Octadecene, reaction products with hexadecene, hydrogenated

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 September 2020 to 23 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)

Version / remarks:

OECD Guideline for the Testing of Chemicals. Test Guideline 218, Sediment-water chironomid toxicity test using spiked sediment. Adopted April 2004.

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

No further details specified

Analytical monitoring:

yes

Details on sampling:

Sediment, overlying water and pore water from the control, solvent control and all test concentrations was sampled throughout the study at the following time points:
On exposure Day 0, from additional dedicated sacrificial replicates.
On exposure Days 7 and 14, from additional dedicated sacrificial replicates. Day 7 and 14 analytical replicates contained larvae.
On exposure Day 28 from one test replicate vessel.

Vehicle:

yes

Details on sediment and application:

The control, solvent control and all test substance concentrations had 1200 g dry weight of sediment prepared.
A proportion (50%) of the dry sand component of the sediment for each treatment was added to glass evaporation dishes; the weight of the vessel and sand was determined. Each test evaporation dish received 10 mL of the test substance in solvent (hexane) such that the nominal sediment concentration of the whole treatment batch was supplied. The stock solutions were distributed evenly over the surface of the sand by pipette. The solvent control sand received the same volume of solvent with no test substance. The solvent was allowed to evaporate within a fume hood for approximately 2.5 hours. The control sand was treated in the same way, but received no addition of solvent or test substance.
After evaporation of the solvent carrier, the remaining quantity of sand, plus the kaolinite clay, conditioned peat and reverse osmosis (RO) water components of the sediment were added to the mixing bottles. The amount of RO water added to the sediment was that which liquefied it sufficiently to obtain good mixing on a tumble mill, using known and equal additions to all bottles. The nominal target moisture content of the sediment was 32% moisture as a percentage of sediment dry weight. RO water was added to the formulated sediment in place of dilution water in error. As this formed only a small proportion of the total liquid in the test system and the validity criteria were met it was not deemed to have affected the outcome of the study.
The contents of the bottles were mixed on a tumble mill overnight before being dispensed to the test vessels the following day.

Test organisms (species):

Chironomus riparius

Details on test organisms:

The test organisms were the first instar larvae of Chironomus riparius, derived from continuous laboratory cultures. The hatch status of the larvae was not recorded in error during the 2 days prior to their use in the study. Other cultures set up around the same time typically hatched approximately 2 days after initiation and the same was assumed for the culture used. As all control and solvent control adult emergence was completed by Day 23 of the exposure, and as the other validity criteria were met, this was not deemed to have affected the outcome of the study.
Laboratory cultures originated from Senckenburg (Molecular Ecology group; Frankfurt, Germany), Covance (Shardlow, UK) and Smithers (Harrogate, UK). The identity of the species was verified by the supplier(s). The cultures, including egg masses and larvae, were maintained under similar conditions of temperature, food type and photoperiod as described for the test. Larvae used to start the test were collected from egg masses that were deposited on the same day.
C. riparius is distributed throughout North America and Europe in a wide variety of freshwater habitats. The larvae construct and live in tubes within the sediment. After 4 larval stages the larvae pupate and the pupae rise to the surface where the adult insects emerge. Adults mate and egg ropes are laid in water.

Study type:

laboratory study

Test type:

not specified

Water media type:

freshwater

Type of sediment:

natural sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Post exposure observation period:

No post exposure observation period specified

Hardness:

71.5 - 80 CaCO3 (mg/L)

Test temperature:

20.3-20.6°C

pH:

7.65-8.27

Dissolved oxygen:

75.5-101.3 (%ASV)

Ammonia:

<1.0-17.9 (NH4+/-N; mg/L0

Conductivity:

335 μS/cm

Nominal and measured concentrations:

Control, solvent control and nominal test substance concentrations of 31.3, 62.5, 125, 250, 500 and 1000 mg/kg sediment dry weight.
Control, solvent control and initial measured concentrations of

Details on test conditions:

Preparation of stock solutions
This study was run with a control, solvent control (hexane) and nominal concentrations of 31.3, 62.5, 125, 250, 500 and 1000 mg/kg sediment dry weight. A series of stock solutions of the test substance were prepared in hexane.
All stocks were observed as clear and colourless after inverting several times to mix.

