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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 November 2014 to 30 January 2015
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)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
See test material information
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Water samples were taken from the control and all surviving test groups (replicates pooled) on Days 0, 1, 6, 7, 9, 10, 13, 14, 16, 17, 20, 21, 23, 24, 27, 28, 31 and 32 for quantitative analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.
- Sampling method: not reported
- Sample storage conditions before analysis: Day 0, 11 and 32 samples were analysed on the day of receipt whilst all other were stored frozen. Frozen samples were allowed to thaw at ambient temperature prior to analysis.
Duplicate samples were prepared at the same time and stored frozen for further testing, if required.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Prior to addition of the test item a glass siphon tube was placed in the test media. Nominal amounts of test item (32, 101, 320, 1010 and 3200 μL, equivalent to 23, 74, 230, 740 and 2300 mg respectively given that 100 μL of test was determined to weigh 73 mg) were each separately added to the surface of 23 litres 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. During the stirring phase the vessels were sealed with a minimal headspace. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A length of Tygon tubing was attached to the top of the glass siphon tube. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or W AF 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.
The concentration and stability of the test item in the test preparations were verified by chemical analysis (replicates pooled) on Days 0, 1, 6, 7, 9, 10, 13, 14, 16, 17, 20, 21, 23, 24, 27, 28, 31 and 32.

- Controls: The control group was maintained under identical conditions but not exposed to the test material.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n/a
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n/a
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): At the start of the mixing period all loading rates was observed to be clear colourless water columns, test item was not visible as the test vessels were completely filled and sealed for the duration of the stirring period. After 23 hours stirring and a 1 Hour standing period all loading rates were observed to remain as at the start of stirring. Microscopic inspection of the W AF showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, all loading rates were observed to be clear colourless solutions.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow
- Strain: not reported
- Source: The in-house breeding stock fish were acquired from Osage Catfisheries on 25 September 2014 and maintained in dechlorinated tap water in glass tanks with an activated carbon and biological filtration system.
- Age at study initiation (mean and range, SD): Freshly laid eggs less than 24 hours old on introduction into the test system.
- Length at study initiation (length definition, mean, range and SD): not reported
- Weight at study initiation (mean and range, SD): not reported
- Method of breeding: Each breeding unit consisted of one male and three females which were supplied with a piece of inverted plastic guttering for the fish to lay eggs on and be fertilised. Fertilised eggs were collected from the breeding tanks on 18 November 2014 and used for the definitive test.
- Feeding during test
- Food type: Trout pellets
- Amount: not reported
- Frequency: two portions per day

ACCLIMATION
- Acclimation period: not reported
- Acclimation conditions (same as test or not): not reported
- Type and amount of food: not reported
- Feeding frequency during acclimation: not reported
- Health during acclimation (any mortality observed): not reported
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
32 d
Hardness:
The water hardness values were observed to range from 106 to 112 mg/L as CaCO3 at the start of the test and from 108 to 126 mg/Las CaCO3 at termination of the test
Test temperature:
24.6 to 25.9 °C
pH:
7.5 - 8.6
Dissolved oxygen:
minimum 4.9 mg/L (equivalent to 59% ASV)
Salinity:
freshwater
Conductivity:
not reported
Nominal and measured concentrations:
Nominal: 1.0, 3.2, 10, 32 and 100 mg/L
Measured: at the 100 mg/L loading rate, the average measured concentration of test item across the test duration was less than the LOQ (0.02 mg/L).
Details on test conditions:
The test was carried out using freshly laid eggs of fathead minnows (Pimephales promelas).
The water temperature was controlled at approximately 25 °C with a dissolved oxygen content of greater than or equal to 7.8 mg O2 /L.
The breeding stock fish were fed with trout pellets.
Each breeding unit consisted of one male and three females which were supplied with a piece of inverted plastic guttering for the fish to lay eggs on and be fertilized. Fertilized eggs were collected from the breeding tanks on 18 November 2014 and used for the definitive test. The eggs were less than 24 hours old on introduction into the test system.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

