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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
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:
read-across source
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
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 toxicity study with D. magna. Neither the survival nor reproduction of D. magna was affected over a 21-day exposure duration of SE7B based on the LOEC, NOEC, IC25, and IC50 test endpoints.

Considering the structural similarity and low soubility of BT4, this result is also considered relevant for the read-across target BT4.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August - October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
See note below
Principles of method if other than guideline:
Deviation: The TOC values for the Day 1 Old test solutions are anomalously high and are not considered in
average calculations. The samples were retested with no changes of results observed. Since these
values are dependent on fluctuations of a live food TOC, the anomaly may be due to a fluctuation in
food growth during the 24 hour interval. The high values were only observed for a single measuring
interval and do not appear to effect the entire test outcome.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
The test substance was SE7B (bio-ester based lubricating oil, CAS # 1365345-64-7).
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:
The WAF solution was produced as per procedures
developed by Harlan Laboratories Ltd. (Harlan, 2013) in compliance with OECD 2000. The WAF
solution was prepared by adding 10,000mg/L of test material to toxicity test control water and gently
mixing for 24 hours before settling, separation, and dosing. Total Organic Carbon (TOC) analysis was
used as a surrogate for determining the presence of soluble product in toxicity test solutions.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test species was the water flea Daphnia magna (D. magna). D. magna is a commonly tested
freshwater cladoceran. It was selected for testing because it is an ecologically important pelagic
crustacean. In addition to its importance in food-web transfer of energy and nutrients, D. magna has
long been used as a standard toxicity test organism and is one of the Daphnia species identified as a
suitable test organism in OECD Method 211. D. magna were obtained from in-house cultures
maintained at Ramboll Environs’ test facilities in Brentwood, Tennessee. D. magna are continuously
cultured in the USEPA reconstituted moderately hard water that also served as the test medium in this
study. Consistent with OECD Guideline 211, D. magna neonates less than 24 hours old were used to
initiate toxicity tests.
D. magna were cultured in the toxicity test medium: 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. In culture, D. magna are fed daily a mixture of green algae
(Pseudokirchneriella subcapitata, formerly Selenastrum capricornutum) and Cerophyl. Parent cultures
are renewed and fed daily.
In the toxicity test, the test organisms were fed at a rate of 1.67 mL per 100 ml of test solution.
Addition of organism food was incorporated into the daily solution renewal procedure. This feeding
density corresponds to a TOC loading of 0.2 mg/L per 60 ml of test solution as per the recommended
food loading rate in OECD guidelines.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
Total Hardness: 80.8 to 88.0 mg CaCO3/L
Test temperature:
Temperature: 24.0 – 24.9 °C
pH:
pH: 7.47 to 8.11 s.u
Dissolved oxygen:
Dissolved Oxygen: 7.1 to 8.7 mg/L
Conductivity:
Conductivity 195 to 271 µS/cm
Nominal and measured concentrations:
Target Water
Concentrations: 0 (control), 3.125%, 6.25%, 12.5%, 25%, 50%, 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 pre-established exposure series of 1, 10, 100, and 1,000 mg/L (nominal loading rate, whole product
basis) of the WAF of the test material was evaluated in February 2015 with a vigorous mixing system
for WAF development. Six D. magna neonates were exposed to each test exposure and control (six
replicates of one organism per exposure condition). Dissolved oxygen, conductivity, and pH were
periodically measured and recorded in each treatment and the control for the 14 day duration of the
range-finding test. No meaningful difference in the above measured parameters for the exposure
series tested was observed compared to control water. However, based on supporting TOC analysis,
the SE7B mixing system for range-finding tests 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. No significant mortality or reproductive inhibition in the 14 day range-finding test was
observed for WAF exposures with a projected TOC concentration comparable to that of the 10,000
mg/L WAF made via the Harlan stirring method.
An experimental trial of the testing system was initiated August 27, 2015 and terminated September
10, 2015 due to direct transfer of insoluble product into the WAF solution from improper separation.
The results of system trial and range-finding work indicated that SE7B would be tested at nominal
WAF concentrations up to 10,000 mg/L.

Definitive Test:
Dosing of the in-life definitive toxicity test was initiated on September 24, 2015 and terminated on
October 15, 2015. The test protocol was based on the OECD 211 guidance for conducting chronic
toxicity tests with D. magna (OECD, 2012). All toxicity testing was conducted following Good
Laboratory Practices (GLP, 40 CFR Part 792), and adhered to the methodologies specified in the
previously authorized Study Protocol.
Nominal WAF SE7B test concentrations were 0 (control), 312.5, 625, 1,250, 2,500, 5,000, and 10,000
mg/L. Test concentrations were selected based on the results of the range-finding tests.