Sediment characterisation
Samples for sediment characterisation (i.e. wet:dry weight, pH and TOC determinations) were taken from the control mixing bottle after the mixing period and prior to being dispensed into the test vessels.
In order to determine the moisture of the formulated sediment, wet:dry determinations were made on the control sediment. Six replicates of approximately 10 g (wet weight) were weighed into beakers of known weight and dried overnight at approximately 105°C. Following drying, the sediment vessels were weighed again and the dry weight of the sediment determined. This data was used to determine the mean moisture content of the sediment.
Two aliquots of approximately 5 g (wet weight) were removed for pH determinations. 25 mL of RO water was added to each aliquot and hand shaken for approximately 2 minutes before leaving to stand for 3.5 hours before measuring.

Sediment equilibration
For each treatment and control, 150 g of the appropriate sediment equivalent to a depth of approximately 20 mm (measured at 19 mm) was weighed into each replicate vessel. The amount of sediment added to each vessel was equal across all replicates. Dilution water was added to each vessel to give a total volume of 400 mL, or a depth of approximately 55 mm (measured at 55 mm). To minimise disturbance of the sediment while adding water, a circular disc was used whilst adding water. The sediment to overlying water depth ratio was approximately 1 in 4 parts sediment:water. The test vessels were fitted with their lids and, after a period of settlement, provided with gentle aeration (as was employed during the animal exposure). The vessels were allowed to stabilise and equilibrate under test conditions (minus aeration which was started on the same day as animal addition) for 48 hours, before addition of the larvae. The overlying water was observed as clear before aeration was initiated. Overlying water levels were checked daily during the equilibration and test exposure phases and topped up with RO water as necessary to replace evaporative losses.

Apparatus
Materials in contact with the dilution water, test systems, sediment, test substance and C. riparius cultures were, where possible, restricted to glass and unplasticized plastics. The test vessels were glass beakers of 450 mL nominal capacity and approximate 8.5 cm internal diameter, fitted with lids consisting of inverted, plastic beakers (400 mL capacity), with meshed air vents acting as traps to retain emerged adults. After a period of settlement, gentle aeration, supplied by a short length of polyetheretherketone (PEEK) tubing was provided. The tubing position and aeration rate was set to minimise disturbance of the sediment and maintain dissolved oxygen (DO) levels ≥60% of air saturation value.
The test system was maintained at 20 ± 1°C by housing the test vessels in a temperature-controlled room. Photoperiod was controlled to provide 16 hours light:8 hours dark with 20 minute dawn and dusk transition periods.

Experimental design
Four test replicate vessels (nominated as A to D), each containing twenty larvae, were employed for each treatment.
After samples for sediment characterisation were removed, there was not enough control sediment remaining for the temperature vessel. Excess solvent control was used for the temperature vessel. This did not affect the outcome of the study.
The positions of the control, solvent control and test concentration groups were randomly allocated within the testing area.

Animal additions
After the 48 hour equilibration period, larvae 2-3 days post-hatch were added to the test vessels. At the start of the test, twenty larvae were impartially allocated to each replicate test vessel. Larvae (of a similar size) were selected and groups of 10 were added to collection vessels containing dilution water using a Pasteur pipette. Two collection vessels were impartially assigned to each test vessel. Larvae were then washed into the test vessels with the minimum volume of dilution water. A small volume of dilution water was used to gently rinse the collection vessel to ensure all animals were transferred. The collection vessels were examined microscopically after each addition to ensure no animals remained. To allow the larvae to settle on the sediment surface, test vessel aeration was stopped immediately prior to larval additions and started again approximately 24 hours post-additions.
No larvae were added to the Day 0 sacrificial replicates or the temperature check vessel.

Feeding
The chironomids were fed a suspension of Aquarian® Tropical Fish Flakes.
The Aquarian® suspension was prepared once a week by dispersing a known weight of (finely ground) Aquarian® in RO water and was stored in a refrigerator in the dark.
During the test, chironomids were fed daily, beginning within 24 hours after addition of larvae. The feeding rates were calculated based on a nominal 20 larvae per vessel.
The concentration of the Aquarian® suspension was adjusted such that a daily feed volume of 0.50 mL was used per replicate vessel, which acted as a practical balance for evaporative losses. There was no feeding on Day 28.

Test procedures
Visual assessments were made on the sediment and overlying water in the replicate vessels of each concentration at least 3 times per week throughout the exposure period. Any abnormal behaviour of the organisms (e.g. larvae leaving sediment or unusual swimming) was also assessed and noted if observed.
During the exposure, fully emerged organisms were counted, sexed and discarded daily. Males were identified by their plumose antennae. At the end of the test any visible pupae that had failed to emerge were recorded. If the total number of emerged adults in a replicate was less than the number initially added, then those organisms were considered dead.
The overlying water was not renewed during the test, however evaporative losses were replaced with RO water.
The test exposure duration was 28 days (post addition of test larvae).

