Hexafluoropropene, oxidized, oligomers, reduced, fluorinated

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

July 2015 to March 2018

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Uncertainty is present about the reproducibility of the results due to several technical difficulties encountered during the set-up and execution of the study.

Remarks:

Several technical difficulties were encountered during the set-up and execution of the study: - Difficulties to generate reproducible test solutions; - Difficulties to maintain test concentrations at acceptable levels within the test period; - Test substance contamination in the control vessels; - Difficulties encountered by the laboratory to maintain healthy cultures of invertebrates; - High maternal mortality in controls and treatment vessels but without dose response relationship;

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2 October 2012

Deviations:

yes

Remarks:

tested species: C.dubia, test duration: 8 days

Qualifier:

according to guideline

Guideline:

other: ISO 20665 Determination of Chronic Toxicity to Ceriodaphnia dubia

Version / remarks:

15 December 2008

Deviations:

yes

Remarks:

OECD 211 parameters used for evaluation

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor / Batch No.: 2021 SP
- Expiration date of the lot/batch: May 2020
- Purity test date: 19 JUne 2015

RADIOLABELLING INFORMATION: not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated 2-8°C
- Stability under test conditions: yes
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: saturated solution in test media prepared and diluted with test media as appropriate..
- Final preparation of a solid: not applicable

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All contrations / All days.
- Sampling method:
Fresh media samples (1 mL) were taken from the additionally prepared vessels, from the control and each test concentration, on Days 0, 1, 2, 3, 4, 5, 6 and 7.
Samples (1 mL) of the 24-hour old media were taken from two replicate test vesselsfrom the control and each test concentration and on Days 1, 2, 3, 4, 5, 6, 7 and 8.
Replicates were sampled alternatively. Samples were taken immediately upon opening the vessels, prior to organism transfer, to minimise potential losses through volatility.
- Sample storage conditions before analysis: Direct analysis. Analysis was carried out immediately upon receipt.

Vehicle:

no

Details on test solutions:

The test substance was known to be of low water solubility and highly volatile. Extensive media preparation trials were required to determine the most appropriate method of preparation for the test substance in order to achieve the maximum saturation concentration in the test medium whist avoiding the formation of emulsions: The trials investigated the use of solvent spike and saturated solution methods of preparation.
A solvent spike method was considered inappropriate due to the high variability observed from the analytical results.
Saturated solutions were prepared by either stirring, sonicating or shaking the initial preparation. A shake method was considered inappropriate due to the high variability observed from the analytical results. A sonication method was considered inappropriate given that analysis showed measured concentrations significantly in excess of the considered water solubility of the test substance. This was considered to be possibly due to emulsions being formed within the preparation which were not fully removed by centrifugation. Stirring periods and initial loading rates were also investigated to determine when saturation was achieved. Increasing stirring periods and using lower loading rates resulted in low and variable results possibly due to losses through volatility.
From the media trials conducted, it was considered that the use of a saturated solution method of preparation was the most appropriate method for the test substance. By stirring an initial loading rate of 676 mg/L for ca 4 hours and removing any undissolved test substance by centrifugation, the resultant supernatant (saturated solution) gave relatively consistent results indicating that saturation was achieved and undissolved test substance or emulsions were removed.

Based on the results of the media preparation trials, it was considered that the most appropriate method of preparation for the tests was a saturated solution method whereby an initial loading rate of 676 mg/L was stirred for ca 4 hours followed by centrifugation at ca 1290 g for ca 5 minutes. The supernatant was then used to prepare the test concentrations.

Test organisms (species):

Ceriodaphnia dubia

Details on test organisms:

JUSTIFICATION FOR THE USE OF THE SPECIES:
The rationale for the selection of this species was based on the small volumes of water required by Ceriodaphnia dubia compared to other species of daphnids. As the test substance is highly volatile from water, the use of small volumes of solutions during preparation, handling and transfer of media allows for minimisation of the possibility of losses of the test substance through volatilisation.

