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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:
24 Feb 2017 - 31 Mar 2017
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:
Adopted April 13, 2004.
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures
Version / remarks:
OECD series on testing and assessment number 23, 2000.
GLP compliance:
yes
Specific details on test material used for the study:
TEST MATERIAL INFORMATION
Identification: Soybean oil, epoxidized, Me ester, reaction products with propylene glycol
Appearance: Yellow liquid (determined by Charles River Den Bosch)
Batch: EM97160711
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions: until 20 July 2017 (expiry date)
Test item: 207811/A
Purity/composition correction factor: No correction factor required
Analytical monitoring:
yes
Remarks:
TOC analysis
Details on sampling:
Single samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 h and t=48 h
Volume: 40 mL from the approximate centre of the test vessels
Storage: Samples were stored in a refrigerator (2-8°C) until analysis.

At the end of the exposure period, the replicates were pooled at each concentration before sampling. Additionally, reserve samples of 40 mL were taken for possible analysis. If not used, these samples were stored in a refrigerator (2-8°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:
no
Details on test solutions:
The batch of Soybean oil, epoxidized, Me ester, reaction products with propylene glycol tested was a yellow liquid UVCB substance and not completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item. Preparation of test solutions started with individually prepared loading rates. A two-day magnetic stirring period was applied to ensure reaching maximum solubility of the test item in medium. The obtained mixtures were then allowed to settle for an overnight period. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. After preparation, volumes of 80 mL were added to each replicate of the respective test concentration. Test solutions were clear and colorless at the end of the preparation procedure, except for the WAF prepared at 100 mg/L that was slightly hazy. Any residual volumes were discarded unless otherwise requested by the Study Director.
Test organisms (species):
Daphnia magna
Details on test organisms:
Test system:
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical 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, young daphnids with an age of < 24 hours were selected from parental daphnids older than two weeks.

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:
no
Total exposure duration:
48 h
Hardness:
180 mg/L expressed as CaCO3
Test temperature:
The temperature continuously measured in a temperature control vessel varied between 19.8 and 20.5°C during the test
pH:
pH t=0h : 7.8 - 8.0
pH t=48h: 7.8
Dissolved oxygen:
O2 t=0h : 8.1-8.9
O2 t=48h: 8.5 - 8.8
Nominal and measured concentrations:
Nominal concentrations: WAFs prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L.

TOC measurements:
WAFs: t=0h: 0.49*, 1.4, 4.3, 7.1,14, 26 mg TOC/L
WAFs: t=48h 0.67*, 1.9, 3.8, 6.8, 12, 24 mg TOC/L

* Values below 1 mg C/L should be considered indicative because of the limit of quantification of the analytical method
Details on test conditions:
Test Concentrations:
Test item: WAFs prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L.
Controls: Test medium without test item or other additives.

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 per 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: Immediately 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, for all concentrations and the control.
Temperature of medium: Continuously in a temperature control vessel, beginning
at the start of the test.

Combined Limit/Range-Finding Test
The project started with a combined limit/range-finding test. Twenty daphnids per concentration (four replicates, 5 daphnids per vessel) were exposed to an untreated control and a WAF prepared at a loading rate of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
•Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to WAFs prepared at 1.0 and 10 mg/L in the combined range-finding test.
•Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration.

- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
K2Cr2O7
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EL0
Effect conc.:
46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Measured concentration:
Measured Carbon concentrations remained fairly stable during the 48-hour test period with average Carbon concentrations at levels corresponding to 36-57% relative to the initial loading rates. Since TOC-analysis is a non-specific method, no further estimations for exposure concentrations were performed. TOC-analysis of the samples taken from the WAF prepared at 100 mg/L showed an initial TOC concentration of 26.0 mg C/L. This concentration remained stable during the 48-hour test period with a measured concentration of 24 mg C/L at the end of the test. Based on this result, it could be anticipated that approximately 36 mg/L of the initial loading of 100 mg/L was actually in solution. Further results will consequently be reported in terms of loading rates and expressed in mg per litre.