Physical and chemical parameters
The pH and DO concentration of the dilution water were measured prior to addition to the sediment at the start of the equilibration phase (Day -2).
The mean TOC in a sample of sediment taken from the control mixing vessel was 0.5%. This is below the range given in the guideline (2.0 ± 0.5%) however as the validity criteria for the study were met this was not deemed to have affected the outcome of the study.
The pH and DO concentration of 20 mL samples from two replicates of each control and treatment were measured on Day 0, before addition of the test organisms and once per week on a sample (20 mL) from one replicate of each treatment. The volumes sampled were not replaced and a new water level mark was made on the vessel for evaporative loss checks. The temperature of one replicate of each control and treatment was measured on Day 0, before addition of the test organisms, and once per week in one replicate of each control and treatment during the test.
Temperature values were continuously measured by a max/min thermometer (checked at the start of the test against a liquid-in-glass thermometer) in an additional, dedicated vessel (without animals). Temperature extremes were recorded daily during the equilibration and test exposure phases. Maximum and minimum temperatures were not recorded on Day -1 in error as the thermometer had not been reset. As the current temperature on Day -1 and the temperatures recorded the day prior and days following were within the test range this was not deemed to have affected the outcome of the test.
At the end of the study (Day 28), temperature, pH and DO concentration measurements were made on all test replicates.
Light intensity was measured at the start of the equilibration phase, exposure Day 0 and Day 28 at the level of solution in the test vessels in a central position.
Total hardness and ammonium concentration of the overlying water for the control, solvent control, and highest test concentration were determined on exposure Day 0, using additional replicates intended for this purpose and in one test replicate of the control, solvent control and highest test concentration on Day 28.

Reference substance (positive control):

not specified

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

62.5 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

emergence rate

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

62.5 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

development rate

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

121 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

emergence rate

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

121 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

development rate

Details on results:

Biological data
The development time and the number emerged after 28 days were calculated for each vessel.
The two lowest concentrations, 31.3 and 62.5 mg/kg sediment dry weight, which were found to be The visual observations made on the test replicate vessels throughout the study showed the sediment of the nominal 1000 mg/kg sediment dry weight concentration remained undisturbed throughout the study, with no burrows or grazing marks visible. No grazing marks were visible in the nominal 500 mg/kg sediment dry weight concentration. There were no appreciable differences in appearance between the remaining treatments, with all showing evidence of burrows by Day 5. The overlying water clarity remained clear in all treatments until Day 7. The overlying water in replicates of the nominal 1000 mg/kg sediment dry weight concentration remained clear until Day 19 (one replicate remained clear until the end of the test). Turbidity of the overlying water was observed in all other treatments from Day 10 to the end of the test.

Emergence ratio
Emergence is expressed as the number of midges which have emerged as adults from each replicate at the end of the test. Since the sex of the midges is unknown at test initiation, emergence ratio is not distinguished by sex.
After 28 days adult emergence ratios in the control and solvent control were both 0.80. The control and solvent control data were compared using a two-tailed Equal Variance t-test. No significant difference (p <0.05) was found between the controls. All subsequent treatment comparisons were therefore made against the solvent control.
The total number emerging within each treatment was compared to the solvent control, using a Cochran-Armitage trend test (p <0.05). Significant differences were found in the nominal 125, 250, 500 and 1000 mg/kg sediment dry weight concentrations when compared to the solvent control. No significant differences were found in the nominal 31.3 or 62.5 mg/kg sediment dry weight concentrations.
ECx values with their associated confidence intervals were calculated using a non-linear regression model.

Development rate
For each replicate, development time and development rate were determined for each gender and as pooled gender. Development time is the time span between the introduction of larvae and the emergence of adults of that gender within the test chamber. The development rate is the reciprocal of the development time and was determined by summing the individual development rate for each emerged chironomid and dividing by the number emerged within each vessel.
The development rate could not be calculated for females in the nominal 1000 mg/kg sediment dry weight concentration as no emergence of females was observed. For all other treatments the development times and development rates were calculated for each gender and as pooled gender for each replicate.
The control and solvent control data were compared for each gender and pooled gender using two tailed Equal Variance t-tests (p <0.05). No significant differences were found between the control and solvent control for the development rate of males, females or pooled gender. All subsequent comparisons were therefore made against the solvent control.

