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Long-term toxicity to fish

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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:
NOELR
Effect conc.:
>= 100 other: mg/l loading rate Water-accommodated fraction
Nominal / measured:
nominal
Conc. based on:
test mat.
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.

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-10-16 - 2009-12-16
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:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of GLP inspection: 15 September2009 Date if signature on GLP certificate: 26 November 2009
Analytical monitoring:
no
Details on sampling:
Chemical analysis of test loading rates
The test material concentrations in the test preparations were not determined by compound specific analysis at the request of the Sponsor.

Total organic carbon analysis
Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from the control and each test group (Replicates R1 and R2 pooled) on Days 0 (fresh media), 4, 7, 11, 14, 18, 21, 25, 28, 32 (old and fresh media) and 33 (old media). Duplicate samples were taken and stored at approximately -20 ºC for further analysis if necessary.

A semi-static test regime was employed in the test involving renewal of the test preparations twice weekly to ensure that the concentrations of the test material remained near nominal and to prevent the build-up of nitrogenous waste products.

Vehicle:
no
Details on test solutions:
Validation of mixing period
Pre-study investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances, in the WAF.
A WAF of nominal loading rate of 100 mg/L was prepared, in duplicate, in dechlorinated tap water. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for Total Organic Carbon analysis.

Definitive test
Information provided by the Sponsor gave an EL/LL50 of greater than 100 mg/L. Therefore, as toxicity was not expected, the number of test concentrations was reduced from five, as recommended in the Test Guideline, to three (10, 32 and 100 mg/L loading rate WAF). This was considered not to affect the validity of the study and reducing the number of test concentrations also reduced the number of fish used which was consistent with the Sponsors Animal Welfare policy.

Experimental Preparation
Due to the low aqueous solubility and complex nature of the test material for the purposes of the definitive test the test material was prepared as a Water Accommodated Fraction (WAF).
Amounts of test material (110, 352 and 1100 mg) were each separately added to the surface of 11 L of dechlorinated tap water to give the 10, 32 and 100 mg/L loading rates respectively. The stirring vessels were sealed with minimal headspace to reduce losses due to the possible volatile nature of the test material. After the addition of the test material, the dechlorinated tap 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 71 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. 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. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present.
Following siphoning the WAFs were dispensed to two test vessels to give replicates R1 and R2.
The control group was maintained under identical conditions but not exposed to the test material.
Total Organic Carbon (TOC) analysis was performed on the fresh media on Day 0, old and fresh media on Days 4, 7, 11, 14, 18, 21, 25, 28, 32 and old media on Day 33.

Vortex depth measurements
The vortex depth was recorded at the start and end of each mixing period.

Total organic carbon analysis
Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from the control and each test group (Replicates R1 and R2 pooled) on Days 0 (fresh media), 4, 7, 11, 14, 18, 21, 25, 28, 32 (old and fresh media) and 33 (old media). Duplicate samples were taken and stored at approximately -20 ºC for further analysis if necessary.


Exposure conditions
Glass exposure vessels containing approximately 300 mL of test media were used for each control and test vessel. The test volume was increased to 2 L on Day 14 of the test to accommodate the growing larvae. The test vessels were filled with minimal headspace and covered to reduce losses through volatility. At the start of the test 30 eggs were placed in each test vessel. Egg baskets were not used due to the small volumes used and to minimise bacterial build up.
The number of fertilised eggs introduced per concentration was 60 ie. 30 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 renewal of the test preparations twice weekly to ensure that the concentrations of the test material remained near nominal and to prevent the build-up of nitrogenous waste products.
The test vessels were maintained at 25ºC ± 2°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 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 5. The larvae were fed protozoan (Paramecia micronucleatum) only from Day 5 to Day 7 as the larvae were too small at this time to feed on brine shrimp nauplii. The protocol stated that the larvae would be fed both protozoan (Paramecia micronucleatum) and brine shrimp nauplii on one day to avoid a sudden change from one food source to another. This was not carried out in error. This was considered not to affect the test as the larvae were considered sufficiently large to eat brine shrimp nauplii and no adverse effects were observed. On Day 8 and throughout the remainder of the test the larvae were fed brine shrimp nauplii only.
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 coloration and lack of reaction to mechanical stimulus.
Test organisms (species):
Pimephales promelas
Details on test organisms:
Test Species
The test was carried out using freshly laid eggs of fathead minnows (Pimephales promelas). The in­house breeding stock fish were from a batch bred on 30 June 2009 and were maintained in dechlorinated tap water in glass tanks 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. The water temperature was controlled at approximately 25 ºC with a dissolved oxygen content of greater than or equal to 8.0 mg O2/L. The breeding stock fish were fed ZM 400 flake food daily.
Each breeding tank was supplied with inverted plastic guttering for the fish to lay eggs on and be fertilised. Fertilised eggs were collected from the breeding tanks on 12 November 2009 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.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
33 d
Hardness:
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 Standard Institution 1993).
The water hardness values were observed to range from 134 to 144 mg/L as CaCO3 at the start of the test and from 132 to 140 mg/L as CaCO3 at termination of the test.
Test temperature:
Temperature was maintained at 25 ± 2 °C throughout the test.
pH:
There were no treatment related differences for pH.
The pH and the dissolved oxygen concentration were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter.
Dissolved oxygen:
There were no treatment related differences for oxygen concentration.
The pH and the dissolved oxygen concentration were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter.
Salinity:
Freshwater used.
Nominal and measured concentrations:
Nominal loading rates of 10, 32 and 100 mg/L.
The test material concentrations in the test preparations were not determined by compound specific analysis at the request of the Sponsor.