Immobility:
The responses recorded at the loading rate of 100 mg/L were slightly below that recorded in the same solution during the combined limit/range-finding test. Possibly, this was due to the fact that a slightly lower Carbon concentration was measured in the final test (25 mg C/L vs 28 mg C/L).

Acceptability of the Test:
1. In the control, no daphnids became immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium.
2. The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.
Results with reference substance (positive control):
The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations (i.e., the 48h-EC50 was between 0.3 and 1.0 mg/L). Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at Charles River Den Bosch.
The 24h-EC50 was 0.64 mg/L with a 95% confidence interval between 0.56 and 0.73 mg/L.
The 48h-EC50 was 0.48 mg/L with a 95% confidence interval between 0.43 and 0.53 mg/L.
Reported statistics and error estimates:
The 24h- and 48h-EL50 could not be determined because the observed effects were below 50%.

Number of Introduced Daphnids and Incidence of Immobility in the Final Test

Time (h)

Replicate

Test item* WAF prep. at loading rate (mg/L)

Control

4.6

10

22

46

100

0

A

5

5

5

5

5

5

B

5

5

5

5

5

5

C

5

5

5

5

5

5

D

5

5

5

5

5

5

Total introduced

20

20

20

20

20

20

24

A

0

0

0

0

0

1

B

0

0

0

0

0

0

C

0

0

0

0

0

0

D

0

0

0

0

0

0

Total immobilised

0

0

0

0

0

1

Effect %

0

0

0

0

0

5

 

 

 

 

 

 

48

A

0

0

0

0

0

 3#

B

0

0

0

0

0

2

C

0

0

0

0

0

2

D

0

0

0

0

0

1

Total immobilised

0

0

0

0

0

8

Effect %

0

0

0

0

0

40

* Test item = Soybean oil, epoxidized, Me ester, reaction products with propylene glycol

#Microscopic observation revealed no test item stuck to the daphnids,

Validity criteria fulfilled:
yes
Remarks:
see details on results
Conclusions:
The 48h-EL50 for Daphnia magna exposed to Soybean oil, epoxidized, Me ester, reaction products with propylene glycol exceeded a WAF prepared at a loading rate of 100 mg/L.
Executive summary:

A full OECDTG 202 GLP test was performed with daphnia magna, based on the results of a preceding range-finding test. Water Accommodated Fractions (WAFs) of Soybean oil, epoxidized, Me ester, reaction products with propylene glycolwere prepared and used as test concentrations. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAFs individually prepared atloading rates of 4.6, 10, 22, 46 and 100 mg/L under static conditions.The total exposure period was 48 hours and samples for Total Organic Carbon (TOC) analyses were taken at the start and at the end of the test. Since TOC-analysis is a non-specific method, no further estimations for exposure concentrations were performed. The results were consequently reported in terms of loading rates and expressed in mg per litre. The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, the 48h EL50 for Daphnia magna exposed to Soybean oil, epoxidized, Me ester, reaction products with propylene glycol exceeded a WAF prepared at a loading rate of 100 mg/L.

Description of key information

A full OECDTG 202 GLP test was performed with Daphnia magna, based on the results of a preceding range-finding test. Water Accommodated Fractions (WAFs) ofSoybean oil, epoxidized, Me ester, reaction products with propylene glycolwere prepared and used as test concentrations. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAFs individually prepared atloading rates of 4.6, 10, 22, 46 and 100 mg/L under static conditions.The total exposure period was 48 hours and samples for Total Organic Carbon (TOC) analyses were taken at the start and at the end of the test. Since TOC-analysis is a non-specific method, no further estimations for exposure concentrations were performed. The results were consequently reported in terms of loading rates and expressed in mg per litre. The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, the 48h EL50 forDaphnia magnaexposed to Soybean oil, epoxidized, Me ester, reaction products with propylene glycol exceeded a WAF prepared at a loading rate of 100 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

The EL50 value is > 100 mg/L and based on loading.