Water quality and physical parameters
As part of the sediment preparation for the test, the pH of the sediment after overnight mixing was determined to be 6.24 (mean of 2 samples). This was outside of the range give in the study plan (7.0 ± 0.5) and the pH was not adjusted prior to use in error. As the validity criteria were met this was not deemed to have affected the outcome of the study.
The moisture content of the sediment, determined by wet to dry analysis was 31.8%. The total organic carbon content of the final sediment mixture was determined to be 0.5%.
pH values ranged from 7.65 – 8.27 and the DO levels ranged from 75.5 – 101.3% ASV (note, 100% ASV for freshwater at 20°C = 9.07 mg/L; Ref 4). Temperature range, recorded during the exposure in an additional temperature vessel was 20.3 to 20.8°C. The batch of dilution water used as overlying water had a pH of 8.21 and DO concentration of 95.8% ASV when sampled immediately prior to addition to the sediment.
Light intensity readings, taken in a central position, at overlying water surface level, on exposure Day -2, Day 0 and Day 28 were 524, 546 and 613 lux (cosine) respectively.

Analytical data
All analytical values are quoted to three significant figures and percentages to the nearest integer. The limit of quantification (LOQ) of SynNova Base Oil was 100 mg/kg sediment dry weight in sediment and 5 mg/L in overlying and pore water, defined as the lowest concentration tested at which an acceptable mean recovery with an acceptable relative standard deviation (RSD) was obtained. These LOQs were determined in a method validation study.
Although every effort was made to analytically support the test concentrations assessed, the LOQ for this study was determined to be higher than the lowest two test concentrations due to the nature of the test substance and the technique used. Therefore, it was known that analysis of the test substance in these concentrations would likely result in values that were At the start of the exposure period (Day 0) the measured concentrations in the sediment ranged from 93 to 105% of the nominal values, with the exception of the 31.3 and 62.5 mg/kg sediment dry weight concentrations which were All overlying water samples from Day 0, 7, 14 and 28 were determined to be The measured concentrations in the pore water were determined to be Analytical calibrations were constructed using a minimum of 5 calibration levels, with a minimum R2 value of 0.995. The maximum percentage difference from nominal concentration for standards at the LOQ is less than 20% and less than 15% at levels greater than the LOQ.
Measured concentrations in the control and solvent control in the sediment, overlying water and pore water were On the basis of the analytical data the initial measured concentration of the sediment (at test start; Day 0) were used in the calculation and reporting of results, as advised in the OECD 218 test guideline.

Validity criteria
The following validity criteria are specified in the OECD 218 test guideline:
1. The emergence in the control and solvent control (if used) will be at least 70% at the end of the test.
- In this study, the emergence was 80% in the control and 80% in the solvent control.
2. Emergence to adults from the control and solvent control (if used) vessels will occur between Days 12 and 23.
- In this study the control emergences occurred between Days 15 and 22 and the solvent control emergences and in this study occurred between exposure Days 15 and 23.
3. Dissolved oxygen concentration will be at least 60% of the air saturation value in all test vessels throughout the test.
- In this study, the DO concentration ranged from 75.5 to 101.3% ASV.
4. pH will be in the range 6 to 9 in all test vessels throughout the test.
- In this study, the pH ranged from 7.65 to 8.27.
5. Water temperature will not vary by more than 1°C throughout the exposure.
- In this study, temperature values recorded during the exposure phase ranged from 20.3 to 20.8°C.

Results with reference substance (positive control):

Postive control not utilised for this study.

Reported statistics and error estimates:

All statistical analyses were conducted using CETIS. Details of the statistical tests used are given with the results. For the reporting of results, the Lowest Observed Effect Concentration (LOEC) is defined as the lowest tested concentration which is observed to have a significant adverse effect (p <0.05), when compared with the solvent control. However, all test concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC. The No Observed Effect Concentration (NOEC) is defined as the test concentration immediately below the LOEC.
Where possible, estimates of the effect concentrations (ECx) along with their associated confidence intervals were calculated.

