(E)-3-methyl-5-cyclopentadecen-1-one

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 24 July 2012 to 11 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD Guideline 212 with GLP statement. All validity criteria were fulfilled. It was considered that this study would provide a more reliable exposure system for the assessment of the chronic effects of the test item on the early-life stages of fish.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 212 (Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages)

Deviations:

yes

Remarks:

See "Principles of method if other than guideline".

Principles of method if other than guideline:

In a Fish, Early-Life Stage Toxicity test conducted on the test item in 2003 (Safepharm Laboratories Project Number 161/309) a number of issues were identified that could have led to artifactual effects on the test organisms that were not directly related to test item effects. These issues included problems in obtaining and maintaining near nominal dissolved concentrations of the test item, poor analytical accuracy, questionable statistical interpretation and systematic variations in test temperature outside of the recommended ranges given in the Test Guideline (OECD 210, 1992) that could have led to positive bias in the growth rates of the control fish. Viewed together these multiple issues were considered to invalidate the original test.
Since this original test was conducted there have been significant advances in solubilisation and analytical techniques for hydrophobic compounds and hence it was considered that the conduct of a new study utilizing these improved techniques was justifiable.
A study was therefore performed to assess the (sub)lethal 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 (1998) No 212, "Fish, Short-term Toxicity on Embryo and Sac-fry Stages” as despite having conducted extensive preliminary work to assess the suitability of the OECD 210 methodology for this test item, it was considered that the OECD 212 method would provide a more reliable exposure system for the assessment of the chronic effects of the test item on the early-life stages of fish.

Based on the results of the pre-study dosing trials and the range-finding tests conducted, the definitive test, a short-term Toxicity test on Embryo and Sac-fry Stages, was conducted with the following amendments to the test design as recommended by the Test Guideline:
1) Concentration generators were employed with specifically designed flow splitters to ensure constant and consistent flow to each replicate test vessel.
2) Three test concentrations were selected rather than the five recommended by the Test Guideline due to the difficulties experienced in the preparation of test concentrations that were distinct from each other using the concentrator generator system. The use of three test concentrations was considered justifiable given that the preliminary range-finding studies conducted indicated that an effect on the test organisms was expected at the highest test concentration without causing complete mortality, whilst the lower concentrations should enable determination of a NOEC.
3) In order to avoid any interference due to adsorbing/desorbing of the test item, the organisms were exposed exclusively in glass beakers, avoiding as much as possible that the test solutions were in contact with any silicone tubing/joints etc during exposure, as its strong affinity for the test item would have impaired the concentration stability.

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 10 July 2012. Signed on 30 November 2012.