In the definitive test, for each control and test concentration 20 eggs were placed in 400 mL of test preparation in 1000 mL glass vessels. At the media renewal from Day 10 onwards the test volume was increased to 800 mL of test preparation in 1000 mL glass vessels and from Day 13 onwards the test volume was increased to 4000 mL of test preparation in 5000 mL glass vessels.
Test vessels were covered to reduce evaporation throughout the exposure period.
The number of fertilized eggs introduced per concentration was 80 i.e. 20 per replicate.
The control group was maintained under identical conditions but not exposed to the test material.
A semi-static test regime was employed in the test involving a daily renewal of the test preparations to ensure that the concentrations of the test item remained near nominal and to prevent the build-up of nitrogenous waste products.
The test vessels were maintained at approximately 25 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods throughout the duration of the test. The test vessels received no auxiliary aeration until Day 17 of the exposure, when aeration was provided to the test vessels via narrow bore glass tubes. The eggs and larvae were not individually identified.
The start of hatching was observed on Day 4 of the test and completion of hatching on Day 6.
The larvae were fed less than 24 hour old brine shrimp nauplii only from Day 6 to Day 15. On Day 16 onwards, the larvae were fed 24-48 hour old brine shrimp nauplii.

TEST SYSTEM
- Test vessel: Glass vessel
- Size of vessel: 1000 ml
- Type (delete if not applicable): closed: Test vessels were covered to reduce evaporation throughout the exposure period.
- Material, size, headspace, fill volume: In the definitive test, for each control and test concentration 20 eggs were placed in 400 mL of test preparation in 1000 mL glass vessels. At the media renewal from Day 10 onwards the test volume was increased to 800 mL of test preparation in 1000 mL glass vessels and from Day 13 onwards the test volume was increased to 4000 mL of test preparation in 5000 mL glass vessels.
- Aeration: The test vessels received no auxiliary aeration until Day 17 of the exposure, when aeration was provided to the test vessels via narrow bore glass tubes
- Type of flow-through (e.g. peristaltic or proportional diluter): n/a
- Renewal rate of test solution (frequency/flow rate): Daily renewal
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): n/a
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the definitive test was laboratory tap water dechlorinated by passage through an activated carbon filter (Elga ACl) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/Las CaCO3 . After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Conductivity: 411.7 μS/cm at 20°C prior to treatment
- Culture medium different from test medium:
- Intervals of water quality measurement: The water temperature, pH, light intensity and dissolved oxygen concentrations were recorded daily throughout the test. The measurements on Day 0, and after each test media renewal, represent those of the freshly prepared test concentrations while the measurements taken prior to each test media renewal and on termination of the test represent those of the used or 24-Hour old test preparations. The temperature was also monitored approximately every hour in control replicate R1.
The water hardness in each vessel was measured at the start and on termination of the test and was determined using the methods described in Fields and On-Site Methods for Analysis of Water (British Standards Institution, 1993).

OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Light intensity: 601 to 636 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The number of dead eggs (up to completion of hatching), dead and live larvae and sub-lethal effects of exposure were recorded daily. The criteria of death for eggs were marked loss of translucency and change in coloration leading to a white opaque appearance. The criteria of death for larvae and juvenile fish were one or more of the following: immobility, absence of respiratory movement, absence of heart beat, white opaque colouration and lack of reaction to mechanical stimulus.
At the end of the test the surviving fish were sacrificed and the length and weight (wet weight) determined for each fish.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: n/a

RANGE-FINDING STUDY
- Test concentrations: none reported
- Results used to determine the conditions for the definitive study: none reported
Reference substance (positive control):
not specified
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
other: hatching, survival and growth.
Details on results:
The number of dead eggs and larvae were observed to be low throughout the duration of the test with no concentration dependent effects being observed.
The start of egg hatching was observed to be 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.
Statistical analysis of the hatching success, post hatch survival and overall survival data showed no significant differences (p>=0.05) between the control and all the test concentrations.
Statistical analysis of the length and wet weight data showed no significant differences (p>=0.05) between the control group data and all the test concentrations.
There were no sub-lethal effects observed in the test.
There were no statistically significant differences (P>=0.05), between the control group data and all test groups in relation to hatching success, overall survival, wet weight and body length at the 100 mg/L loading rate WAF, therefore the "Lowest Observed Effect Loading Rate" (LOEL) was not determined.
As the LOEL was not determined it was not possible to determine the "No Observed Effect Loading Rate" (NOEL), this was therefore regarded to be equal to or greater than 100 mg/L loading rate WAF.
Reported statistics and error estimates:
For the estimation of the "Lowest Observed Effect Loading Rate" (LOEL) and the "No Observed Effect Loading Rate" (NOEL) the length and wet weight control and each test group data obtained on termination of the test were compared using Williams Multiple Sequential t-test respectively. The hatching and survival data for the control and each test group were compared using the Chi Squared 2x2 Table Test with Bonferroni Correction. All results were calculated using the ToxRat Professional computer software package (ToxRat).