Total organic carbon analysis
Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from the control and each test group (Replicates R1 and R2 pooled) on Days 0 (fresh media), 4, 7, 11, 14, 18, 21, 25, 28, 32 (old and fresh media) and 33 (old media). Duplicate samples were taken and stored at approximately -20 ºC for further analysis if necessary.
Details on test conditions:
Physico-chemical measurements
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 old test preparations. The pH and the dissolved oxygen concentration were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The temperature was also monitored approximately every hour in Control Replicate R1 using a Testo temperature logger. The light intensity was measured using a digital luxmeter.
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 Standard Institution 1993).

The test was carried out using freshly laid eggs of fathead minnows (Pimephales promelas). The in­house breeding stock fish were from a batch bred on 30 June 2009 and were maintained in dechlorinated tap water in glass tanks with an activated carbon and biological filtration system.

Exposure conditions
Glass exposure vessels containing approximately 300 mL of test media were used for each control and test vessel. The test volume was increased to 2 L on Day 14 of the test to accommodate the growing larvae. The test vessels were filled with minimal headspace and covered to reduce losses through volatility. At the start of the test 30 eggs were placed in each test vessel. Egg baskets were not used due to the small volumes used and to minimise bacterial build up.
The number of fertilised eggs introduced per concentration was 60 ie. 30 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 renewal of the test preparations twice weekly to ensure that the concentrations of the test material remained near nominal and to prevent the build-up of nitrogenous waste products.
The test vessels were maintained at 25ºC ± 2°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 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 5. The larvae were fed protozoan (Paramecia micronucleatum) only from Day 5 to Day 7 as the larvae were too small at this time to feed on brine shrimp nauplii. The protocol stated that the larvae would be fed both protozoan (Paramecia micronucleatum) and brine shrimp nauplii on one day to avoid a sudden change from one food source to another. This was not carried out in error. This was considered not to affect the test as the larvae were considered sufficiently large to eat brine shrimp nauplii and no adverse effects were observed. On Day 8 and throughout the remainder of the test the larvae were fed brine shrimp nauplii only.
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 coloration and lack of reaction to mechanical stimulus.

Test Water
The test water used for the definitive test was laboratory tap water dechlorinated by passage through an activated carbon filter (Elga AC1) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
Reference substance (positive control):
no
Duration:
33 d
Dose descriptor:
NOELR
Effect conc.:
100 other: mg/l loading rate Water-accommodated fraction
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: There were no significant reductions (P≥0.05) in terms of fish length and dry weight when compared to the control at the end of the test.
Duration:
33 d
Dose descriptor:
LOELR
Effect conc.:
> 100 other: mg/l loading rate Water-accommodated fraction
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: There were no significant reductions (P≥0.05) in terms of fish length and dry weight when compared to the control at the end of the test.
Details on results:
The “Lowest Observed Effect Loading Rate” (LOEL), based on nominal loading rates, was considered to be greater than 100 mg/L loading rate WAF on the basis that there were no significant reductions (P≥0.05) in terms of fish length and dry weight when compared to the control at the end of the test.
The “No Observed Effect Loading Rate” (NOEL), based on nominal loading rates, was considered to be 100 mg/L loading rate WAF on the basis that there were no significant reductions (P≥0.05) in terms of fish length and dry weight when compared to the control at the end of the test.