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Water samples were taken daily throughout the exposure period from the control and all test groups (individual replicates sampled) for quantitative analysis. Samples were also taken directly from the concentration generators on Days -4 through to Day 10 for quantitative analysis. Samples were taken from the test vessel and introduced into a graduated cylinder up to the required sampling volume using a standard glass beaker. They were then poured in a bottle already containing 15 mL of hexane for direct extraction . An aliquot of the hexane layer was taken for analysis.
- Sample storage conditions before analysis: The samples were prepared and vialled for analysis on the day of receipt. If not analysed immediately, the prepared samples were stored for a maximum of 7 days at -20°C prior to analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test system was dosed using three Concentration Generators. The principal of this column generator system was that the test item was placed inside silicone tubing and would diffuse through the walls into surrounding water within a closed cylinder. Water was pumped through the cylinder at a known rate (and this was kept constant for each test concentration) and so the test item concentration was controlled by changing the length of the silicone tubing in contact with the water phase inside the cylinder. To prepare each generator column, a length of silicone tubing with a 1.5 mm wall thickness and 5 mm bore was completely filled with test item. The tubing was then wrapped around stainless steel cages in a double layer and placed in the glass generator column. Tubing lengths of 1.5, 6 and 9 m were used in the different generator columns in order to produce three separate dissolved concentrations of the test item. The generator containing the 9 m length of tubing also had a ‘pre-generator’ vessel containing a further 2 x 5 m lengths of silicone tubing filled with the test item in order to boost the dissolved test item concentration. The generator columns were covered with aluminium foil to minimise any potential photo-degradation of the test item and discourage growth of photosynthetic organisms.
Pre-filtered dechlorinated tap water was pumped through the columns at a rate of 100 mL/min to provide solutions of the test item. The generator system was cleaned using a chemical sterilant (chlorine dioxide, nominal concentration 500 mg/L) on Days -4, -2, 3 and 6 to destroy and then prevent growth of biofilms. The cleaning cycle involved pumping the sterilant solution into the test system, turning off the flow for approximately 1½ hours to allow the sterilant solution to remain in the test system, then flushing the sterilant out of the test system by pumping the diluent through the test system for approximately 3½ hours. This protection was considered necessary further to numerous pretests in which concentrations of the test item very quickly decreased after manual cleaning. Chlorine dioxide was chosen due to its high efficacy as a disinfectant and its well-known degradation pathway to highly soluble substances (chlorite and chlorate) that could be rapidly flushed from the system and thus avoid influencing the outcome of the study. After flushing the absence of chlorine ions in the eluting flow was systematically checked using appropriate analytical means (limit of detection = 0.03 mg/L) after each cleaning.
On exit from the generator, the flow was split using a 4-way stainless steel splitter to feed the four replicate vessels. Each replicate vessel therefore received a nominal and constant flow rate of 25 mL/min.
- Controls: The control group received test water of the same quality as that for the test groups however the control water was not passed through a generator column and hence the control group was not exposed to the test item. The control system was also cleaned using chlorine dioxide as described above.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnows
- Strain: no data
- Source: The in­house breeding stock fish were acquired from Osage Catfisheries, Osage Beach, Missouri, USA on 31 May 2012 and maintained in dechlorinated tap water in glass tanks with an activated carbon and biological filtration system.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
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 25 °C ± 2 °C with a dissolved oxygen content of greater than or equal to 7.5 mg O2/L. The breeding stock fish were fed ZM 400 flake food daily and brine shrimp nauplii and blood worm as a supplement.
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 1 February 2013 and used for the definitive test. The eggs were less than 24 hours old on introduction into the test system.
Analysis of the diet, and data supplied by the water company for the diluent water showed no contaminants that were considered to affect the integrity and outcome of the study.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

10 d

Remarks on exposure duration:

none

Post exposure observation period:

None

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 Standards Instituted, 1993).
Hardness = between 134 to 156 mg/L as CaCO3.

Test temperature:

The water temperature was recorded daily throughout the test, 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.
Temperature was maintained at 25 ± 1°C throughout the test. A decline in recorded temperatures was observed at 72 and 144 hours, this was during the cleaning of the test system to prevent the buildup of biofilm. Given the relatively short duration of these lower temperatures and that they did not fall below the lower limit of 23°C, these low temperatures are considered to have had no adverse effect on the study or the results obtained.

pH:

The pH was recorded daily throughout the test, using a Hach HQ30d Flexi handheld meter.
pH = between 8.1 and 8.2.

Dissolved oxygen:

The dissolved oxygen concentration was recorded daily throughout the test, using a Hach HQ30d Flexi handheld meter.
Dissolved oxygen = 7.7 to 8.4 mg O2/L, corresponding to 93% to 104% ASV.