Table: Hatching Rates, Survival Rates and Number of Healthy Fish at the End of the Test in the Definitive Test

 Nominal Loading Rate (mg/l)   Mean Hatching Rate (%)   Mean Survival Rate (%)  Total No. Healthy Fish at End of Test
 Control  91  100  73
 1.0  93  95  74
 3.2  95  100  76
 10  93  96  74
 32  95  96  76
 100  96  99  77

Table: Mean Length and Wet Weight of Fish per Concentration at the End of the Exposure Period

Nominal Loading Rate (mg/l)  Mean Length (mm)  Mean Wet Weight (mg)
 Control  19.4  55
 1.0  20.15  61.35
 3.2  19.67  58.3
 10  19.5  59.03
 32  19.71  59.05
 100  19.61  55.4
Validity criteria fulfilled:
yes
Conclusions:
Measured toxicity data are available for Shell GTL Solvent GS190 (Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics) to the freshwater fish Pimephales promelas. The test was conducted under semi-static (daily renewal of the test media) conditions in accordance with OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test) and EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test). Appropriate modifications to the test and media preparation procedures were made to take account of the test substance containing multiple constituents, having low solubility in water and being potentially volatile. No significant effect on the hatching, survival or growth of Pimephales promelas were observed after 32 days exposure to the test medium prepared as a water-accommodated fraction (WAF) at a loading rate of 100 mg/l; 32 day NOEC value was ≥100 mg/l. The results of the test are considered to be reliable.
Executive summary:

Introduction

A study was performed to assess the effects of the test item on freshly hatched larvae 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" and the US EPA Draft Ecological Effects Test Guideline OCSPP 850.1400.

 

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 (W AF). Based on data supplied by the Sponsor, newly laid eggs were exposed to a WAF 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 32 days at a temperature of approximately 25 °C under semi-static test conditions. The test solutions were renewed daily throughout the test.

The number of mortalities or any sub-lethal effects of exposure in each test and control vessel were recorded daily until termination of the test (28 days post-hatch). At test termination the length and wet weight of the surviving fish were measured.

 

Results

Chemical analysis of the test preparations showed measured test concentrations to range from less than the limit of detection (LOD) of the analytical method employed, which was determined to be 0.0059 mg/L, to 0.0369 mg/L. The majority of the measured concentrations determined were between the LOD and the limit of quantification (LOQ) of the analytical method employed which was determined to be 0.023 mg/L. Measured concentrations between the LOD and LOQ should be interpreted with caution as the recovery data at this level supports the conclusion that whilst test item can be detected, this is only indicative of test item in solution and is not accurately quantifiable. Given that toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results are typically expressed based on nominal loading rates. Additionally, at the 100 mg/L loading rate, the average measured concentration of test item across the test duration was less than the LOQ (0.02 mg/L).

There were no significant effects resulting from the exposure of fathead minnow (Pimephales promelas) eggs and larvae throughout the test including the highest loading rate of 100 mg/L, therefore the Lowest Observed Effect Loading Rate was not determined. Consequently, it was not possible to determine the No Observed Effect Loading Rate was not determined, this was therefore regarded to be equal to or greater than 100 mg/L loading rate W AF.

 

Conclusion

The application of the test item to fathead minnow eggs and larvae was considered to have no significant effect on the hatching, survival or growth.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Description of key information

The Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics measured 32 day NOEC value was ≥100 mg/l based on hatching, survival and growth of Pimphales promelas.

Key value for chemical safety assessment

Additional information

No measured long-term toxicity to fish data are available for Hydrocarbons, C11-C16, n-alkanes, isoalkanes, <2% aromatics, but data are available for a closely related Fischer-Tropsch process-derived substance.

Measured toxicity data are available for Shell GTL Solvent GS190 (Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics) to the freshwater fish Pimephales promelas. The test was conducted under semi-static (daily renewal of the test media) conditions in accordance with OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test) and EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test). Appropriate modifications to the test and media preparation procedures were made to take account of the test substance containing multiple constituents, having low solubility in water and being potentially volatile. No significant effect on the hatching, survival or growth of Pimephales promelas were observed after 32 days exposure to the test medium prepared as a water-accommodated fraction (WAF) at loading rates up to 100 mg/l; 32 day NOEC value was ≥100 mg/l. The results of the test are considered to be reliable.