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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
March-June 2015
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:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test item: SE7B
Chemical name: Estolide Dimer, Trimer and Tetramer
Origin: Biosynthetic Technologies
Lot No.: 0000140602
CAS-No.: 1365345-64-7
Purity: 98%
Analytical monitoring:
yes
Details on sampling:
Chemical analysis of the test item was performed at the test site BiochemA GmbH. Samples
were taken in duplicate from the test loading rate and the control at 0 h, 24 h, 48 h, 72 h and
96 h. With every sampling 2 x 4 mL were taken out of the control and test vessels and filled
into glass vials. Samples were kept in the refrigerator at 5 ± 3°C until being sent to the
analytical laboratory. Since the test item adsorbs to glass surfaces, acetone was used to
rinse-off the test item from the glass vial directly prior analytical analysis. A retain sample
was kept in the refrigerator until finalization of the study. Proper departure and arrival of the
samples was documented
Vehicle:
not specified
Details on test solutions:
Stock solutions (synthetic water)
DF1 Calcium chloride dihydrate e.p. CaCl2 x 2 H2O 294 g/L (2.0 mol/L)
DF2 Magnesium sulfate heptahydrate p.a MgSO4 x 7 H2O 123.3 g/L (0.5 mol/L)
DF3 Potassium chloride p.a. KCl 2.3 g/L (0.03 mol/L)
DF4 Sodium hydrogen carbonate p.a. NaHCO3 25.9 g/L (0.3 mol/L)
The synthetic water is prepared by adding 10 mL of solutions DF1 and 2, 25.5 mL of
solutions DF3 and DF4, and adding deionised water up to 10 litres (final concentrations:
DF1: 2.0 mmol/L, DF2: 0.5 mmol/L, DF3: 0.07 mmol/L and DF4: 0.76 mmol/L)
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
The test fish were healthy and showed no abnormal appearance. They had a total length of 2 - 3 cm. The fish were held in the Hydrotox laboratory since 27 January 2015. The fish were purchased from Aquarium Dietzenbach Fischzucht, van-Hevesy Str. 1a, D-63128 Dietzenbach.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Test temperature:
Between 20.4 to 23.0°C
pH:
pH Range 7.3 - 7.7
Dissolved oxygen:
The oxygen concentration in the test and control beakers ranged from 8.1 to 8.5 mg/L.
Nominal and measured concentrations:
A static limit test with the nominal loading rate of 1000 mg/L was performed. As the test item is a mixture of not readily soluble substances,
results are reported as nominal loading rates and no conversion to effective concentration can be given.
Details on test conditions:
A limit test with the loading rate 1000 mg/L was performed. The loading rate was prepared by adding 3500 mg of the test item into 3500 mL synthetic fish water (prepared according to OECD 203). To obtain 7000 mL of the control and test solutions, three times 3500 mL were set up for the 1000 mg/L loading rate and two times 3500 mL were set up for the control (synthetic fish water). The loading rate and control medium were stirred for 24 h at 21.0 –22.8°C with an overhead high-grade stainless steel radial impeller (about 500 rpm) obtaining a depth of the vortex of about 1/3 of the total height of the liquid. After stirring, 7000 mL of the test solution was transferred into a glass aquarium by siphoning from the middle fraction of the loading rate. Seven fish each were added to the control and test aquarium. Oxygen supply was ensured by aeration with air pumps.
The oxygen and temperature of the test and the control treatments were measured daily, the pH at the beginning and end of the test. The behavior of the fish was recorded on a daily basis. In the first 3 h of the test two additional observations were performed.
During the test the fish were kept at a light/dark cycle of 14:10. Temperature was in the range of 21.8 – 23.3°C. The fish were not fed during the course of the study.
Reference substance (positive control):
not specified
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
All fish survived the exposure period of 96 h. The LCx values are thus
LLR50 (96h, nominal) > 1000 mg/L
LLR0 (96h, nominal) > 1000 mg/L
LLR100 (96h, nominal) > 1000 mg/L
Substance-specific chemical analysis was based on the detection of the Estolide Dimer component of the test item, which makes up about 94% of the mixture. Measured test concentrations of the dimer compound ranged between 2.10 mg/L to 9.91 mg/L in the
loading rate of 1000 mg/L. As the test item is a mixture of not readily soluble substances, results are reported as nominal loading rates and no conversion to effective concentration can be given.
Sublethal observations / clinical signs:

Results table attached

Validity criteria fulfilled:
yes
Conclusions:
A static limit test with the nominal loading rate of 1000 mg/L was performed with Danio rerio.
All fish survived the exposure period of 96 h. The LCx values are thus
LLR50 (96h, nominal) > 1000 mg/L
LLR0 (96h, nominal) > 1000 mg/L
LLR100 (96h, nominal) > 1000 mg/L
Executive summary:

A static limit test according to OECD guideline 203 with the nominal loading rate of 1000 mg/L was performed withDanio rerio.