Salinity:

Not applicable

Nominal and measured concentrations:

See "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
Four glass beakers (nominal capacity 3 litres) fitted with a stainless steel mesh rim to allow flow through of the test medium were used as test vessels for the control and each test concentration. The test vessels were placed in a water bath to maintain constant temperature and covered to reduce losses by evaporation.
At the start of the test (Day 0), 40 fertilized eggs were randomly distributed to the four replicate test vessels of each treatment (10 eggs per replicate). The fertilized eggs were held in egg containers consisting of a glass tube (approximately 5 cm (diameter) by 8 cm (height)) fitted with a stainless steel mesh to retain the eggs. The loading rate of the eggs (biomass per volume of test medium in each test vessel) was lower than 0.5 g/L.
The test was started within 8 hours of the eggs having been fertilized. Microscopic inspection of a representative sample of eggs confirmed that the eggs were immersed in the test solutions prior to cleavage of the blastodisc.
The test was conducted with continuous medium renewal (dynamic test) whereby the test media were renewed continuously at a constant flow rate of 25 mL/min/replicate, equivalent to approximately 12 test chamber volumes per 24 hours.

TEST MEDIUM / WATER PARAMETERS
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. Downstream from the above described purification devices, the water supply to the control and test vessels was sequentially filtered (10 µm), subjected to UV sterilization and finally passed through two 0.2 µm cartridge filters (each filter containing a 0.45 µm pre-filter) in order to minimize passage of bacteria into the control and test vessels.

OTHER TEST CONDITIONS
The test vessels were maintained at 25 °C ± 1 °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; the diluent supply only was aerated.

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 embryos were absence of body movement and/or absence of heart beat and/or opaque coloration. The criteria of death for larvae 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.

VEHICLE CONTROL PERFORMED: not applicable

RANGE-FINDING STUDY
See Appendix 5 of the study report in "Attached background material"

Reference substance (positive control):

no

Duration:

10 d

Dose descriptor:

NOEC

Effect conc.:

0.23 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Hatching

Duration:

10 d

Dose descriptor:

NOEC

Effect conc.:

0.13 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Survival and Length

Duration:

10 d

Dose descriptor:

LOEC

Effect conc.:

> 0.23 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Hatching

Duration:

10 d

Dose descriptor:

LOEC

Effect conc.:

0.23 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Survival and Length

Duration:

10 d

Dose descriptor:

EC10

Effect conc.:

> 0.23 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Hatching and Length

Key result

Duration:

10 d

Dose descriptor:

EC10

Effect conc.:

0.18 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Survival

Duration:

10 d

Dose descriptor:

EC50

Effect conc.:

> 0.23 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Hatching, Survival and Length

Details on results:

See Table 6.1.2/2 in "Any other information on results incl. tables" and Tables in "Attached background material".
Throughout the test, all test concentrations were observed to be clear, colorless solutions by visual inspection.

The CV for the length data ranged from 2.1% to 10.7% between replicates and from 3.4% to 7.7% between the concentrations thereby indicating high confidence in the reliability of the length data. However the CV for the pooled wet weight data ranged from 6.8% to 43.6% with both the lowest and highest test concentrations having CV’s in excess of 40%. The wet weight data was therefore viewed as having a high amount of dispersion / uncertainty.
In view of the uncertainties on the wet weight measurements brought by the experimental bias and the high CV observed in both the lowest and highest concentrations, the Tukey test, a conservative test procedure for multiple comparisons was also applied on the dataset (see Appendix 11).
The outcome of this statistical test confirmed that the experimental error (associated with the larvae preparation for weighing) directly impacts the robustness of the data. Therefore it is concluded that the weight is a much less reliable endpoint than length and as such it was considered justifiable to base the growth parameter results on the effect that the test item had on length alone.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The hatchability, survivability, length (individual larvae) and wet weight (pooled larvae weight) data obtained 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). The “Lowest Observed Effect Concentration” (LOEC) and the “No Observed Effect Concentration” (NOEC) were determined where possible.
The ECx values of the above endpoints were determined by analysis of the data where possible.

Table 6.1.2/2: Results

	Mean Measured Concentration (mg/L)
	Control	0.040	0.13	0.23
Mean Hatching Rate	85	87.5	82.5	87.5
Mean Survival Rate	100	93.8	96.9	64.6*
Body Length (mean±standard deviation, mm)	6.1±0.5	6.0±0.3	6.0±0.2	5.6±0.3

Validity criteria fulfilled:

yes

Conclusions:

The lowest EC10 value was determined at 0.18 mg test item/L (measured concentration), based on survival.