All exposures contained ten individual replicates for determination of test endpoints. Each replicate
was initiated with a single D. magna neonate. 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 test exposures. Test vessels consisted of 200 mL glass
beakers, each containing 100 mL of control or test solution. Tests were conducted in a temperature
and light-controlled test room to provide water temperatures of 24 to 26 oC, and a 16:8 L:D
photoperiod at a light intensity of approximately 500 to 1,000 lux. Daphnid feeding was accomplished
by daily addition of dilute food suspension to renewal waters prior to daily water
renewal. Feeding during the definitive test was the same as that administered during organism
acclimation and during the range-finding test (i.e., 16.7 ml of food stock solution per liter control or
test solution).
Test solutions were prepared for initiating dosing and every 48-hours thereafter until test termination.
Three and a half liters (3.5 l) of control water were placed in two separate 1 gallon (approximately 4
liter) 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 allowed to
separate for one hour. Since SE7B floats, the WAF was obtained by carefully siphoning near the
bottom of the gallon jar. Solutions were refrigerated at 4 ⁰C when not in use. Each batch of water was
used for two daily renewal events.
At test initiation, a single neonate D. magna was added randomly to each test replicate. Test system
observations were recorded with each daily solution renewal. Physical/chemical test conditions were
documented (i.e., water temperature, pH, dissolved oxygen, etc.) on freshly prepared and post exposure
solutions. Any observations of abnormal test organism activity (e.g., floating, pale organisms, or
incomplete shedding) were noted on the test forms and in the log book.
Observations of mortality and neonate production were noted daily up to test termination (day 21). Test
acceptability criteria for D. magna chronic tests stipulate no more than 20 percent mortality and the
cumulative reproduction of control organisms is greater than 60 neonates produced per female.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
The experimental conditions utilized in this study maintained dissolved oxygen concentrations in the
control exposures above 30 percent saturation (i.e., above 2.5 mg/L at 25 oC), and maintained test
water pH between 6.0 and 9.0 s.u. The average control organism survival exceeded 80 percent, and
the average cumulative neonate production for controls exceeded 60 neonates (185.3). Thus, all test
acceptability criteria specified in the Study Protocol were met.

All chemical and physical parameters for the 21-day study were within specified ranges.
The test-water temperatures ranged from 24.0 to 24.9 ºC, which is
within the 25 ± 1 ºC temperature range specified by the study protocol and OECD methodologies
(OECD, 20012). Dissolved oxygen concentrations ranged from 7.1 to 8.7 mg/L (84 to 104 percent of
air saturation at test temperatures), and test solution pH ranged from 7.47 s.u. to 8.11 s.u. in all
control and SE7B exposures throughout the study. Light intensity ranged from 853 to 874 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 88.0 mg CaCO3/L, and total alkalinity values ranged from 41 to 47 mg CaCO3/L

Survival for the control replicates was 100 percent. This meets the protocol test acceptability
criterion of at least 80 percent survival. The survival of parent D. magna was 100, 100, 90, 100, 78,
and 90 percent in the T1 (313 mg/L), T2 (625 mg/L), T3 (1,250 mg/l), T4 (2,500 mg/L), T5
(5,000 mg/L), and T6 (10,000 mg/L) nominal WAF treatments, respectively. The mortality-based IC
values show no effect and are all greater than 10,000 mg/L nominal WAF concentration. The NOEC
value also shows no effect to survival at the 10,000 mg/L nominal WAF exposure.
The average neonate production (Table 5) for D. magna based on the test-initiating number of
organisms was 185.3, 187.3, 184.1, 166.7, 190.9, 158.8, and 166.5 in the T0 (control), T1 (313
mg/L), T2 (625 mg/L), T3 (1,250 mg/l), T4 (2,500 mg/L), T5 (5,000 mg/L), and T6 (10,000 mg/L)
nominal WAF treatments, respectively. Since there was no statistically significant mortality, the
average neonate production for D. magna was also calculated based on the surviving number of test
organisms as summarized above. The IC25, IC50, NOEC, and LOEC values demonstrate no
reproductive inhibition to D. magna at the 10,000 mg/L nominal WAF dose. The statistical results upon
which these test endpoints are based are summarized in Table 5 (attached)

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 toxicity study with D. magna. Neither the survival nor reproduction of D. magna
was affected over a 21-day exposure duration of SE7B based on the LOEC, NOEC, IC25, and IC50 test
endpoints.

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 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 toxicity study with D. magna. Neither the survival nor reproduction of D. magna was affected over a 21-day exposure duration of SE7B based on the LOEC, NOEC, IC25, and IC50 test endpoints.

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