Fish did not show any abnormal behavior in the control and test treatment. The oxygen concentration in the test and control beakers ranged from 8.1 to 8.5 mg/L. The maximal difference in pH between start and end of the experiment was 0.3 pH units. The temperature ranged from 21.8 to 23.3°C during the test period.

All fish survived the exposure period of 96 h. The LCx values are thus

LLR50 (96h, nominal) > 1000 mg/L

LLR0 (96h, nominal) > 1000 mg/L

LLR100 (96h, nominal) > 1000 mg/L

Substance specific analysis was performed at the test site and was based on the detection of dimer component, which makes up about 94% of the original test item.Measured test concentrations were low ranging from 2.10 mg/L to 9.91 mg/L in the loading rate of 1000 mg/L. This confirms the poor solubility of the test substance in the test medium.

The variability of measured values was high probably due to adsorption effects in the test vessels and the application system of the MS, the latter one leading to memory effects in the control medium.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 April 2013 - 24 June 2013
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:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from the control and the 100 mg/L loading rate WAF at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media). Duplicate samples, and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media), were taken and stored at approximately -20 ºC for further analysis if necessary.
Vehicle:
yes
Details on test solutions:
The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in-house since 10 April 2013. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 9 May 2013 to 21 May 2013. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

The water temperature was controlled at approximately 14 °C with a dissolved oxygen content of greater than or equal to 9.5 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 4.4 cm (sd = 0.3) and a mean weight of 1.09 g (sd = 0.28) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.38 g bodyweight/liter.

The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.
Hardness:
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3.
Test temperature:
The test was conducted ata temperature of between 14 and 15 deg C.
pH:
The pH was measured to be between 7.9 and 8.3 during the cours of the study, in both the control and test vessels.
Dissolved oxygen:
The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.
Salinity:
Not applicable as a fresh water study was conducted
Nominal and measured concentrations:
A limit test was conducted with the test substance being tested at a nominal concentration of 100 mg/L, please note WAFs were used throughout the study.

Samples of the control and the 100 mg/L loading rate WAF were taken at 0 and 72 hours (fresh media) and 24 and 96 hours (old media) for Total Organic Carbon (TOC) analysis. Analysis of the fresh media for the 100 mg/L loading rate WAF showed measured concentrations of between less than the control and 0.19 mg C/L. Analysis of the old or expired media showed measured concentrations of between less than the control and 0.21 mg C/L. The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.
Details on test conditions:
Experimental preparation:
An amount of test item (2100 mg) was added to the surface of 21 liters of dechlorinated tap water to give the 100 mg/L loading rate. After the addition of the test item, the dechlorinated tap water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF.

Total Organic Carbon (TOC) analysis was performed on the test solutions at 0 and 72 hours (fresh media) and 24 and 96 hours (old media).

TEST SYSTEM
As in the range-finding test, 20 liter glass exposure vessels were used for each test concentration. At the start of the test 7 fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at 14 °C to 15 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.

The control group was maintained under identical conditions but not exposed to the test item.

Data from the control group was shared with similar concurrent studies.

A semi-static test regime was employed in the test involving a daily renewal of the test preparations to ensure that the concentrations of the test item remained near nominal and to prevent the build-up of nitrogenous waste products.

TEST MEDIUM / WATER PARAMETERS
Information on water quality can be found within appendix 2 which has been attached in the attached background

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable
- Justification for using less concentrations than requested by guideline: In the range-finding test fish were exposed to a single nominal loading rate of 100 mg/L as the results of the Acute Toxicity to Daphnia (Harlan Study Number 41301081) indicated that toxicity was not expected at this level.

Range finding study
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test a "Limit test" was conducted at a single loading rate of 100 mg/L to confirm that no mortalities or sub-lethal effects of exposure were observed.
Reference substance (positive control):
yes
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
Range finding test:
Cumulative mortality data from the exposure of rainbow trout to the test item during the range-finding test are given below:

cumulative mortality
Nominal Loading Rate (mg/L) 3 hours 6 hours 24 hours 48 hours 72 hours 96 hours
Control 0 0 1 1 2 2
100 0 0 0 0 0 0

The results showed no mortalities at 100 mg/L loading rate WAF. Whilst mortalities were observed in the control, this was not considered to affect the integrity of the result obtained as no mortalities were observed in the 100 mg/L loading rate WAF.
Based on this information, a single loading rate of 100 mg/L using a stirring period of 23 hours followed by a 1-Hour standing period was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no mortalities or sub-lethal effects of exposure were observed.
Sublethal observations / clinical signs:

Definitive Test:

Mortality Data:

Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in the table below:

Nominal

Loading Rate

(mg/L)

Cumulative Mortality (initial population = 7)

% mortality

3 hours

6 hours

24 hours

48 hours

72 hours

96 hours

96 h0urs

Control

0

0

0

0

0

0

0

100

0

0

0

0

0

0

0

 

There were no mortalities in 7 fish exposed to a 100 mg/L loading rate WAF for a period of 96 hours. Inspection of the mortality data gave the following results:

Time (h)

LL*50 (mg/L Loading Rate (WAF)

3

>100

6

>100

24

>100

48

>100

72

>100

96

>100

There were no sub-lethal effects of exposure observed in the test.

