Hexanoic acid, 3,5,5-trimethyl-, 1,1'-[2-ethyl-2-[[(3,5,5-trimethyl-1-oxohexyl)oxy]methyl]-1,3-propanediyl] ester

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-10-31 to 2014-01-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 5, 9, 14 and 20 (fresh media) and on Days 2, 7, 12, 16 and 21 (old media)
Water samples were taken from the control and each surviving test group (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 5, 9, 14 and 20 and of the expired test preparations on Days 2, 7, 12, 16 and 21. Samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.
Samples at the No Observed Effect Loading Rate and above only were analyzed.

Vehicle:

no

Details on test solutions:

Amounts of test item (10, 32, 100, 320 and 1000 mg) were each separately added to the surface of 10 liters of test water to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively.
After the addition of the test item, the test 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 each 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 WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
Each daphnid received approximately 2 to 7 |iL of an algal suspension {Desmodesmus subspicatus) and approximately 10 to 55 |iL of Tetramin® flake food suspension daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

None

Hardness:

220 to 524 mg/L as CaCO3. Some of the water hardness measurements were above the 250 mg /L as CaCOs stated in the study plan. However, given that no adverse effects were observed in the control group, this was considered not to have had an effect on the outcome or integrity of the study

Test temperature:

19 °C to 21 °C

pH:

8.0 ± 0.2

Dissolved oxygen:

>7.4 mg O2/L

Salinity:

not applicable

Nominal and measured concentrations:

Amounts of test item (10, 32, 100, 320 and 1000 mg) were each separately added to the surface of 10 liters of test water to give nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L respectively.

Details on test conditions:

For each concentration a single daphnid was placed in 100 mL of the test preparation in 150 mL glass flasks which were then covered with a plastic lid to reduce evaporation. For each test and control group ten replicate test vessels were prepared. The flasks were maintained at approximately 20 0C with a photoperiod of 16 hours light (641 to 787 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. Each vessel was randomly assigned to a position in the laboratory. The test vessels were not aerated. The diluent water only was aerated prior to use.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were renewed 3 times per week on Days 0, 2, 5, 7, 9, 12, 14, 16 and 20. The adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted before being discarded.
On a daily basis the numbers of live and dead of the "Parental" (Pi) generation, the numbers of live and dead "Filial" (Fi) Daphnia and the number of discarded unhatched eggs were counted.
An assessment was also made of the general condition and size of the parental Daphnia as compared with the controls.
The number of Daphnia with eggs or young in the brood pouch was determined daily. Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Daphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilization criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring
produced in the flasks.
At the end of the test, the length of each surviving parent animal was determined.
Information on water and media can be found in the attached document.

Study period: 2013-07-16 to 2013-09-19

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Details on results:

Lethal Effects on the Parental Generation (Pi)
No significant mortalities occurred at any of the loading rates throughout the test. Immobilized parent daphnia were observed in the control, 3.2 and 10 mg/L loading rate WAF test groups, however, as no more than 20% immobilization was observed, this was considered to be due to natural causes.
Sub-lethal Effects on the Parental Generation (Pi)
The 32 and 100 mg/L loading rates showed a statistically significant difference from the control and the remaining test groups after 21 days in terms of producing fewer numbers of live young per adult.
The EL50 (reproduction) value based on nominal loading rates was estimated to be greater than 100 mg/L loading rate WAF.
After 21 days the length of each surviving adult was determined. The results showed that there were no statistically significant differences (P> 0,05) between the control and the 1.0, 3.2 and 10 mg/L loading rate WAF test groups in terms of length of the daphnids after 21 days exposure to the test item.
However, there was a significant difference between the control and the 32 and 100 mg/L loading rate WAF test groups.
Effects on the Filial Generation (Fi)
Information on the effects of the test item on the Fi generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test.
Young were first produced in the control test group on Day 8 of the test. There were no unhatched eggs or dead young for the duration of the test.
Lowest Observed Effect Loading Rate
The "Lowest Observed Effect Loading Rate" (LOEL) was 32 mg/L loading rate WAF as this test group produced significantly fewer live young per adult (P<0.05) than the control group.
No Observed Effect Loading Rate
The "No Observed Effect Loading Rate" (NOEL) was 10 mg/L loading rate WAF as there were no significant mortalities (immobilization) observed in the parental generation (Pi) and there were no significant differences (P>0.05) in terms of the number of live young produced per adult when compared to the control after 21 days.
Maximum Acceptable Toxicant Loading Rate
The "Maximum Acceptable Toxicant Loading Rate" (MATL) was calculated to be 18 mg/L loading rate WAF.
Verification of Test Concentrations
Chemical analysis of control samples throughout the test showed a peak was present, the profile of which was consistent with that of the test item. It was assessed as a possible trace contaminant which had a characteristic profile similar to that of the test item.
Given that no adverse effects were observed in the control group, , it was considered appropriate to correct the test results for the control.
Analysis of the 10, 32 and 100 mg/L loading rates on days 0, 5, 9, 14 and 20 (fresh media) and on days 2, 7, 12, 16 and 21 (old media) showed that measured concentrations of between 0.00023 and 0.33 mg/L were obtained. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Validation of Mixing Period
Pre-study investigational work indicated that there was no significant increase in the amount of dissolved test item when the preparation period was extended for longer than 24 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.
Water Quality Criteria
Temperature was maintained at approximately 20°C throughout the test, while there were no treatment related differences for oxygen concentration or pH. Temperatures were maintained at 19°C to 21°C for the duration of the test.
Throughout the test the light intensity was observed to be in the range 641 to 787 lux.
The water hardness was observed to be in the range 220 to 524 mg/L as CaCO3 in the control and the highest surviving test group throughout the test. Some of the water hardness measurements were above the 250 mg /L as CaCO3 stated in the study plan. However, given that no adverse effects were observed in the control group, this was considered not to have had an effect on the outcome or integrity of the study.
Observations on Test Item Solubility
At the start of the mixing period the 1,0, 3.2, 10, 32, and 100 mg/L loading rates were observed to be clear colourless water columns with test item floating at the surface. After 23 hours stirring and a 1-Hour standing period the 1.0, 3.2, 10, 32, and 100 mg/L loading rates were observed to remain as at the start of stirring. Microscopic inspection of the WAFs showed no microdispersions or undissolved test item to be present. After siphoning and for the duration of the
test, the loading rates were observed to be clear, colorless solutions.
Validation Criteria
The following validation criteria were achieved during the test
Required Actual
a. Control mortality <20% 10%
b. Dissolved oxygen > 3 mg O2/L >7.4 mg O2/L
c. pH (control group)deviation <1.5 0.6
d. pH (control group)between 6-9 7.5-8.1
e. Mean number of live young per
surviving adult (control group) > 60 after 21 days 134
f. Coefficient of variation for control
group <25% 3.8%
g. First offspring in the control group born after a Day 8
maximum of 9 days

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

See details on results.

Table 1: Summary of findings following the exposure of Daphnia magna for 21 days.

Nominal Loading Rate (mg/L)	% Survival of P1	Number of Live Young*		Number of Dead Young		Number of Unhatched Eggs
		Total	Per Female (cumulative)	Total	Per Female (cumulative)	Total	Per Female (cumulative)
Control	90	1207	134	0	0	0	0
1.0	100	1315	132	0	0	0	0
3.2	80	1120	140	0	0	0	0
10	80	1090	136	0	0	0	0
32	100	995	100	0	0	0	0
100	100	1096	110	0	0	0	0

* The number of live young per live adult at Day 21

See attached document for additional tables of findings.