CULTURING
Ephippia were originally obtained from MicroBioTests Inc., Belgium. The Ceriodaphnia dubia are cultured in 600 mL glass beakers containing 500 ml of Elendt M4 medium. Each vessel and its contents are referred to as a 'culture'. New cultures are initiated with juvenile Ceriodaphnia dubia (less than 24 hours old), at a density of approximately 10-20 individuals per vessel. The cultures are fed daily with 6 x 106 cells/mL of Chlorella vulgaris, 3 x 106 cells/mL of Pseudokirchneriella vulgaris and 250 μL/500 mL of fish flake food suspension prepared in accordance with standard operating procedures.

The water in each culture was renewed or partially renewed at least once a week.
Juveniles were removed when present in cultures using either a sieve or pipette.
Cultures were maintained, generally, for up to 14 days. Juveniles for use in tests were collected from the second brood onwards. Approximately 24 hours before a test was set up, juveniles present in the cultures were removed and discarded. Over the next 24 hours, juveniles for use in the test were removed from the culture using a wide bore pipette and transferred to fresh culture medium. The juveniles were then left for at least 1 hour before selecting actively swimming individuals for use. All juveniles used to initiate a test were less than 24 hours old.
All cultures, prior to and during tests, are maintained under fluorescent lighting on a 16-hour light:8-hour dark photoperiod with an approximate 30 minute dawn/dusk period.

FEEDING DURING TESTING
The Ceriodaphnia dubia were fed daily with 12 × 106 cells/L of Chlorella vulgaris, 6 × 106 cells/L of Pseudokirchneriella subcapitata and 500 μL/L of a 5 g/L fish food suspension. The feed was added to individual vessels at the time of preparation.

SENSITIVITY CONTROL
A reference test, using sodium chloride, was conducted alongside the definitive test to demonstrate that the organisms were suitable for use in testing.
Reproduction tests and acute tests using potassium dichromate were conducted prior to the definitive test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

8 d

Remarks on exposure duration:

The test ended at 8 day when 60%of the control organisms have produced their third brood.

Post exposure observation period:

not applicable.

Hardness:

Standard Elendt M4 medium

Test temperature:

Temperature was maintained at 25 ± 2ºC

The temperature was determined in each freshly prepared test medium at each renewal. At the end of each exposure period, water quality measurements were determined using pooled replicate samples of test media at each of the nominal test concentrations. Pooled replicates were used as the small volume of media in each test vessel was insufficient for accurate measurements.
The minimum and maximum temperature, maintained in an additional vessel in the test area, was recorded daily.
All measurements were conducted using calibrated equipment and performed to internal Standard Operating Procedures.

pH:

The pH of the freshly prepared media ranged from 6.89 to 7.98 and from 6.80 to 7.62 in the 24-hour old media. The pH was, in general, outside the 8.0 ± 0.3 as specified in the ISO 20665 standard. However, the pH range was within the 6 – 9 range specified in the OECD 211 test guideline and did not vary by more than 1.5 units throughout the test. In addition, the pH was within the acceptable parameters for the test organism.

The pH was determined in each freshly prepared test medium at each renewal. At the end of each exposure period, water quality measurements were determined using pooled replicate samples of test media at each of the nominal test concentrations. Pooled replicates were used as the small volume of media in each test vessel was insufficient for accurate measurements.
All measurements were conducted using calibrated equipment and performed to internal Standard Operating Procedures.

Dissolved oxygen:

The oxygen concentration in the freshly prepared media ranged from 77% to 100% air saturation value (ASV) thereby satisfying the requirement of the ISO 20665 standard of the oxygen concentration not exceeding 100% ASV. The concentration range in the freshly prepared 100% saturated solution was 77% to 99% ASV. The oxygen concentration in the 24-hour old media ranged from 53% to 85% ASV. A value of 53% ASV was the lowest recorded during the test which was equivalent to 4.46 mg O2/L thereby satisfying the requirement of the OECD 211 guideline that oxygen concentration should be maintained at > 3 mg O2/L. The oxygen concentration did generally decrease in the test vessels over each 24-hour period. This was considered to be due to the use of completely filled and sealed vessels which prevented gaseous exchange between the air and media as oxygen from the water was consumed by the test organisms.

