Alkenes, C15-18 α-, sulfurized

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 07 January 2021 Experimental completion date: 14 October 2021

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:

please refer to "Any other information on Materials and Methods".

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: ALKENES, C15-18 ALPHA-, SULFURIZED
CAS Number: 67762-55-4
EC Number: 267-023-8
Batch: 0720HI434
Purity: 100% UVCB
Physical state / Appearance: Dark red/brown liquid
Expiry date: 01 July 2022
Storage conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and all surviving test groups from the freshly prepared bulk test preparation on Days 0, 6, 13, 20, 27 and 32 and from the old or expired media on Days 1, 7, 14, 21, 28 and 33 taken from one replicate (cycling between replicates throughout the test) for quantitative analysis. The Day 0 and Day 27 samples were stored frozen prior to analysis, all other samples were sent for immediate quantitative analysis.

Vehicle:

no

Details on test solutions:

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a WAF of the test item.

Nominal amounts of test item (20, 64, 200, 640 and 2000 mg) were each separately added to the surface of 20 L 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 mixtures allowed to stand for 1 hour. The aqueous phase or WAF was removed by mid depth siphoning (the first 75 to 100 mL discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro dispersions or undissolved test item to be present. Photographic images were taken weekly of the control and 100 mg/L loading rate WAF (Fresh) when being viewed under the microscope.

The control group was maintained under identical conditions but not exposed to the test item.

Test organisms (species):

Pimephales promelas

Details on test organisms:

The test was carried out using newly fertilized eggs of fathead minnows (Pimephales promelas). The adult fathead minnows that produced the eggs for the test were obtained from Osage Catfisheries Inc. Missouri, USA and maintained in house since 13 May 2021 and maintained in dechlorinated tap water with an activated carbon and biological filtration system.

The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. In the 7 days preceding the start of the test, the water temperature was controlled at approximately 24 to 26 °C with a dissolved oxygen content of greater than or equal to 6.6 mg O2/L. The breeding stock fish were fed daily with frozen brine shrimp and/or ZM small granular fish feed.

Each breeding tank was supplied with inverted plastic guttering for the fish to lay eggs on and be fertilized. Fertilized eggs were collected from the breeding tanks on 17 June 2021 and used for the definitive test. The eggs were visually inspected before introduction into the test system and were identified as being at early blastodisc stage.

The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Remarks:

The test water used for the definitive test was laboratory tap water dechlorinated by passage through an activated carbon filter (Fleck 2750 Duplex Dechlorination unit) and partly softened (Elga Nimbus 1248D Duplex Water Softener).

Limit test:

no

Total exposure duration:

33 d

Hardness:

The water hardness values were observed to range from 156 to 180 mg/L as CaCO3 at the start of the test and from 140 to 158 mg/L as CaCO3 at termination of the test.

Test temperature:

The temperature measurements recorded in a surrogate vessel by a Testo temperature logger and were shown to have been maintained at between 24 and 25 °C.

pH:

The pH ranged from 7.5 to 8.5.

Dissolved oxygen:

The dissolved oxygen concentration was maintained at least at 5.1 mg/L (equivalent to approximately 62% ASV at 25 ºC) .

Salinity:

Freshwater study.

Nominal and measured concentrations:

Nominal amounts of test item (20, 64, 200, 640 and 2000 mg) were each separately added to the surface of 20 L of test water to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively.

Details on test conditions:

Exposure Conditions
In the definitive test 1 L glass vessels were used from Day 0 to Day 14 and from Day 15 to the end of the test 5 L glass vessels were used. The approximate volume of test preparation in each vessel was 400 mL from Day 0 to Day 6, 800 mL from Day 7 to Day 14 and 4000 mL from Day 15 to Day 32. Four replicate flasks were used for each control and test concentration.
A semi static test regime was employed in the test, this involved renewing the test preparations daily from the start of the test, with the exception of Day 3 where there was no water change to avoid causing premature hatching of the eggs.

Twenty eggs were placed into each replicate test vessel and the vessels covered to reduce evaporation. The test vessels were maintained at 25 °C with a maximum deviation of ± 1.5 °C between test chambers or between successive days with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 33 days.
The test vessels were aerated via narrow bore glass tubes from Day 8 onwards. The eggs and larvae were not individually identified.

The larvae were fed freshly hatched special grade brine shrimp nauplii from Day 7 to the end of the test.

Reference substance (positive control):

no

Key result

Duration:

33 d

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Post-hatch survival

Remarks on result:

not determinable

Key result

Duration:

33 d

Dose descriptor:

NOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Post-hatch survival

Remarks on result:

not determinable

Key result

Duration:

33 d

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks on result:

not determinable

Key result

Duration:

33 d

Dose descriptor:

NOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks on result:

not determinable

Details on results:

Number of Eggs Hatching
The number of dead eggs observed was low throughout the test with no concentration dependent effects being observed.

The start of egg hatching was observed on Day 4 of the test and completion of hatching was observed on Day 6 of the test. There were no significant mortalities or sub lethal effects of exposure observed in any of the test concentrations.

There were no statistically significant differences (P≥ 0.05), between the control and each loading rate WAF test group and therefore the NOEL based on the number of eggs hatching was 100 mg/L loading rate WAF. Correspondingly the LOEL based on body length could not be determined as the NOEL was at the highest leading rate tested.

Post-hatch Survival
The number of dead larvae were observed to be low throughout the duration of the test with no concentration dependent effects being observed.
There were no significant mortalities or sub-lethal effects of exposure observed in any of the test concentrations.

There were no statistically significant differences (P≥ 0.05), between the control and each loading rate WAF test group and therefore the NOEL based on post-hatch survival was 100 mg/L loading rate WAF. Correspondingly the LOEL based on body length could not be determined as the NOEL was at the highest leading rate tested.