Validation of Mixing Period
Pre-study investigational work indicated that there was an increase in the amount of total organic carbon by extending the preparation period from 24 to 96 hours. At the request of the Sponsor a preparation period of 72 hours was used to maintain consistency with other studies conducted on the test material.

Definitive Test

Observations
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 mean hatching success rate for the control group was 94% thereby satisfying the validation criterion of greater than 75% hatching rate. The mean hatching rate for the test loading rates was 92% to 95%.
The mean survival rate of the larvae for the control group was 98% thereby satisfying the validation criterion for post-hatch survival success rate of greater than 70%. The mean survival rates for the test loading rates ranged between 93% and 97% .
The start of egg hatching was observed to be on Day 4 of the test and completion of hatching was observed on Day 5 of the test.
There were no significant mortalities or sub-lethal effects of exposure observed in any of the test concentrations.

Sub-lethal Effects
There were no sub-lethal effects observed in the test.

Length and dry weight data
Statistical analysis of the length and dry weight data by analysis of variance showed the 32 and 100 mg/L loading rate WAFs not to be significantly different (P≥0.05) from the control group.
However, the 10 mg/L loading rate WAF test group was significantly different (P<0.05) from the control group in that the larvae were greater in length and dry weight compared to the control.
The observed significant differences between the control group and the 10 mg/L loading rate WAF test group were considered to be due to the presence of a small number of larger fish in the 10 mg/L loading rate WAF test group. This was considered to be due to the normal variation observed in a natural population of organisms. The larval length and dry weight of the 10 mg/L loading rate WAF test group was not significantly reduced compared to the control group therefore these differences were considered not to affect the outcome of the study.
Given this information and data assessment above it was considered that no effect on survival or growth attributable to the test material was observed.

Lowest observed effect loading rate
The “Lowest Observed Effect Loading Rate” (LOEL), based on nominal loading rates, was considered to be greater than 100 mg/L loading rate WAF on the basis that there were no significant reductions (P≥0.05) in terms of fish length and dry weight when compared to the control at the end of the test.

No observed effect loading rate
The “No Observed Effect Loading Rate” (NOEL), based on nominal loading rates, was considered to be 100 mg/L loading rate WAF on the basis that there were no significant reductions (P≥0.05) in terms of fish length and dry weight when compared to the control at the end of the test.

Vortex depth measurements
The vortex depth was recorded at the start and end of each mixing period and was observed to be a dimple at the water surface on each occasion.

Observations on test material solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of each mixing period the 10, 32 and 100 mg/L loading rates were observed to be clear, colourless water columns with test material floating on the surface. At the end of each mixing period, and after the 1-Hour settlement period the 10, 32 and 100 mg/L loading rates were observed to be clear, colourless water columns with a thin layer of test material at the surface. After siphoning and for the duration of the test, the 10, 32 and 100 mg/L loading rates were observed to be clear, colourless solutions. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present.

Physico-chemical measurements
Temperature was maintained at 25ºC ± 2°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.
The water hardness values were observed to range from 134 to 144 mg/L as CaCO3 at the start of the test and from 132 to 140 mg/L as CaCO3 at termination of the test.

Total organic carbon analysis
Total Organic Carbon (TOC) analysis of the test preparations showed no significant differences in the amount of carbon present within the 10, 32 and 100 mg/L loading rate WAF test vessels when compared to the control vessels. This was expected due to the low aqueous solubility of the test material. Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that the majority of the results were around the limit of quantitation of the analytical method.
The dissolved test material may have been one or several components of the test material. Given that toxicity cannot be attributed to a single component or mixture of components but to the test material as a whole, the results were based on nominal loading rates only.
Reported statistics and error estimates:
Evaluation of data
For the estimation of the “Lowest Observed Effect Loading Rate” (LOEL) and the “No Observed Effect Loading Rate” (NOEL) the length and dry weight data obtained on termination of the test for the control and each test group were compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Please see Attachment section for:

Table 1          Number of Dead Eggs in the Definitive Test                                                

Table 2          Number of Dead Larvae in the Definitive Test                                             

Table 3          Cumulative Number of Hatched (Live) Larvae in the Definitive Test      

Table 4          Number of Hatched (Live) Larvae (Non-cumulative) in the Definitive Test

Table 5          Hatching and Survival Rates in the Definitive Test                                    

Table 6          Vortex Depth Measurements at the Start and End of Each Mixing Period  

Figure 1        Temperature Measurements Recorded Over the Duration of the Test Using a Using a Testo Data Logger.