The Oxygen Concentration was determined in each freshly prepared test medium at each renewal. At the end of each exposure period, water quality measurements were determined using pooled replicate samples of test media at each of the nominal test concentrations. Pooled replicates were used as the small volume of media in each test vessel was insufficient for accurate measurements.
All measurements were conducted using calibrated equipment and performed to internal Standard Operating Procedures.

Salinity:

Standard Elendt M4 medium

Conductivity:

not applicable

Nominal and measured concentrations:

Nominal: 10, 18, 32, 56 and 100% of the saturated solution.
Measured conc.: 0.025, 0.037, 0.062, 0.11 and 0.17 mg/L (based on time-weighted mean measured test concentrations).

Details on test conditions:

TEST SYSTEM
The test vessels were glass scintillation vials containing ca 25 mL of test medium. Due to the volatile nature of the test substance, the vessels were completely filled and sealed with minimal headspace.
- Type: completely closed
- Material, size, headspace, fill volume: glass, 25 mL, completely filled, minimezed headspace
- Aeration: none
- Renewal rate of test solution: 24 h
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): not applicable (no vehicle used)
An additional test vessel was prepared for the control and each test concentration for analytical sampling only of the freshly prepared media. This was conducted to avoid any possible losses due to volatilisation from the test vessels due to sampling.

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light:8-hour dark cycle and an approximate 30 minute dawn/dusk transition period.
- Light intensity: light intensity (measured in the test area) of 911 to 993 LUX.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The adult Ceriodaphnia dubia were observed daily for immobility, the presence or absence of eggs developing in the brood pouch (gravid or non-gravid) and mortality throughout the duration of the test. In addition, the number of juveniles present (alive or dead) were recorded, removed from the vessels and then discarded.
Carapace lengths of all surviving parental C dubia measured at the end of the test.

STATISTICAL ANALYSIS
Statistical analysis was conducted on the data using the CETIS program v 1.8.6.8. To determine the No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC), the total number of juveniles produced by surviving adults by Day 8, final adult lengths and final adult survival were analysed using the Dunnett Multiple Comparison Test, Bonferroni adjusted t-Test and Fisher Exact/Bonferroni-Holm Test, respectively. Due to the observed variability in adult survival, the data were also analysed using the Tukey-Kramer Test to determine significant differences between treatments.
The EC10, EC20 and EC50 values are defined as the concentrations which result in 10%, 20% and 50% effect, compared to the control, respectively. The EC10, EC20 and EC50 values, along with corresponding 95% confidence limits where appropriate, were calculated using linear interpolation.

RANGE FINDING STUDY
An eight-day range-finding test, with duplicate vessels per concentration, was conducted at nominal concentrations of 10, 18, 32, 56 and 100% saturated solution (0.059, 0.038, 0.060, 0.12 and 0.18 mg/L, respectively, based on time-weighted mean measured test concentrations). The test design was semi-static with daily renewal of test media.
A control treatment was prepared by the addition of Elendt M4 medium only to the test vessels. Chemical analysis of the test solutions was conducted daily, on a single replicate per control and test concentration, with the exception of the weekend.

After the range-finding study, the culture of C. dubia died, and so another batch of C. dubia was used. Acute and Chronic sensitivity tests were launched but chronic sensitivity tests on this new batch were not good. Indeed in 5 on 9 instances the chronic sensitivity test with teh reference suibstance Socium Chloride conducted according the TG ISO 20665 on the new batch failed in fulffiling the validity criteria defined in the guideline. Therefore sensitivity tests were repeated several times, and the main study was launched after obtaining two consecutive valid sensitivity tests. Considering the issue on the culture, it was requested to add a sensitivity test in parallel to the main study with Galden LMW.

Reference substance (positive control):

yes

Remarks:

A reference test, using sodium chloride, was conducted alongside the definitive test to demonstrate that the organisms were suitable for use in testing.