Executive summary:

In a Fish, Early-Life Stage Toxicity test conducted on the test item in 2003 (Safepharm Laboratories Project Number 161/309) a number of issues were identified that could have led to artifactual effects on the test organisms that were not related to test item toxicity. These issues included problems in obtaining and maintaining near nominal dissolved concentrations of the test item, poor analytical accuracy, questionable statistical interpretation and systematic variations in test temperature outside of the recommended ranges given in the Test Guideline (OECD 210, 1992) that could have led to positive bias in the growth rates of the control fish. Viewed together these multiple issues were considered to invalidate the original test.

Since this original test was conducted there have been significant advances in solubilisation and analytical techniques for hydrophobic compounds and hence it was considered that the performance of a new study utilizing these improved techniques was justified.

A study was therefore performed to assess the (sub)lethal 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 (1998) No 212, "Fish, Short-term Toxicity on Embryo and Sac-fry Stages” as despite having conducted extensive preliminary work to assess the suitability of the OECD 210 methodology for this test item,

it was considered that the test item was extremely difficult to maintain at stable aqueous concentrations over such a long study period even under flow-through conditions. In fact the substance is so rapidly degraded that significant loss of the substance occurs within a few days. It was therefore decided that the OECD 212 method would provide a more reliable exposure system for the assessment of the chronic effects of the test item on the early-life stages of fish.

Newly laid eggs were exposed to an aqueous solution of the test item over a range of test concentrations of 0.040, 0.13 and 0.23 mg/L (mean measured) for a period of 10 days at a temperature of approximately 25 ºC under dynamic (flow-through) test conditions.

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. At test termination the length and wet weight of the surviving fish were measured.

Statistical analysis of the length data showed the 0.040 and 0.13 mg/L test concentrations not to be significantly different (p≥0.05) from the control group. However, the 0.23 mg/L test group was significantly different (p<0.05) from the control group.

Over the duration of the test there were no significant mortalities or sub-lethal effects resulting from the exposure of fathead minnow (Pimephales promelas) larvae to mean measured test concentrations of 0.040 and 0.13 mg/L.

However, mortalities and sub-lethal effects of exposure were observed at a mean measured test concentration of 0.23 mg/L.

Given the above results and information it was considered that the application of the test item to newly laid eggs of fathead minnows had an effect on growth and survivability. The 10d-EC10 based on hatching, survival and length were >0.23, 0.18 and >0.23 mg/L, respectively. The 10d-EC50 based on hatching, survival and length were >0.23 mg/L for all parameters. The 10d-NOEC based on hatching, survival and length were 0.23, 0.13 and 0.13 mg/L, respectively. The 10d-LOEC based on hatching, survival and length were >0.23, 0.23 and 0.23 mg/L, respectively.

Given the inadequacies of the previous study conducted in the test item as previously discussed, it is considered that the above results provide a more reliable assessment of the chronic effects of the test item on the early life stages of fish.

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

From 12 June to 15 July 2003

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

This study was performed according to the OECD Guideline 210 and the US EPA Draft Ecological Effects Test Guideline OPPTS 850.1400 with GLP statement. This study does not meet the validity criteria and should be considered as invalid due to multiple deviations from the guideline. A position paper is available in "Attached background material" for justified these observations and the laboratory that originally performed the study has issued a statement invalidating the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

yes

Remarks:

. Validity criteria were not fulfilled.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 2nd December 2002. Signed on 13 February 2003.