                                                                                               

Appendix 1   Typical Water Quality Characteristics                                                            

Appendix 2   Validation of Mixing Period                                                                                           

Appendix 4   Statistical Analysis                                                                                            

Appendix 5   Fish Length and Weight Values                                                                     

Appendix 6   Physico-Chemical Measurements                                                                 

Appendix 7   Water Hardness Values                                                                                   

Appendix 3     Total Organic Carbon Analysis

RESULTS

Samples

Nominal Loading Rate

(mg/L)

Concentration of TOC

(mg C/L)

Concentration of TOC Corrected for Control

(mg C/L)

Day 0

(Fresh Media)

Control

2.03

-

10

2.67

0.64

32

2.93

0.90

100

2.27

0.24

Day 4

(Old Media)

Control

3.55

-

10

3.81

0.26

32

2.87

<Control

100

3.40

<Control

Day 4

(Fresh Media)

Control

1.74

-

10

1.61

<Control

32

2.10

0.36

100

2.44

0.70

Day 7

(Old Media)

Control

2.32

-

10

2.59

0.27

32

2.23

<Control

100

2.75

0.43

Day 7

(Fresh Media)

Control

1.37

-

10

1.89

0.52

32

1.72

0.35

100

2.43

1.06

 


 Appendix 3 (continued)  Total Organic Carbon Analysis

Samples

Nominal Loading Rate

(mg/L)

Concentration of TOC

(mg C/L)

Concentration of TOC Corrected for Control

(mg C/L)

Day 11

(Old Media)

Control

6.21

-

10

5.79

<Control

32

6.33

0.12

100

5.83

<Control

Day 11

(Fresh Media)

Control

1.17

-

10

1.71

0.54

32

1.34

0.17

100

2.69

1.52

Day 14

(Old Media)

Control

8.87

-

10

8.34

<Control

32

7.95

<Control

100

7.86

<Control

Day 14

(Fresh Media)

Control

3.32

-

10

6.65

3.33

32

5.61

2.29

100

5.89

2.57

Day 18

(Old Media)

Control

7.24

-

10

8.07

0.83

32

8.15

0.91

100

6.92

<Control

Day 18

(Fresh Media)

Control

2.95

-

10

2.45

<Control

32

3.66

0.71

100

4.18

1.23

Day 21

(Old Media)

Control

5.74

-

10

5.30

<Control

32

5.97

0.23

100

6.25

0.51

 


Appendix 3 (continued)   Total Organic Carbon Analysis

Samples

Nominal Loading Rate

(mg/L)

Concentration of TOC

(mg C/L)

Concentration of TOC Corrected for Control

(mg C/L)

Day 21

(Fresh Media)

Control

3.00

-

10

24.26

21.26[1]

32

7.47

4.47

100

8.05

5.05

Day 25

(Old Media)

Control

9.18

-

10

7.86

<Control

32

7.43

<Control

100

8.43

<Control

Day 25

(Fresh Media)

Control

3.18

-

10

7.36

4.18

32

8.23

5.05

100

5.27

2.09

Day 28

(Old Media)

Control

7.83

-

10

7.11

<Control

32

8.10

0.27

100

8.28

0.45

Day 28

(Fresh Media)

Control

3.64

-

10

3.99

0.35

32

2.26**

<Control

100

3.46

<Control

Day 32

(Old Media)

Control

6.73

-

10

6.62

<Control

32

5.74

<Control

100

7.04

0.31

Day 32

(Fresh Media)

Control

2.99**

-

10

2.80

<Control

32

2.78

<Control

100

3.77

0.78

Day 33

(Old Media)

Control

4.39

-

10

3.92

<Control

32

3.70

<Control

100

3.57

<Control

[1]High value considered to be due to contamination from an unknown source. Frozen duplicate sample gave a similar result.

**Duplicate sample analysis as original sample result was deemed to be anomalous.

   
Validity criteria fulfilled:
yes
Conclusions:
The application of the test material to newly laid eggs of fathead minnows was considered to have no effect on the survival or growth of the larvae. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.
Executive summary:

Introduction.

