Fatty acids, linseed-oil, reaction products with bisphenol A-epichlorohydrin polymer and glycidyl neodecanoate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 23, 2016 to November 18, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

on the study plan but considered uncritical, signed and assessed by the study director

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

yes

Remarks:

on the study plan but considered uncritical, signed and assessed by the study director

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch no.: #210162718
Purity: 100% as per definition of UVCB
Appearance: brown liquid
Composition: reaction products of linseed-oil fatty acids, 4,4'Methylendiphenyldiglycidylether with neodecanoic fatty acid, oxiranylmethylester

Analytical monitoring:

yes

Remarks:

DOC measurement

Details on sampling:

Because of the low solubility of the test substance in the fish medium, the main experiment could not be performed as it was planned.
A first testing design using the water-accommodated fraction with a nominal loading of 100 mg/L was planned. After shaking the solution for 24 hours and having removed undissolved test substance by filtration over a 0.45 µm Cellulose ester filter, it turned out to be impossible to obtain the necessary volume of 7L of test solution. Several pre-tests for the determination of a more suitable and meaningful testing design were performed.
In order to have a better estimation of the maximal solubility in the test medium, the water accommodated fraction was prepared by weighing the nominal load of 10 g/L, adding the corresponding amount of dilution water and shaking vigorously for 7 days. After membrane filtration through 0.45 µm PTFE filters, the DOC of this solution was measured. This resulted in a measured concentration of 81 mg/L. This value was stated as limit of solubility in the test medium. In the next experiment, it was the intention to use a loading rate of 81 mg/L as high dose. In reality, the solution had been prepared at a concentration of 102 mg/L (because of a calculation error) and was shaken for 7 days. The resulting solution should have been used unfiltered. However, this solution was cloudy and membrane filtration was not possible. Therefore, the test was not started. This wrong concentration doesn’t affect the outcome of the study, as at even lower loading rate (20 mg/L) the resulting solution was still cloudy and membrane filtration was not possible.
An attempt was made to increase the solubility of the test substance by slightly changing the pH value. However, different pH values in the test solutions showed no difference in solubility of the test substance at the loading rates 10 mg/L and 20 mg/L.
In another pre-test, the loading rates 10 mg/L and 20 mg/L were shaken for 7 days. The solution at the loading rate of 20 mg/L was still strongly turbid whereas the solution at the loading rate 10 mg/L was clear. In this solution the DOC was measured in filtered and unfiltered solutions. The measured DOC at the loading rate 10 mg/L was 4.1 mg/L in the filtered solution and 6.8 mg/L in the unfiltered solution. Because of the similar DOC concentration in filtered and unfiltered solutions, it can be assumed, that the test substance was nearly completely dissolved at the loading rate 10 mg/L. As last one, a fish pre-test with the unfiltered solution containing 10 mg/L nominal load was performed. In this pre-test, 3 fishes were used in the treatment and in the control. The solution showed 100 % mortality after 24h. In the control none of the animal died.

Vehicle:

no

Details on test solutions:

The water-accommodated fractions were prepared for the test. This was done by weighing the nominal loads 10, 4.6, 2.2, 1.0 and 0.46 mg/L (real loads 10.1, 4.7, 2.2, 1.0 and 0.5 mg/L), adding the corresponding amount of dilution water and shaking vigorously for 7 days. The resulting solutions were used as test solution without membrane filtration.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species: Danio rerio HAMILTON-BUCHANAN
Origin: The animals were obtained from a commercial supplier. Before being used for the test, the fish were kept for 26 days under test conditions. During this period, mortality did not surpass 5 %.
Supplier: Dehner Garten Center, 67433 Neustadt Date of arrival: 19. Oct. 2016
Age: sexually immature young fish
Length: ca. 2 cm
Husbandry:
Vessels: polyethylene aquaria Medium chlorine-free tap water
Feeding: three times a day with warmwater fishfood and daphnia, totalling about 1-2% of body weight per day
Photo period: 12/12 hours, using neon tubes
Temperature: ca. 23°C

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

1.03 mmol/L

Test temperature:

21.7 - 22.6°C

pH:

7.3 - 8.1

Dissolved oxygen:

7.8 - 9.1 mg O2/L

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

0, 0.46, 1.0, 2.2, 4.6 and 10.0 mg/L (nominal)

Details on test conditions:

- The main experiment for toxicity against Danio rerio was tested using a static test design. The water-accommodated fraction was prepared by weighing the nominal loads 10, 4.6, 2.2, 1.0 and 0.46 mg/L, adding the corresponding amount of dilution water and shaking vigorously for 7 days. The resulting solution was not cloudy and therefore used as test solution without membrane filtration to ensure maximal exposure. The gradient of the concentration-response relationship was very steep. Mortality was observed only in the highest test concentration.
- Experimental Conditions
Loading: 1 fish/L
Vessels: glass aquaria, maximal volume 10L
Aeration: accomplished with glass tubes
Feeding: none
Photo period: 12/12 hours using neon tubes
Temperature: 21.7 – 22.6 °C
pH adjustment: none
Replicates (Treatments): 1 vessel, each containing 7L test solution and 7 fish
Replicates (Blank Control): 1 vessel, containing 7L tap water and 7 fish Observations were made every 24 hours.
- The pH and the concentration of dissolved oxygen (mg/l and % saturation) was measured every 24 hours. The content of the DOC in the test solutions was measured at the beginning and at the end of the test.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mortality (fish)