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LL*50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.
Executive summary:

Introduction:

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Methods:

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, seven fish were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L for a period of 96 hours at a temperature of 14 °C to 15 ºC under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results:

The 96-Hour LL*50 based on nominal loading rates was greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Samples of the control and the 100 mg/L loading rate WAF were taken at 0 and 72 hours (fresh media) and 24 and 96 hours (old media) for Total Organic Carbon (TOC) analysis. Analysis of the fresh media for the 100 mg/L loading rate WAF showed measured concentrations of between less than the control and 0.19 mg C/L. Analysis of the old or expired media showed measured concentrations of between less than the control and 0.21 mg C/L.

The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Endpoint:
short-term toxicity to fish
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Read-Across Justification is attached below.
Reason / purpose for cross-reference:
read-across source
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Conclusions:
Read-Across is claimed between BT4 (target) and SE7B (Source), according to the justification attached to the target record, and based on structural and physical/chemical similarities.

Analogue diesters (SE7B, SE6B) and BT4 contain the same functional groups, i.e the ester group adjacent to the ethylhexane side chain, and the ester group at the opposite end of the molecules. The carbon range in the main backbone of the molecules is all the same (C18) though the acetate moiety is attached at slightly different positions (C12 for BT4, C9/10 for the analogue diesters). The analogue diesters have the additional alkane chain attached to the acetate cap. The alkane chains themselves are not typically considered to be functional groups, per se, as they are relatively inactive biologically. Thus the parent molecules BT4 and the analogue diesters are similar enough to allow for read across in that there are no differences with respect to functional groups, and their only real difference is number of, and length of, saturated hydrocarbon chains.

The Water Solubility of SE7B was measured as 0.778 mg/L @ 30 °C, whereas BT4 is found to be not stable in water but forms micelle structures resulting in suspension.

Hydrolysis of BT4 would yield acetic acid plus 12-hydroxystearic acid (C18), versus either lauric acid (C12) or the coconut oil fatty acid mixture (C8-18) for SE6B and SE7B, respectively.
These substances are not expected to persist in the environment, consistent with the hazard assessment presented in the OECD SIDS (2009) for the category “Aliphatic Acids Category” where aliphatic fatty acids with a carbon chain length in the range of C8 – C22 were judged to be readily biodegradable.

The acute toxicity of the test item SE7B to the freshwater fish rainbow trout (Oncorhynchus mykiss) gave a 96-Hour LC50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF. Considering the structural similarity and low soubility of BT4, this result is also considered relevant for the read-across target BT4.

Description of key information

Key value for chemical safety assessment

Additional information

Read-Across is claimed between BT4 (target) and SE7B (Source), according to the justification attached to the target record, and based on structural and physical/chemical similarities.

Analogue diesters (SE7B, SE6B) and BT4 contain the same functional groups, i.e the ester group adjacent to the ethylhexane side chain, and the ester group at the opposite end of the molecules. The carbon range in the main backbone of the molecules is all the same (C18) though the acetate moiety is attached at slightly different positions (C12 for BT4, C9/10 for the analogue diesters). The analogue diesters have the additional alkane chain attached to the acetate cap. The alkane chains themselves are not typically considered to be functional groups, per se, as they are relatively inactive biologically. Thus the parent molecules BT4 and the analogue diesters are similar enough to allow for read across in that there are no differences with respect to functional groups, and their only real difference is number of, and length of, saturated hydrocarbon chains.

The Water Solubility of SE7B was measured as 0.778 mg/L @ 30 °C, whereas BT4 is found to be not stable in water but forms micelle structures resulting in suspension.

Hydrolysis of BT4 would yield acetic acid plus 12-hydroxystearic acid (C18), versus either lauric acid (C12) or the coconut oil fatty acid mixture (C8-18) for SE6B and SE7B, respectively.

These substances are not expected to persist in the environment, consistent with the hazard assessment presented in the OECD SIDS (2009) for the category “Aliphatic Acids Category” where aliphatic fatty acids with a carbon chain length in the range of C8 – C22 were judged to be readily biodegradable.

The acute toxicity of the test item SE7B to the freshwater fish rainbow trout (Oncorhynchus mykiss) gave a 96-Hour LC50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.

A static limit test with the nominal loading rate of 1000 mg/L was performed with Danio rerio. All fish survived the exposure period of 96 h. The LL50 (96h, nominal) was > 1000 mg/L

Considering the structural similarity and low solubility of SE7B and BT4, these result is also considered relevant for the read-across target BT4.