A study was performed to assess the effects of the test material on freshly hatched larvae of the fathead minnow (Pimephales promelas). The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 210, "Fish, Early-Life Stage Toxicity Test”, US Code of Federal Regulations, Title 40, Part 797, Section 1600 and the US EPA Draft Ecological Effects Test Guideline OPPTS 850.1400.

Methods.

Based on data supplied by the Sponsor, newly laid eggs were exposed to Water Accommodated Fractions (WAFs) of the test material over a range of nominal loading rates of 10, 32 and 100 mg/L for a period of 33 days at a temperature of 25ºC±2°C under semi-static test conditions. Due to the possible volatile nature of the test material, the test was conducted using completely filled test vessels which were then covered. 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 dry weight of the surviving fish were measured.

Results.

Over the duration of the test there were no significant mortalities or sub-lethal effects of exposure resulting from the exposure of fathead minnow (Pimephales promelas) larvae to nominal loading rates of 10, 32 and 100 mg/L loading rate WAF.

The mean hatching rate ranged from 92% to 95% and the mean survival rate ranged from 93% to 98%. The fish length and dry weight data obtained at termination of the test are summarised as follows:

 

Nominal Loading Rate (mg/L)

Control

10

32

100

Body Length (mean ± standard deviation, mm)

17.13 ± 1.29

17.90 ± 1.17

17.47 ± 1.05

17.55 ± 1.29

Dry Weight

(mean ± standard deviation, mg)

18.0 ± 4.4

20.4 ± 4.7

19.9 ± 4.4

19.6 ± 5.1

Statistical analysis of these data showed there were no significant reductions (P≥0.05) between the control and all the test groups in terms of fish length or dry weight.

Conclusion.

Given the above results and information it was considered that the test material had no effect on the survival or growth of newly laid eggs of fathead minnows.

The “Lowest Observed Effect Loading Rate” (LOEL), based on nominal loading rates, was considered to be greater than 100 mg/L loading rate WAF on the basis that there were no significant reductions (P≥0.05) in terms of fish length and dry weight when compared to the control at the end of the test.

The “No Observed Effect Loading Rate” (NOEL), based on nominal loading rates, was considered to be 100 mg/l loading rate WAF on the basis that there were no significant reductions (P≥0.05) in terms of fish length and dry weight when compared to the control at the end of the test.

Total Organic Carbon (TOC) analysis of the test preparations showed no significant differences in the amount of carbon present within the 10, 32 and 100 mg/L loading rate WAF test vessels when compared to the control vessels. This was expected given the extremely low aqueous solubility of the test material. Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that the majority of the results were around the limit of quantitation of the analytical method.

The dissolved test material may have been one or several components of the test material. Given that toxicity cannot be attributed to a single component or mixture of components but to the test material as a whole, the results were based on nominal loading rates only.

The application of the test material to newly laid eggs of fathead minnows was considered to have no effect on the survival or growth of the larvae. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Description of key information

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

The GTL Gasoil measured 33-day NOELR value was 100 mg/l (OECD 210) based on fish length and dry weight of Pimphales promelas.

Key value for chemical safety assessment

Additional information

No measured long-term toxicity to fish data are available for Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics, but data are available for closely related Fischer-Tropsch process-derived substances.

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; the 32-day NOELR value was ≥100 mg/l. The results of the test are considered to be reliable. This study is included to demonstrate lack of toxicity of the lower chain length constituents which may be present at low concentrations in Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics.

A Fish Early Life Stage study has been conducted for GTL Gasoil using the freshwater species Pimephales promelas (Fathead minnow) (Priestly, 2010a). The test was conducted in accordance with OECD 210 (1992) and in compliance with GLP.

Newly laid eggs from laboratory cultures were exposed for a period of 33-days to water accommodated fractions (WAFs) of the test material over a range of nominal loading rates of 10, 32, 100 mg/L.  The study was conducted at the specified temperature conditions for this species (25°C ± 2°C) under semi-static test conditions. As the test material is volatile, the test was conducted using completely filled test vessels which were covered. Exposure to the WAFs confirmed using total organic carbon (TOC) analysis.

Throughout the duration of the study, no significant (P ≤0.05) effects on mortality or sub-lethal effects (fish length, dry weight) as a result of exposure of fathead minnow to any of the nominal loading rate WAFs compared to the control were observed. The NOELR, based on nominal loading rate, was determined to be 100 mg/L.