Analytical monitoring:

yes

Details on sampling:

The concentration and stability of the test material in the test preparations were verified by chemical analysis (replicates pooled) on days 0, 5, 7, 12, 14, 19, 21, 26, 28, 32 and 33. In addition samples of the solvent stock solutions used to prepare the test concentrations were taken on days 0, 5, 7, 12, 14, 19, 21, 26, 28, 32 and 33.
- Sampling method: A volume of test sample was eluted through the cartridge. The test material was eluted from the cartridge with acetonitrile, made to volume with acetonitrile to give a final theoretical concentration of approximately 0.5 to 10 mg/L.
- Sample storage conditions before analysis: duplicate samples were taken and stored frozen (approximately -20°C) for further analysis if necessary.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test material (315 and 100 mg) were each dissolved in tetrahydrofuran and the volume adjusted to 50 ml to give solvent stock solutions of 315 and 100 mg/50 mL. Serial dilutions were made from these solvent stock solutions to prepare further solvent stock solutions of 31.5, 10, 3.15 and 1.0 mg/50 ml. Each of the stock solutions was inverted several times to ensure adequate mixing and homogeneity. Each of the 1.0, 3.15, 10, 31.5 and 100 mg/50 ml solvent stock solutions were pumped at a rate of 0.36 ml/hour and mixed with diluent pumped at a rate of 60 ml/minute to give test concentrations of 0.0020, 0.0063, 0.020, 0.063 and 0.20 mg/I, in duplicate such that individual replicates received their own individually prepared test concentration.
- Eluate: laboratory tap water dechlorinated
- Differential loading: 3.15
- Controls: control and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Tetrahydrofuran
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): the solvent control group was exposed to 100 µL/L of tetrahydrofuran.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no data

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnows
- Strain: no data
- Source: Osage Catfisheries Inc, Osage Beach, Missouri, USA.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): no data
- Method of collection of fertilised eggs: each breeding tank was supplied with inverted plastic guttering for the fish to lay eggs on and be fertilised.
- Subsequent handling of eggs: no data

POST-HATCH FEEDING
- Start date: fertilised eggs were collected from the breeding tanks on 12 June 2003
- Type/source of feed: Tetramin flake food
- Amount given: no data
- Frequency of feeding: daily

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

33 d

Remarks on exposure duration:

= 28 days post-hatch

Post exposure observation period:

No data

Hardness:

the water hardness values were observed to range from 132 to 134 mg/L as CaCO3 at the start of the test and from 139 to 142 mg/L as CaCO3 at termination of the test.

Test temperature:

From 20.5 to 26.7°C.

pH:

From 7.4 to 8.2

Dissolved oxygen:

From 41 to 108 % of air saturation value.

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: 0.0020, 0.0063, 0.020, 0.063 and 0.20 mg/L.
Measured concentrations: 0.00098, 0.00265, 0.00495, 0.0157 and 0.0316 mg/L, corresponding to 49, 42, 25, 25 and 16% of nominal concentration.

Details on test conditions:

TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): egg baskets = glass tube approximately 5 cm diameter and 8 cm in height and fitted with a stainless steel mesh to retain the eggs.
- Test vessel: no data
- Material, size, headspace, fill volume: 12 litre exposure vessels containing 10 litres of test media were used for each control and test vessel.
- Aeration: no auxiliary aeration; the diluent supply only was aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter): Peristaltic diluter (dynamic, continuous flow test conditions (see Figure 2) were employed in the test. The solvent stock solutions were pumped at a rate of 0.36 ml/hour by two Harvard 22 syringe pumps and mixed with diluent pumped at a rate of approximately 60 ml/minute by two Watson Marlow 603 peristaltic pumps. The solvent stock solutions and diluent were mixed in glass mixing vessels fitted with magnetic stirrers prior to flowing to the test vessels.)
- Renewal rate of test solution (frequency/flow rate): flow rate (oscillated by a variable speed rocker arm system at a rate of approximately 1 oscillation per 2 minutes to induce non-turbulent flow of water through the egg baskets).
- No. of fertilized eggs/embryos per vessel: 30
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2
- Biomass loading rate: no data