The emergence results, based on initial measured concentrations were:

	SynNova Base Oil (mg/kg sediment dry weight)	95% confidence limits	Method of determination
NOEC	62.5 (nominal concentration)	-	Cochran-Armitage Trend Test
LOEC	121	-
EC10	29.4	10.2-49.9	Nonlinear regression
EC20	62.2	43.4-82.2
EC50	193	160-231

 

Results for development rate (pooled males and females)

	SynNova Base Oil (mg/kg sediment dry weight)	95% confidence limits	Method of determination
NOEC	524	-	Dunnett Multiple Comparison Test (p <0.05)
LOEC	932	-
EC10	685	574-739	Linear interpolation
EC20	904	808-N/A
EC50	>932	N/A

 

Results for development rate (males)

	SynNova Base Oil (mg/kg sediment dry weight)	95% confidence limits	Method of determination
NOEC	524	-	Dunnett Multiple Comparison Test (p <0.05)
LOEC	932	-
EC10	579	276-660	Linear interpolation
EC20	820	657-886
EC50	>932	N/A

 

Results for development rate (females)

	SynNova Base Oil (mg/kg sediment dry weight)	95% confidence limits	Method of determination
NOEC	524	-	Dunnett Multiple Comparison Test (p <0.05)
LOEC	>524	-
EC10	>524	N/A	Linear interpolation
EC20	>524	N/A
EC50	>524	N/A

Validity criteria fulfilled:

yes

Conclusions:

The reported overall NOEC for the study, based on initial measured concentrations (where possible) and including emergence ratio and development rates was 62.5 mg/kg sediment dry weight (nominal concentration used due to high LOQ of analytical method) with an overall LOEC of 121 mg/kg sediment dry weight.

Executive summary:

At the request of the sponser, the toxicity of SynNova Base Oil on the larval development and emergence of the freshwater midge, Chironomus riparius, was determined. The study consisted of two separate runs.

The procedures employed were in accordance with the OECD 218 test guideline.

 

Test concentrations: Control, solvent control and nominal test substance concentrations of 31.3, 62.5, 125, 250, 500 and 1000 mg/kg sediment dry weight.

Control, solvent control and initial measured concentrations of <LOQ (nominally 31.3 mg/kg sediment dry weight), <LOQ (nominally 62.5 mg/kg sediment dry weight), 121, 232, 524 and 932 mg/kg sediment dry weight.

Although every effort was made to analytically support the test concentrations assessed, the LOQ for this study was set at 100 mg/kg sediment dry weight due to the nature of the test substance and the technique used. The LOQ was taken from the method validation study (Ref 3).

Sediment: Formulated sediment with an organic carbon content of 0.5%, equilibrated with overlying water for 2 days prior to exposure of organisms.

Length of test: 2 days sediment equilibration plus 28 days exposure

 

The reported overall NOEC for the study, based on initial measured concentrations (where possible) and including emergence ratio and development rates was 62.5 mg/kg sediment dry weight (nominal concentration used due to high LOQ of analytical method) with an overall LOEC of 121 mg/kg sediment dry weight.

Description of key information

The reported overall NOEC for the study, based on initial measured concentrations (where possible) and including emergence ratio and development rates was 62.5 mg/kg sediment dry weight (nominal concentration used due to high LOQ of analytical method) with an overall LOEC of 121 mg/kg sediment dry weight.

Key value for chemical safety assessment

EC50 or LC50 for freshwater sediment:

193 mg/kg sediment dw

EC10, LC10 or NOEC for freshwater sediment:

62.5 mg/kg sediment dw

Additional information

SynNova Base Oil on the larval development and emergence of the freshwater midge, Chironomus riparius, was determined. The study consisted of two separate runs.

The procedures employed were in accordance with the OECD 218 test guideline.

 

Test concentrations: Control, solvent control and nominal test substance concentrations of 31.3, 62.5, 125, 250, 500 and 1000 mg/kg sediment dry weight.

Control, solvent control and initial measured concentrations of <LOQ (nominally 31.3 mg/kg sediment dry weight), <LOQ (nominally 62.5 mg/kg sediment dry weight), 121, 232, 524 and 932 mg/kg sediment dry weight.

Although every effort was made to analytically support the test concentrations assessed, the LOQ for this study was set at 100 mg/kg sediment dry weight due to the nature of the test substance and the technique used. The LOQ was taken from the method validation study (Ref 3).

Sediment: Formulated sediment with an organic carbon content of 0.5%, equilibrated with overlying water for 2 days prior to exposure of organisms.

Length of test: 2 days sediment equilibration plus 28 days exposure

 

The reported overall NOEC for the study, based on initial measured concentrations (where possible) and including emergence ratio and development rates was 62.5 mg/kg sediment dry weight (nominal concentration used due to high LOQ of analytical method) with an overall LOEC of 121 mg/kg sediment dry weight.