evaluation: This had no effect on the results. The study integrity was not adversely affected by this deviation.
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
In the vessel A of the control treatment, seven, instead of five, daphnids were exposed. Evaluation: This had no effect on the results. The study integrity was not adversely affected by this deviation.
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
None specified
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter containing undissolved residue was kept for possible analysis.

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.
Samples were sent to the analytical site on dry ice.
Vehicle:
no
Details on test solutions:
Preparation of test solutions
The batch of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] consisted of white to off-white pellets with a purity of 97.8%. The test substance was not completely soluble in test medium at the loading rate initially prepared. The test substance was ground to powder before preparation of test solutions.
Water Soluble Fraction (WSF) was prepared at a loading rate of 100 mg/l applying a two-day period of magnetic stirring. The obtained mixture containing undissolved material was filtered through a 0.45 μm membrane filter (rs55, Whatman) and the filtrate was used as the highest concentrations. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.

Test concentrations
2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]: Solutions containing 1.0, 10 and 100% of WSF prepared at a loading rate of 100 mg/l.
Controls: Test medium without test substance or other additives
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
Post exposure observation period:
No post exposure observation period specified in the study report.
Hardness:
180 mg/l expressed as CaCO3 and the pH: 7.7 ± 0.3.
Test temperature:
The temperature continuously measured in a temperature control vessel varied between 19 and 21°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).
pH:
The results of measurement of pH is 7.9 – 8.1. The test conditions remained within the limits prescribed by the protocol (pH: 6.0-8.5, not varying by more than 1.5 units).
Dissolved oxygen:
The results of measurement oxygen concentrations (mg/l) is 8.7 – 8.9. The test conditions remained within the limits prescribed by the protocol (oxygen: ≥3 mg/l at the end of the test).
Salinity:
Not applicable; freshwater
Nominal and measured concentrations:
Samples taken from the limit concentrations were analysed. The actual concentration measured at the start of the test was 0.037 mg/l. This concentration remained stable during the exposure, i.e. was 0.035 mg/l at the end of the exposure period. Based on these results, the effect parameters were expressed in terms of measured concentrations.
Details on test conditions:
Test procedure and conditions
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 concentration 10 each for the remaining concentrations
Loading: 5 per vessel containing 80 ml of test solution
Light: 16 hours photoperiod daily (05:00 – 21:00)
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnids: Within 7 minutes after preparation of the test solutions.

Measurements and recordings
Immobility (including mortality): At 24 hours and at 48 hours.
pH and dissolved oxygen: At the beginning and at the end of the test, in the control and the highest concentration.
Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.037 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Water Soluble Fraction (WSF) was prepared at a loading rate of 100 mg/l. Concentration relates to this.
Details on results:
Measured concentrations
Samples taken from the limit concentrations were analysed. The actual concentration measured at the start of the test was 0.037 mg/l. This concentration remained stable during the exposure, i.e. was 0.035 mg/l at the end of the exposure period. Based on these results, the effect parameters were expressed in terms of measured concentrations.

Immobility
The responses recorded in this test allowed for reliable determination of an EC50. No immobility was observed in the control and the exposure groups. Therefore, this test was considered a limit.
Results with reference substance (positive control):
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.
Reported statistics and error estimates:
None specified

Number of introduced daphnids and incidence of immobility

Time (h)

Replicate

2,2-Bis[[3-(dodecylthio)-1-oxopropxy]methyl]propane-1,2-diyl bis[3-(dodecylthio)propionate]

%WSF prepared at 100 mg/l

Control

1.0

10

100

0

A

B

C

D

7

5

5

5

5

5

5

5

5

5

5

5

Total introduced

22

10

10

20

24

A

B

C

D

0

0

0

0

0

0 (2)

0

0

0

0

0

0 (1)

Total immobilised

Effect %

0

0

0

0

0

0

0

0

48

A

B

C

D

0

0

0

0

0

0 (2)

0

0

0

0

0

0

Total immobilised

Effect %

0

0

0

0

0

0

0

0

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were re-immersed into the respective solutions before recording of mobility.

 

pH and oxygen concentrations during the final test

2,2-Bis[[3-dodecylthio)-1-oxopropoxy]propane-1,3-diyl bis[3-(dodecylthio)propionate]

Measured concentration (mg/l)

Start (t=0 h)

End (t=48 h)

pH

O2

pH

O2

Control

0.037

7.9

7.9

8.7

8.7

8.1

8.1

8.9

8.9

 

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

0.10

0.18

0.32

0.56

1.0

1.8

20

20

20

20

20

20

20

0

0

0

0

10

95

100

0

0

0

10

100

100

100

0

0

0

0

0

40

100

102

10

10

30

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.

Validity criteria fulfilled:
yes
Conclusions:
The 48h-EC50 was beyond the range tested, i.e. exceeded a measured concentration of 0.037 mg/l being the maximum soluble concentration in medium.
Executive summary:

Acute Toxicity Study in Daphnia magna with 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diylbis[3-(dodecylthio)propionate].

 

The study procedures described in this report were based on the OECD guideline No. 202, 2004. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.2, 2008 and the OECD series on testing and assessment number 23, 2000.

 

The batch of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] consisted of white to off-white pellets with a purity of 97.8%. The test substance was not completely soluble in test medium at the loading rate initially prepared. The test substance was ground to powder before preparation of test solutions.

 

A Water Soluble Fraction (WSF) was prepared at a loading rate of 100 mg/l and used as the highest concentration. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.

 

Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to the undiluted WSF, in a limit test. In addition ten daphnids per group (5 per replicate, duplicate) were exposed to solutions containing 1.0 and 10% of WSF in the combined range-finding test. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

 

Samples taken from the limit concentrations were analysed. The actual concentration measured at the start of the test was 0.037 mg/l and remained stable during the exposure, i.e. was 0.035 mg/l at the end of the exposure period. Based on these results, the effect parameters were expressed in terms of measured concentrations.

 

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

The 48h-EC50 was beyond the range tested, i.e. exceeded a measured concentration of 0.037 mg/l being the maximum soluble concentration in medium.

Description of key information

Key value derived in a GLP accredited laboratory study in accordance with OECD Guideline 202 and EU Method C.2

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

The batch of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] consisted of white to off-white pellets with a purity of 97.8%. The test substance was not completely soluble in test medium at the loading rate initially prepared. The test substance was ground to powder before preparation of test solutions.

 

A Water Soluble Fraction (WSF) was prepared at a loading rate of 100 mg/l and used as the highest concentration. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.

 

Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to the undiluted WSF, in a limit test. In addition ten daphnids per group (5 per replicate, duplicate) were exposed to solutions containing 1.0 and 10% of WSF in the combined range-finding test. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

 

Samples taken from the limit concentrations were analysed. The actual concentration measured at the start of the test was 0.037 mg/l and remained stable during the exposure, i.e. was 0.035 mg/l at the end of the exposure period. Based on these results, the effect parameters were expressed in terms of measured concentrations.

The 48h-EC50 was beyond the range tested, i.e. exceeded a measured concentration of 0.037 mg/l being the maximum soluble concentration in medium.