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 ACI) and partly softened (Elga Nimbus 1248D Duplex Water Softener). After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Total organic carbon: 1.200mg C/L
- Pesticides: <0.010 µg/L
- Chlorine: chlorine free = 0.151 mg/L ; chlorine total = 0.233 mg/L
- Alkalinity: 111 mg CaCO3/L
- Conductivity: 415.85 µS/cm
- Salinity: not applicable
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data

OTHER TEST CONDITIONS
- Adjustment of pH: no data
- Photoperiod: the lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Light intensity: 18 to 338 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 coloration and lack of reaction to mechanical stimulus.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: nominal concentrations of 0.0020, 0.020 and 0.20 mg/L.
- Results used to determine the conditions for the definitive study:
At concentrations in excess of 0.20 mg/L indissolved test material was observed in the test vessels.
No significant mortalities or sub-lethal effect were observed at all test concentrations tested. Statistical analysis of the length and dry weight data showed no significant differences (P>= 0.05) between the solvent control and 0.0020 mg/L test groups in terms of length and dry weight. However, the 0.020 and 0.20 mg/L test groups were shown to be significantly different (P < 0.05) from the solvent control in terms of both length and weight. Based on these results the following test concentration range was assigned to be definitive test: 0.0020, 0.0063, 0.020, 0.063 and 0.20 mg/L.
Therefore, the range-finding test was carried out using dimethylformamide as the auxiliary solvent used in the preparation of the test concentrations. However during initial tests performed a bacterial growth was observed to form in the test system causing death of the eggs. It was considered that the bacterial growth may have been due to the solvent used acting as a substrate for the bacteria. A dosing trial was performed using tetrahydrofuran as the auxiliary solvent. No bacterial growth was observed.

POST-HATCH DETAILS
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 8 as the larvae were too small at this time to feed on brine shrimp nauplii. On Day 8 the larvae were fed protozoan (Paramecia micronucleatum) and brine shrimp nauplii as they were considered sufficiently large to eat brine shrimp nauplii. Both food types were fed on Day 8 to avoid a sudden change from one food source to another. On Day 9 and throughout the remainder of the test the larvae were fed brine shrimp nauplii only.

Reference substance (positive control):

no

Key result

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

0.001 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

length

Remarks:

and weight

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

0.003 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

length

Remarks:

and weight

Details on results:

- Observations: The number of dead eggs and larvae were observed to be low throughout the duration of the test and in all test groups. Analysis of the data using the corrected chi-squared statistic (Breslow and Day 1980) showed the number of dead eggs observed in the 0.20 mg/l test concentration to be significantly different from the solvent control. The mean hatching success rate for the solvent control group was 97% thereby satisfying the validation criterion of greater than 66% hatching rate. The mean survival rate of the larvae for the control and solvent control groups were 99% and 97% respectively thereby satisfying the validation criterion for post-hatch survival success rate of greater than 70%. The mean survival rates for the test concentrations ranged between 97% and 100%. 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.

- Sub-lethal effects: Some larvae were observed to have bent spines. This observation was recorded from Day 4 until Day 19 in both replicates of the 0.20 mg/l test concentration. The overall number of larvae with bent spines was very low (8 in total). This effect was considered to be due to the effect of the test material as this effect was not observed at any other test concentration. Also from Day 15 onwards the surviving organisms at the test concentration of 0.20 mg/l were observed to be paler in colour than the controls.

- Length and weight data: See table in "Attached background material". Statistical analysis of the length data by analysis of variance showed the control and 0.0020 mg/l test concentration not to be significantly different (P>=0.05) from the solvent control group. However, all other test groups were significantly different (P<0.05) from the
solvent control group. Statistical analysis of the dry weight data showed no significant differences (P>=0.05) between the solvent control group and the 0.0020 mg/l test concentration. However, all other test groups were significantly different (P<0.05) from the solvent control group. Critical examination of the data, means and standard deviations, of the control and solvent control groups, showed the standard deviations to overlap each other as well as the overall means of length and dry weight. The observed significant difference between the solvent control group and the control group was considered to be due to the presence of a small number of larger fish in the solvent control group and a small number of smaller fish in some of the other test groups. This was considered to be due to the normal variation observed in a natural population of organisms and hence considered
not to be biologically significant.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

No data

None

Validity criteria fulfilled:

no

Conclusions:

Based on the mean measured test concentrations of the centrifuged test media, the NOEC was 0.00098 mg/l (no significant differences in terms of length and weight at the end of the test compared to the solvent control). The LOEC was 0.00265 mg/I (significant effects in terms of length and weight at the end of the test). The current study is not valid, therefore the results are not reliable and this study should be reperformed using more up-to-date methodologies.

