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 fish

Link to relevant study record(s)

Reference

Endpoint:

adult fish: sub(lethal) effects

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-10-14 to 2014-02-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)

Deviations:

yes

Remarks:

21-day exposure period. Transfer of one fish from control group into 10 mg/L dose group on Day 16.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and all surviving test groups on Days 0, 3, 6, 10, 13, 17 and 20 (fresh media) and on Days 1, 4, 7, 11, 14, 18 and 21 (old media) for quantitative analysis. The samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen should further analysis be required.
Additional samples were taken on Days 2, 5, 8, 9, 12, 15, 16 and 19 (fresh and old media) and on Days 4, 7, 11 and 18 (fresh media) and on Days 3, 6, 10, 13, 17 and 20 (old media). These samples were stored frozen should further analysis be required.
Only samples at the No Observed Effect Loading Rate and above were analyzed.

Vehicle:

no

Details on test solutions:

Amounts of test item (210, 672 and 2100 mg) were each separately added to the surface of 21 liters of test water to give the 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 mixtures 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 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 75-100 mL discarded) to give the 10, 32 and 100 mg/L loading rate WAFs.
At the start and end of each mixing period, and following a 1-Hour standing period, each of the loading rate WAFs were observed to have formed clear colorless water columns with oily globules / an oily slick of test item floating on the surface. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the 10, 32 and 100 mg/L loading rates were observed to be clear, colorless solutions.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Age at study initiation: Juvenile
- Length at study initiation: mean standard length 3.7 cm (sd = 0.32)
- Weight at study initiation (mean and range, SD): mean weight of 0.76 g (sd = 0.20)
- Feeding during test: commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test.

ACCLIMATION
- Acclimation period: 10 November 2013 to 22 November 2013
- Acclimation conditions (same as test or not): same as test; maintained in a glass fiber tank with a "single pass" water renewal system.
- Type and amount of food: as above
- Health during acclimation (any mortality observed): No mortalities in 7 days prior to start of definitive test.

Experimental Starting Date: 21 November 2013
Experimental Comnletion Date: 15 December 2013

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

None

Hardness:

total hardness of approximately 140 mg/L as CaCOs.

Test temperature:

approximately 14 °C

pH:

7.3 - 8.2

Dissolved oxygen:

9.2 - 10.7 mg O2/L

Salinity:

not applicable

Details on test conditions:

0, 10, 32 and 100 mg/L loading rates

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Concentration based on Loading Rate

Details on results:

There were no mortalities in 10 fish exposed to 10, 32 and 100 mg/L loading rate WAFs for a period of 21 Days.
The results of the definitive test showed the highest loading rate resulting in 0% mortality to be 100 mg/L. The No Observed Effect Loading rate (NOEL) was 100 mg/L loading rate WAF.
It was noted on Day 16 of the test that only 9 fish remained in the control tank with all other tanks containing 10 fish. However, it was then noted on Day 18 of the test that there were 11 fish in the 10 mg/L loading rate WAF test group. It was considered therefore that on Day 16 one of the fish from the control vessel was accidentally transferred into the 10 mg/L loading rate WAF test vessel but was not recorded in error. This was considered not to impact the validity of the
test as the No Observed Effect Loading Rate was 100 mg/L and sufficient fish remained in the control group to satisfy the validation criteria.
It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.
There were no sub-lethal effects observed during the test.
Analysis of the freshly prepared media for the 100 mg/L loading rate WAF on Days 0, 3, 6, 10, 13, 17 and 20 showed measured concentrations of between 0.0097 and 0.13 mg/L. Analysis of the old or expired media on Days 1, 4, 7, 11, 14, 18 and 21 showed measured concentrations of between 0.0093 and 0.081 mg/L. 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, and after discussion with the Sponsor, it was considered appropriate to correct the test results for the control.
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.
After 21 days the lengths and weights of the surviving fish were determined. The results showed that there were no statistically significant
differences (p<0.05) between the control and the test groups in terms of either length or weight at Day 21.
Temperature was maintained at approximately 14 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH. No fresh media observations were conducted on Day 14 as the media was not renewed in error. This was considered not to impact the integrity of the study as no toxicity was observed.
Validation Criteria
The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was >60% of ASV (6.2 mg O2/L) in the control and test vessels.

Results with reference substance (positive control):

Not applicable

Validity criteria fulfilled:

yes

Conclusions:

The prolonged toxicity of the test item to the freshwater fish rainbow trout {Oncorhynchus mykiss) has been investigated and gave a 21 Day L L 5 0 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.
The threshold levels for lethal and observed effects were considered to be greater than 100 mg/L loading rate WAF.

Executive summary:

Test Guidance

A study was performed to assess the prolonged 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 (1984) No 204 "Fish, Prolonged Toxicity Test: 14 Day Study".

Methods

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF). Based on data from an Acute Toxicity to Fish study (Parr 2014), fish were exposed, in groups of seven, to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 10, 32 and 100 mg/L for a period of 21 days at a temperature of approximately 14 0C under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined daily throughout the test until termination after 21 days.

Results

Analysis of the freshly prepared media for the 100 mg/L loading rate WAF on Days 0, 3, 6, 10, 13, 17 and 20 showed measured concentrations of between 0.0097 and 0.13 mg/L. Analysis of the old or expired media on Days 1, 4, 7, 11, 14, 18 and 21 showed measured concentrations of between 0.0093 and 0.081 mg/L. 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, and after discussion with the Sponsor, it was considered appropriate to correct the test results for the control. 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.

Conclusion

Exposure of rainbow trout to the test item gave LL 50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF. The threshold levels for lethal and observed effects were considered to be greater than 100 mg/L loading rate WAF.

Description of key information

One reliable study is available. In this study Rainbow trout (Oncorhynchus mykiss) were exposed to a Water Accommodated Fraction of the substance at nominal loading rates of 10, 32 or 100 mg/L under semi-static conditions for 21 days. The LL50 (21 d) was > 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF. The substance is not harmful to fish after prolonged exposure.

Additional information

In a reliable study performed under GLP and based on OECD TG 204 (Parr, 2014) Rainbow trout (Oncorhynchus mykiss) were exposed, in groups of seven, to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 10, 32 or 100 mg/L for a period of 21 days at a temperature of approximately 14 °C under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined daily throughout the test until termination after 21 days. No mortalities were observed during the study. Exposure of rainbow trout to the test item gave LL 50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF. The threshold levels for lethal and observed effects were considered to be greater than 100 mg/L loading rate WAF.