2-ethylhexyl 12-(acetoxy)octadecanoate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20th October - 21st November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

yes

Remarks:

See remarks below

Principles of method if other than guideline:

Deviation:
Pathogen in test presumably introduced from Artemia. Changed all test vessels on Day 16. The
pathogen caused increased mortality in individual replicates (though insufficient invalidate the control
TAC), and reduced statistical precision. Overall no effect on test outcome since mortality and growth
indicates same results for both control and test exposure.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Substance: SE7B (bio-ester based lubricating oil) Lot # 0000140602
CAS# 13635345-64-7

Analytical monitoring:

yes

Details on sampling:

Samples were collected from “new” and “old” test solution for TOC analyses at test initiation (i.e.,
T=0) and old test solutions 24 hours later, and likewise at week 1 and 2, and at test termination.
“New” test solution samples were obtained from the prepared test exposure concentrations prior to
food addition. The “old” test solution samples were composited directly from several of the exposure
test vessels for the appropriate dosing concentration. A 200 mL sample was composited from
randomly-selected replicates in the control and all test exposures. Samples were placed into sulfuricacid preserved sample bottles and submitted to the contract laboratory for TOC analysis.

Vehicle:

not specified

Details on test solutions:

Reconstituted moderately hard laboratory water (US EPA, 2002) served as the control water in all test
and control exposures. Control and test exposures were constantly renewed at the rate of two test
chamber volume exchanges per day using calibrated peristaltic pumps transferring water to
standpiped test chambers. Fresh control or test solution came from stocks prepared every 24 hours.

Test organisms (species):

Pimephales promelas

Details on test organisms:

The test species was the freshwater fish Pimephales promelas (fathead minnow). It was selected for
testing because it is a common fish species in US freshwater ecosystems. In addition to its importance
freshwater ecosystems, P. promelas has long been used as a standard toxicity test organism and is
one of the fish species identified as a suitable test organism in OECD Method 210. Consistent with
OECD Guideline 210, fertile eggs (within 48 hours of fertilization) were used to initiate the toxicity
test. P. promelas eggs were purchased from cultures maintained at Environmental Consulting and
Testing (ECT) production facilities in Superior, Wisconsin. P. promelas are continuously cultured at ETC
in a de-chlorinated tap water with chemistry parameters consistent with the USEPA reconstituted
moderately hard water that served as the test medium in this study. Culture conditions are
documented at ECT to provide information related to test organism health. Reference toxicant tests
are regularly conducted at Ramboll Environ’ s toxicity testing laboratory with P. promelas larvae
obtained from ECT, therefore establishing a database documenting organism health and consistency of
chemical tolerance of outsourced P. promelas.
P. promelas at ECT are cultured in a de-chlorinated tap water with hardness and alkalinity
concentrations consistent with specifications for USEPA moderately hard water (USEPA, 2002).
Cultures were maintained at approximately 25 ±1 oC, and pH 7.0 to 8.0 s.u. for a minimum of 48
hours prior to test initiation. No eggs were used from brood cultures with more than 20 percent
culture stock mortality occurring during the two days preceding the test. Prior to test initiation,
dilution / test water was sent from Ramboll Environ to ECT for acclimation and transport of newly laid
eggs. Upon receipt at Ramboll Environ, the eggs were held at test temperature (25 ±1.5 oC) to insure
proper acclimation prior to test initiation.

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

32 d

Hardness:

Total Hardness: 80.8 to 94.5 mg CaCO3/L

Test temperature:

Temperature: 24.0 – 24.6 °C

pH:

pH: 7.63 to 8.05 s.u

Dissolved oxygen:

Dissolved Oxygen: 7.8 to 8.6 mg/L

Nominal and measured concentrations:

Target Water Concentrations: 0 (control), and 100% of Nominal 10,000 mg/L WAF preparation.
Measured Water Concentrations: Total Organic Carbon (TOC) as surrogate for product in WAF solution.