Executive summary:

This 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" and the US EPA Draft Ecological Effects Test Guideline OPPTS 850.1400 with GLP statement.

However, this study comprises multiple issues that, either individually or combined, could have led to purely artifactual effects on the test organisms during this study. Achieving solubility of the substance in water already poses significant problems and the use of solvents to facilitate the preparation and use in a flow-through regime may have significantly contributed to the problem. The use of solvents has previously been demonstrated to contribute to ecotoxicological effects which are not found when test organisms are exposed to the substances without the use of this solvent when used in flow-through regimes. Analytical results exhibit unsatisfactory accuracy, and reprocessing of the results by appropriate statistical methods lead to different conclusions than those expressed by Safepharm. Moreover, some biological aspects of the study also lead to the conclusion that the study performed by Safepharm sufferered from multiple drawbacks that impaired the results.

However, since 2003 when this study was conducted, significant improvements in water solubilisation techniques of hydrophobic compounds and in their quantification have been made that are likely to drastically improve the reliability of the OECD 210 test.

Four points invalidate the conclusion of this study.

1.     The analytical method employed was not fit for purpose

2.     The method of solubilisation of the substance in water and the biological impact of solvent use

3.     The control of the temperature (one of the validity criteria of the OECD 210 Guideline which in itself invalidates this study, but this was not mentioned in the report) and lack of consideration of the resulting and potentially serious biological consequences

4.     The statistical interpretation of the results

There were no observed effects at any concentration up to 0.2 mg/L nominal on hatch, time to hatch or mortality although mean hatch was slightly lower at this concentration than in the controls or solvent control. This was also noted in the range-finding test where hatch at 0.2 mg/L was 97% and in the solvent control 100%. The only recorded sub-lethal effects were of bent spines recorded from day 4 until day 19 in 8 larvae, although apparently this phenomenon corrected itself on day 20, and from day 15 a paler colour was observed at 0.2 mg/L. The effect was not observed at lower concentrations. However, this cannot be considered as an effect under OECD Guideline 210.

The only significant effects were observed on growth rate and biomass. However, these effects may have been directly related to the difference in temperature between the controls and the concentrations at which an effect was observed. The statistical significance of these results can also be questioned further to in-depth consideration of the appropriateness of the statistical test used and differences between the controls.

In any case the study does not meet the validity criteria for temperature and should be considered as invalid due to multiple deviations of this parameter from the guideline.

A further argument to consider reperforming this study is the poor concentration maintenance. No explanation is really provided justifying the range of results. The study Director applied a time weighted mean to the results including multiple LOQ results leading to a measured NOEC that is orders of magnitude lower than the nominal.

Based on the way the experiment has been conducted and on the analytical results, we conclude it is not possible to determine whether the observed effects are due to the specific operating conditions that may generate bias or to the studied compound. Moreover, the statistical interpretation of the result also gives rise to question.

This test should be reperformed using reliable temperature controlled chambers, appropriate analytical methods and adapted techniques of solubilisation. Conclusions must be deduced from robust statistical analysis. 

Description of key information

OECD Guideline 212, GLP, key study, validity 1:

10d-EC10 (Pimephales promelas) = 0.18 mg/L based on survival and measured concentrations.