Length and Weight Data
Statistical analysis of the fish body length data was carried out for the control and all loading rates. There were no statistically significant differences (P ≥ 0.05), between the control and each loading rate WAF test group and therefore the NOEL based on body length was 100 mg/L loading rate WAF. Correspondingly the LOEL based on body length could not be determined as the NOEL was at the highest leading rate tested.

Statistical analysis of the fish wet weight data was carried out for the control and all loading rates. There were no statistically significant differences (P≥ 0.05), between the control and each loading rate WAF test group and therefore the NOEL based on wet weight was 100 mg/L loading rate WAF. Correspondingly the LOEL based on body length could not be determined as the NOEL was at the highest leading rate tested.

Critical examination of the data, means and standard deviations, of all the test groups, showed the standard deviations of all groups to overlap each other as well as the overall means of length, wet weight.

Given this information and data assessment above it was considered that no effect on survival or growth attributable to the test item was observed.

Observations
The abnormal observations were seen in the control, 1.0, 32 and 100 mg/L test groups and included the following observations; kinked spine, curved spine and small.

There were no sub-lethal effects observed in the 1.0, 3.2 and 10 mg/L test groups throughout the test.

Range-finding Test

The results showed there was no difference between the control and test concentrations of  1.0 and 10 mg/L loading rate WAF in terms of hatching, survival and growth, however, slight differences in terms of eggs hatched and fish surviving may have been apparent at a test concentration of 100 mg/L loading rate WAF. A dose relationship was not apparent in relation to fish weights at the end of the test.

Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF were selected for the definitive test.

Chemical analysis of the fresh test preparations on Days 0, 7 and 14 showed measured test concentrations of 0.32 to 3.7 mg/L.  Measured test concentration of the corresponding aged test preparations on Days 1, 8 and 15 showed measured test concentrations of none detected to 3.7 mg/L indicating that the test item was not stable under test conditions.

 

Definitive Test

Chemical Analysis of Test Loading Rates

Analysis of the fresh test preparations on days 0, 6, 13, 20, 27 and 32 showed measured test concentrations to range from 0.034 to 0.74 mg/L.  A decline in measured test concentration of the aged test preparations on days 1, 7, 14, 21, 28 and 33 was observed to be between none detected and 0.20 mg/L. 

The dissolved test item may have been one or several components of the test item.  Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only (OECD GD 23, 2019 s 7.9.2.5).

 

Validation Criteria

The following validity criteria were achieved during the test:

	Required	Achieved
1)    Dissolved oxygen	Equal to or greater than 60% ASV	62% ASV
2)    Water temperature
Between test chambers	± 1.5 ^oC	± 2.6 ^oC
Between successive days	± 1.5 ^oC	± 2.5 ^oC
3)    Hatching success rate*	>70%	95%
4)    Post-hatch survival*	>75%	95%

 

* In control vessels

 

 

Vortex Depth Measurements

The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

Observations on Test Item Solubility

Observations on the test media were carried out during the mixing and testing of the WAFs.

After dosing all control and test concentrations were observed to be clear, colorless solutions by visual inspection.

 

Photographic Image of Treatment Groups

For illustrative purposes only, collective photographic images of the control and 100 mg/L loading rate WAF treatment groops are given.

The images illustrate that the 100 mg/L loading rate WAF preparation did not contain any undissolved test item and as such the images at 100 mg/L loading rate were comparable to the control.

Validity criteria fulfilled:

yes

Conclusions:

Over the duration of the test there were no significant mortalities or sub-lethal effects of exposure resulting from the exposure of the early-life stages of fathead minnow (Pimephales promelas) larvae up to a test concentration of 100 mg/L loading rate WAF.

Executive summary:

Introduction

A study was performed to assess the effects of the test item on the early life stages of the fathead minnow (Pimephales promelas).  The method followed that described in the OECD Guidelines for Testing of Chemicals (2013) No 210, "Fish, Early-Life Stage Toxicity Test”.

Results

Analysis of the fresh test preparations on days 0, 6, 13, 20, 27 and 32 showed measured test concentrations to range from 0.034 to 0.74 mg/L.  A decline in measured test concentration of the aged test preparations on days 1, 7, 14, 21, 28 and 33 was observed to be between none detected and 0.20 mg/L. 

The dissolved fraction of the WAF test solution may have been comprised of one or several components of the test item. 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 (OECD GD 23, 2019 s 7.9.2.5).

Conclusion

Over the duration of the test there were no significant mortalities or sub-lethal effects of exposure resulting from the exposure of the early-life stages of fathead minnow (Pimephales promelas) larvae up to a test concentration of 100 mg/L loading rate WAF. 

Description of key information

A study was performed to assess the effects of the test item on the early life stages of the fathead minnow (Pimephales promelas). The method followed that described in the OECD Guidelines for Testing of Chemicals (2013) No 210, "Fish, Early-Life Stage Toxicity Test”.

Results

Analysis of the fresh test preparations on days 0, 6, 13, 20, 27 and 32 showed measured test concentrations to range from 0.034 to 0.74 mg/L. A decline in measured test concentration of the aged test preparations on days 1, 7, 14, 21, 28 and 33 was observed to be between none detected and 0.20 mg/L.

The dissolved fraction of the WAF test solution may have been comprised of one or several components of the test item. 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 (OECD GD 23, 2019 s 7.9.2.5).

Conclusion

Over the duration of the test there were no significant mortalities or sub-lethal effects of exposure resulting from the exposure of the early-life stages of fathead minnow (Pimephales promelas) larvae up to a test concentration of 100 mg/L loading rate WAF.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

> 100 mg/L