Details on test conditions:

Range-Finding Test:
A seven-day screening trial at the 10,000 mg/L (nominal loading rate, whole product basis) WAF limit
concentration was evaluated in February 2015 with a vigorous mixing system for WAF development.
Forty newly hatched p. promelas larvae were exposed to the test exposure and control (four replicates
of ten fish per exposure condition). Dissolved oxygen, specific conductance (conductivity), and pH
were periodically measured and recorded in the test treatment and the control for the 7 day duration
of the short term limit test. No significant mortality or growth inhibition in the seven-day range-finding
test was observed. Also, no meaningful difference in the above measured parameters for the exposure
treatment was observed compared to control water.
Based on supporting TOC analysis, the SE7B mixing system for initial trials was determined to leave
excess insoluble material in solution. Subsequent testing with a less vigorous mixing system,
replicating the mixing system developed by Harlan Laboratories (Harlan, 2013) for acute toxicity
testing, provided TOC results for a 10,000 mg/L WAF solution that were comparable to the TOC
concentrations projected for the 1 and 10 mg/L WAF solutions prepared via the vigorous mixing
system used for the February 2015 range finding test with Daphnia magna. Since no significant
mortality or growth inhibition in the seven-day range-finding test was observed for WAF exposures
with vigorous mixing, the seven-day trial was considered adequate to predict effects of a 10,000 mg/L
WAF made via the Harlan stirring method for a 32 day trial.

Limit Test:
Nominal WAF SE7B test concentrations were 0 (control) and 10,000 mg/L. The limit test was based on the results of the range-finding limit test.

The egg hatchability portion of the test was initiated with four replicates each containing 25 fertile
eggs. After maximum hatching (Day 5), the count of hatched and unhatched eggs was assessed for
test acceptability of the control exposure (70 percent) and statistical differences in exposed eggs. As
no significant difference in hatch was noted, 15 larvae from each control and test replicate were
transferred from the hatching vessels (already suspended in the test chambers) to the larvae test
chambers. Exposure vessels were labelled to identify the percentage of the nominal SE7B WAF
concentration, and identify individual test vessels. Organisms were assigned randomly to control and
the limit test exposure. Test vessels consisted of 4 L glass tanks, standpiped to maintain 2 L of control
or test solution on a constant flow basis. Tests were conducted in a temperature- and light-controlled
test room to provide water temperatures between 23.5 and 26.5oC, and a 16:8 L:D photoperiod at a
light intensity of approximately 500 to 1,000 lux. Feeding was accomplished by direct addition of
Artemia nauplii to the test vessels 2 times daily. As per OECD 210, feeding was paced with growth,
starting with a rate of 0.2 ml of concentrated Artemia per test vessel per feeding, and going up to 0.4
ml per feeding by week 3.
Fresh test solutions were prepared every 24-hours for installation into the flow through system. Four
and a half liters (4.5 L) of control water were placed in four separate 1 gallon (approximately 5 L)
glass jars, and 10 gram per liter of SE7B was added directly to the jars to prepare the appropriate
10,000 mg/L WAF stock concentration. The mixing vessels were loosely covered and stir bars were
added to spin the solution to cause a 0.2 cm vortex at the water surface. WAF stock solutions were
mixed for 23 hours (at ambient room temperature and lighting) and then installed (while still in the
mixing vessel) into the flow through pumping system, and allowed to separate for one hour. After 1
hour of settling the flow through system was turned on to transfer the test solution from the mixing
vessel to the test vessel at the rate of 2.8 ml/minute. The flow rate was calculated to achieve a
minimum of two test vessel turnovers per day. Since SE7B floats, the WAF was obtained by carefully
extracting the test solution from near the bottom of the mixing jar over the course of 24 hours while a
new volume of WAF was being prepared.
At test initiation, 25 eyed P. promelas eggs were added randomly to a screened jar suspended in each
test replicate. P. promelas eggs are normally fanned and cleaned by a tending male during incubation, so
each suspended jar containing eggs also contained a small airline to clean/agitate the eggs during the
incubation period. Test system observations were recorded with each daily solution renewal.
Physical/chemical test conditions were documented (i.e., water temperature, pH, dissolved oxygen, etc.)
in the test vessels. Any observations of abnormal test organism activity (e.g., fungus on eggs, growths on
fish, erratic swimming) were noted on the test forms and in the log book.
Hatchability was assessed on Days 3, 4, and 5. Maximum hatch was noted on Day 5. No significant
difference was noted between the control and test exposure and the minimal acceptable hatch rate for the
control was noted. Subsequently, a total of 15 hatched larvae were moved into the test vessels (60 fish
total per control and WAF exposure), and the remaining fish were used to determine hatch weight.
Observations of mortality were noted daily up to test termination (day 32). Test acceptability criteria for
P. promelas chronic tests stipulate a hatch rate of 70 percent, and no more than 25 percent mortality of
control fish by test termination.