The OECD 212 Fish Short-Term Toxicity Test on Embryo and Sac-Fry Stages performed on the registered substance was conducted in 2013 without testing proposal submission for the following reasons, also detailed in the attached document:

- The test is a short term test performed after invalidation of the OECD 210 (2003);

- The test was a repeat test and therefore not a new test requiring a testing proposal;

- The OECD 212 (1998) is not an animal test.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.18 mg/L

Additional information

In a Fish, Early-Life Stage Toxicity test conducted on the test item in 2003 (Safepharm Laboratories Project Number 161/309) a number of issues were identified that could have led to artifactual effects on the test organisms that were not related to test item toxicity. These issues included problems in obtaining and maintaining near nominal dissolved concentrations of the test item, poor analytical accuracy, questionable statistical interpretation and systematic variations in test temperature outside of the recommended ranges given in the Test Guideline (OECD 210, 1992) that could have led to positive bias in the growth rates of the control fish. Viewed together these multiple issues were considered to invalidate the original test.

Since this original test was conducted there have been significant advances in solubilisation and analytical techniques for hydrophobic compounds and hence it was considered that the performance of a new study utilizing these improved techniques was justified.

A study was therefore 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 (1998) No 212, "Fish, Short-term Toxicity on Embryo and Sac-fry Stages” as further to conducting extensive preliminary work to assess the suitability of the OECD 210 methodology for this test item, it was considered that the test item was extremely difficult to maintain at stable aqueous concentrations over such a long study period even under flow-through conditions. In fact the substance is so rapidly degraded that significant loss of the substance occurs within a few days. It was therefore decided that the OECD 212 method would provide a more reliable exposure system for the assessment of the chronic effects of the test item on the early-life stages of fish.

This study was conducted in 2013 without testing proposal submission for the following reasons, also detailed in the attached document:

- The test is a short term test performed after invalidation of the OECD 210 (2003);

- The test was a repeat test and therefore not a new test requiring a testing proposal;

- The OECD 212 (1998) is not an animal test.

In this study, newly laid eggs were exposed to an aqueous solution of the test item over a range of test concentrations of 0.040, 0.13 and 0.23 mg/L (mean measured) for a period of 10 days at a temperature of approximately 25 ºC under dynamic (flow-through) test conditions.

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. At test termination the length and wet weight of the surviving fish were measured.

Statistical analysis of the length data showed the 0.040 and 0.13 mg/L test concentrations not to be significantly different (p≥0.05) from the control group. However, the 0.23 mg/L test group was significantly different (p<0.05) from the control group.

Over the duration of the test there were no significant mortalities or sub-lethal effects resulting from the exposure of fathead minnow (Pimephales promelas) larvae to mean measured test concentrations of 0.040 and 0.13 mg/L.

However, mortalities and sub-lethal effects of exposure were observed at a mean measured test concentration of 0.23 mg/L.

Given the above results and information it was considered that the application of the test item to newly laid eggs of fathead minnows had an effect on growth and survivability. The 10d-EC10 based on hatching, survival and length were >0.23, 0.18 and >0.23 mg/L, respectively. The 10d-EC50 based on hatching, survival and length were >0.23 mg/L for all parameters thus suggesting that the original acute study on fish with an 96 h LC50 of 0.22 mg/L suffered from similar technical flaws as the original ELS study. The 10d-NOEC based on hatching, survival and length were 0.23, 0.13 and 0.13 mg/L, respectively. The 10d-LOEC based on hatching, survival and length were >0.23, 0.23 and 0.23 mg/L, respectively.

Given the inadequacies of the previous study conducted in the test item as previously discussed, it is considered that the above results provide a more reliable assessment of the chronic effects of the test item on the early life stages of fish. Indeed, even if the study period is shorter (10 days) than a real chronic study, the substance was well maintained at stable aqueous concentrations during the test and therefore the study provide a more reliable exposure system. In addition, as the substance has a non-specific narcotic mode of action, smaller differences in sensitivity between this study and the full early-life stage test (OECD Guideline 210) is expected, as mentionned in the OECD Guideline 212 (1998).