Validity criteria fulfilled:

yes

Conclusions:

The 21-Day EL50 (immobilization) value, based on nominal test concentrations, for the parental Daphnia generation (Pi) was estimated to be greater than 100 mg/L loading rate WAF.
The 21-Day E L 5 0 (reproduction) value based on nominal test concentrations was estimated to be greater than 100 mg/L.
The "Lowest Observed Effect Loading Rate" was considered to be 32 mg/L loading rate WAF on the basis that at this test concentration significantly fewer live young per adult (P<0.05) were produced when compared to the control.
The "No Observed Effect Loading Rate" was considered to be 10 mg/L loading rate WAF on the basis that at this test concentration there were no significant mortalities (immobilization) observed in the parental generation (Pi) and that there were no significant differences (P>0.05) between the control and the 10 mg/L loading rate WAF test group in terms of numbers of live young produced per adult by Day 21.
The "Maximum Acceptable Toxicant Loading Rate" was calculated to be 18 mg/L loading rate WAF.

Executive summary:

Guideline

A study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2008) No 211, "Daphnia magna Reproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008 and the US EPA Draft Ecological Effects Test Guidelines OPPTS 850.1300 "Daphnid Chronic Toxicity Test".

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).

Based on the results of an acute toxicity test to Daphnia Magna (Harris 2013), Daphnia magna were exposed (10 replicates of a single daphnid per group) to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L for a period of 21 days. The test solutions were renewed 3 times per week. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with a mixture of algal suspension and Tetramin® flake food suspension.

Results

The 21-Day EL50 (immobilization) value, based on nominal test concentrations, for the parental Daphnia generation (Pi) was estimated to be greater than 100 mg/L loading rate WAF.

The 21-Day EL50 (reproduction) value based on nominal test concentrations was estimated to be greater than 100 mg/L.

The "Lowest Observed Effect Loading Rate" was considered to be 32 mg/L loading rate WAF on the basis that at this test concentration significantly fewer live young per adult (P<0.05) were produced when compared to the control.

The "No Observed Effect Loading Rate" was considered to be 10 mg/L loading rate WAF on the basis that at this test concentration there were no significant mortalities (immobilization) observed in the parental generation (Pi) and that there were no significant differences (P>0.05) between the control and the 10 mg/L loading rate WAF test group in terms of numbers of live young produced per adult by Day 21.

The "Maximum Acceptable Toxicant Loading Rate" was calculated to be 18 mg/L loading rate WAF.

Description of key information

One reliable study is available. In this study Daphnia magna were exposed to a Water Accommodated Fraction of the substance at nominal loading rate of 1.0, 3.2, 10, 32 or 100 mg/L under semi-static conditions for 21 days. The EL50 (21 d, immobilization) value was > 100 mg/L loading rate WAF. The EL50 (21 d, reproduction) value was > 100 mg/L loading rate WAF. The Lowest Observed Effect Loading Rate was 32 mg/L loading rate WAF (based on production of live young per adult) and the No Observed Effect Loading Rate  was 10 mg/L  loading rate WAF (based on parental mortality and production of live young per adult).

Additional information

One reliable study is available. In this study (Harris, 2014) performed under GLP according to OECD TG 211, EC Method C.20 and draft OPPTS 850.1300, Daphnia magna were exposed to the substance under semi-static conditions for 21 days. 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). Based on the results of the acute toxicity test (Harris, 2013) daphnids (10 replicates of a single daphnid per group) to WAFs of the test item at nominal loading rates of 1.0, 3.2, 10, 32 or 100 mg/L. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily.

The 21-Day EL50 (immobilization) value, based on nominal test concentrations, for the parental Daphnia generation (Pi) was > 100 mg/L loading rate WAF. The 21-Day E L 5 0 (reproduction) value based on nominal test concentrations was > 100 mg/L.

The "Lowest Observed Effect Loading Rate" was considered to be 32 mg/L loading rate WAF on the basis that at this test concentration significantly fewer live young per adult (P<0.05) were produced when compared to the control.

The "No Observed Effect Loading Rate" was considered to be 10 mg/L loading rate WAF on the basis that at this test concentration there were no significant mortalities (immobilization) observed in the parental generation (Pi) and that there were no significant differences (P>0.05) between the control and the 10 mg/L loading rate WAF test group in terms of numbers of live young produced per adult by Day 21. The "Maximum Acceptable Toxicant" Loading Rate was calculated to be 18 mg/L loading rate WAF.