Reference substance (positive control):

no

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The experimental conditions utilized in this study maintained dissolved oxygen concentrations in the
control exposures above 60 percent saturation (i.e., above 4.8 mg/L at 25 oC), and maintained test
water pH between 6.0 and 9.0 s.u. The average control organism survival exceeded 75 percent. The
average length of control fish at test termination was approximately 12 mm, which is 67 percent of
the “typical” P. promelas final length recommendation in OECD 210. Control growth was sufficient for
demonstrating adequate control performance.

There is no analytical standard for SE7B. TOC was therefore used as a surrogate for determining the
presence of material in solution under a standardized mixing system. The precedent for this
methodology was derived from previous studies conducted by Harlan Laboratories Ltd.
According to the Harlan study, the 10,000 mg/L nominal WAF preparation contained 1.69 mg/L TOC
for a freshly prepared sample and less than detectable TOC after 48-hours of exposure
after accounting for background TOC from the dilution water.

All chemical and physical parameters for the 32-day study were within specified ranges.
The test-water temperatures ranged from 24.0 to 24.6 ºC, which is
within the 25 ± 1.5 ºC temperature range specified by the study protocol and OECD methodologies.
Dissolved oxygen concentrations in test vessels ranged from 7.8 to 8.6 mg/L (91 to
100 percent of air saturation at test temperatures), and test vessel pH ranged from 7.63 s.u. to 8.05
s.u. in all control and SE7B exposures throughout the study. Light intensity ranged from 796 to 822
lux.

Dilution water parameters were consistent. Total ammonia concentration and total residual chlorine
concentrations were always at non detectable levels. Total hardness throughout the study ranged from
80.8 to 94.5 mg CaCO3/L, and total alkalinity values ranged from 41 to 48 mg CaCO3/L

The average egg hatch rate for the control replicates was 95 percent. This meets the
protocol test acceptability criterion of at least 70 percent hatchability. Control survival was 83.3
percent. This meets the protocol test acceptability criterion of at least 75 percent survival. The
hatchability and survival of P. promelas was 85 percent and 88.3 percent, respectively for the T1
(10,000 mg/L) nominal WAF treatment. The NOEC values show no statistically significant effect tohatchability or survival at the 10,000 mg/L nominal WAF exposure.
Growth was assessed by both weight and length measurements (Table 4). The average weight of the
control (T0) and WAF exposed (T1) fish based on surviving fish was 1.941 mg and 1.875 mg
respectively. The average weight of the control (T0) and WAF exposed (T1) fish based on initial
number of fish was 1.651 mg and 1.601 mg respectively. The difference in fish weights based on
either initiating or surviving fish is not statistically significant (i.e. NOEC is 10,000 mg/L WAF) (Table
5). There was also no statistically significant difference in total fish length of surviving fish. The
average length of the T0 control fish was 11.63 mm, and the average length of T1, 10,000 mg/L WAF
exposed fish was 11.39 mm. The statistical results upon which these test endpoints are based are
summarized in Table 5, and the original statistical analyses are presented in Attachment 4.

Results tables attached

Validity criteria fulfilled:

yes

Conclusions:

The test material (SE7B) is poorly soluble in test water. Using the introduction method standardized
by Harlan Laboratories, less than 2 mg/L of TOC was consistently dosed from a nominal 10,000 mg/L
WAF solution in this flow-through toxicity study with P. promelas. Neither the survival nor growth of P.
promelas was affected over a 32-day exposure duration of SE7B based on the LOEC and NOEC test
endpoints.

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Read-Across Justification is attached below.

Reason / purpose for cross-reference:

read-across source

GLP compliance:

yes (incl. QA statement)

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

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 test material (SE7B) is poorly soluble in test water. Using the introduction method standardized by Harlan Laboratories, less than 2 mg/L of TOC was consistently dosed from a nominal 10,000 mg/L WAF solution in this flow-through toxicity study with P. promelas. Neither the survival nor growth of P. promelas was affected over a 32-day exposure duration of SE7B based on the LOEC and NOEC test endpoints.
Considering the structural similarity and low soubility of BT4, this result is also considered relevant for the read-across target BT4.

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 test material (SE7B) is poorly soluble in test water. Using the introduction method standardized by Harlan Laboratories, less than 2 mg/L of TOC was consistently dosed from a nominal 10,000 mg/L WAF solution in this flow-through toxicity study with P. promelas. Neither the survival nor growth of P. promelas was affected over a 32-day exposure duration of SE7B based on the LOEC and NOEC test endpoints.

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