Remarks on result:

other: based on mortality observed at the highest concentration

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

9.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mortality (fish)

Details on results:

- The toxicity against Danio rerio was tested using a static test design. In the original study design, testing of the water-accommodated fraction using the nominal loading of 100 ± 1 mg/L was planned. After shaking the solution for 24 hours and having removed undissolved test substance by filtration over a 0.45 µm Cellulose ester filter, it turned out to be impossible to obtain the necessary volume of 7L of test solution. Therefore, the study could not be performed with this testing design.
- For determination of a meaningful test design several pre-tests were performed. In the main test the water-accommodated fraction was prepared by weighing the nominal loads 10, 4.6, 2.2, 1.0 and 0.46 mg/L, adding the corresponding amount of dilution water and shaking vigorously for 7 days. The resulting solution was used as test solution without membrane filtration. Mortality was observed only in the highest test concentration. At the start and at the end of the test, the content of the test substance in the test solutions was determined using measurement of dissolved organic carbon (DOC). The measured concentration based on DOC measurement was very low, in line with the low water solubility of the test substance that was observed during the pre-tests. Therefore, no calculation of the test substance concentration based on the organic carbon content of the test substance and DOC measurement was possible. Only in the 2 highest test concentrations (4.6 and 10 mg/L (nominal)), the measured DOC concentration showed the presence of the test substance in the test solutions. Therefore, the determination of the results was based on the nominal concentration. The result of the test was considered valid.
- Fish mortality: 3/7 at 72h and 5/7 at 96h.

Results with reference substance (positive control):

-

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software. The estimation of the biological results was accomplished using the software ToxRat® Professional, version 3.2.1. Biological results were stated using two significant digits.

Sublethal observations / clinical signs:

Validity:

- The mortality in the blank control may not exceed 1 fish at the end of the test. No mortality occurred in the blank control.

- The dissolved oxygen concentration must be at least 60% throughout the test. The concentration of dissolved oxygen was at least 7.6 mg/L or 89 % throughout the test

- The pH-value in the test solutions should not vary by more than 1 unit during the test. The highest variation was 0.7 units.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 96 h LC50 and NOEC (Danio rerio) for the test substance were determined to be 9.2 and 4.6 mg/L (nominal).

Executive summary:

A study was conducted to determine the short-term toxicity of the test substance to fish according to OECD Guideline 203 and EU Method C.1, in compliance with GLP. Danio rerio were exposed to water accomodated fractions (WAFs) prepared at concentrations of 0 (control), 0.46, 1.0, 4.6 and 10 mg/L (nominal load) for 96 h under static conditions. The test concentrations for the main test were determined based on pre-tests, where solubility of the test substance in the fish medium was observed as the main challenge. The samples for analytical confirmation of actual exposure concentrations were taken at the start and at the end of the test and were measured for dissolved oxygen contents. The measured concentration based on DOC measurement was very low, in line with the low water solubility of the test substance. Therefore, no calculation of the test substance concentration based on the organic carbon content of the test substance and DOC measurement was possible. Only in the 2 highest test concentrations (4.6 and 10 mg/L (nominal)), the measured DOC concentration showed the presence of the test substance in the test solutions. Therefore, the determination of the results was based on the nominal concentration. The result of the test was considered valid. Mortality was observed only in the highest test concentration. Three out of seven fish and five out of seven fish were found dead after 72 and 96 h, respectively. Under the study conditions, the 96 h LC50 and NOEC (Danio rerio) for the test substance were determined to be 9.2 and 4.6 mg/L (nominal) (Mucke, 2017).

Description of key information

The 96 h LC50 (Danio rerio) for the test substance was determined to be 9.2 mg/L (nominal).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

9.2 mg/L

Additional information

A study was conducted to determine the short-term toxicity of the test substance to fish according to OECD Guideline 203 and EU Method C.1, in compliance with GLP.Danio rerio were exposed to water accomodated fractions (WAFs) prepared at concentrations of 0 (control), 0.46, 1.0, 4.6 and 10 mg/L (nominal load) for 96 h under static conditions. The test concentrations for the main test were determined based on pre-tests, where solubility of the test substance in the fish medium was observed as the main challenge. The samples for analytical confirmation of actual exposure concentrations were taken at the start and at the end of the test and were measured for dissolved oxygen contents. The measured concentration based on DOC measurement was very low, in line with the low water solubility of the test substance. Therefore, no calculation of the test substance concentration based on the organic carbon content of the test substance and DOC measurement was possible. Only in the 2 highest test concentrations (4.6 and 10 mg/L (nominal)), the measured DOC concentration showed the presence of the test substance in the test solutions. Therefore, the determination of the results was based on the nominal concentration. The result of the test was considered valid. Mortality was observed only in the highest test concentration. Three out of seven fish and five out of seven fish were found dead after 72 and 96 h, respectively. Under the study conditions, the 96 h LC50 and NOEC (Danio rerio) for the test substance were determined to be 9.2 and 4.6 mg/L (nominal) (Mucke, 2017)