Duration:

8 d

Dose descriptor:

NOEC

Effect conc.:

0.17 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 0.17 mgL = highest tested concentration

Duration:

8 d

Dose descriptor:

LOEC

Effect conc.:

> 0.17 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other:

Remarks:

0.17 mgL = highest tested concentration

Duration:

8 d

Dose descriptor:

NOEC

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Juvenile production

Duration:

8 d

Dose descriptor:

LOEC

Effect conc.:

0.17 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Juvenile production

Remarks on result:

other: 0.17 mgL = highest tested concentration

Duration:

8 d

Dose descriptor:

NOEC

Effect conc.:

0.17 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks on result:

other: 0.17 mgL = highest tested concentration

Duration:

8 d

Dose descriptor:

LOEC

Effect conc.:

> 0.17 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks on result:

other: 0.17 mgL = highest tested concentration

Details on results:

EC10 and EC20 were calculated but due to the wide 95% confidence limits are were not considered relevant. In addition, a 20% effect for a chronic study is not generally considered to be biologically significant given the complexity of the test system. It is therefore considered that the NOEC and LOEC values are the most relevant endpoints.

ADULT MORTALITY
In the control group, 20% parental mortality was observed.
Total parental Ceriodaphnia dubia survival on Day 8 is presented in the table below.
The OECD 211 test guideline states that the 20% mortality validity criteria can be used for accidental parental mortality for the controls as well as for each of the test concentrations. The statement was added to the guideline to remove ‘a source of error’ if 20% of the adults were lost due to accidental or inadvertent issues, for example, killed on transfer to new media. The stipulation of 20% is provided to suggest that if more than 20% of the adults in a treatment group are accidentally or inadvertently lost, the results of the test may be affected.
For this test no adults were accidentally killed however, it should be noted that the test was conducted with daily renewal of the test media therefore the organisms were handled daily. Whilst every care was taken to minimise damage to the organisms during transfer, the added stress of daily handling could result in unexpected mortality.
The transfer at the moment of the daily renewal may exert a significant stress on the organisms. The mortality observed in the negative control and at the different test concentrations was considered more likely to be due to a mechanical stress during daily renewals rather than to be due to a toxic effect of the test substance or to the bad health status of the C. dubia culture.
The 40% mortality observed at the 18% saturated solution test concentration was inconsistent with the remaining test groups. At this concentration the health of the organism seemed to be good as demonstrated by the high reproduction rate. It was therefore considered that the mortality > 20% (actual:40%) observed may be due to the mechanical stress due to the daily renewal.
On comparison to the control group, there were no statistically significant effects on the mortality of the adult Ceriodaphnia at any of the concentrations employed. In addition, no significant differences were observed between treatments. The NOEC and LOEC values for adult survival were considered to be 0.17 and >0.17 mg/L.

C.DUBIA JUVENILE PRODUCTION
At the highest concentration a variable effect on reproduction was observed between the test organisms (i.e. 4 of the 7 surviving organisms experienced a market effect on reproduction while the remaining 3 organism did not. See Table 7). This would suggest the beginning of a toxic response.
The observed effect on reproduction at the highest concentration is considered to be not affected by the 30% mortality.
On comparison to the control group, there were no statistically significant effects on
the total numbers of juveniles produced at nominal concentrations of 56% saturated solution and below. A significant reduction (p >0.05) in the number of juveniles produced was observed at the 100% saturated solution test concentration compared to the control. The NOEC and LOEC values for reproduction were considered to be 0.11 and 0.17 mg/L, respectively.

GROWTH MEASUREMENTS
On comparison to the control group, there were no statistically significant effects on the length of the adult Ceriodaphnia surviving to Day 8 at any of the concentrations employed. The NOEC and LOEC values for growth (based on final length) were considered to be 0.17 and >0.17 mg/L.

Results with reference substance (positive control):

A test with Sodium Chlorine was conducted in parallel to the main study, according to the the requirements of the ISO 20665 Determination of Chronic Toxicity to Ceriodaphnia dubia (15 December 2008).

Treatment Rates: Control, 320, 560, 1000, 1800 and 3200 mg/L
Preparation Method: Direct addition of the reference substance to test media followed by serial dilution.
Replication: 10 replicates per control and each test concentration
Study Duration: 8 days
Test Design: Semi-static with daily renewal of the test media except weekend
Test System: A single Ceriodaphnia dubia, <24 hours old, per test vessel
Test Medium: Elendt M4
Test Vessels: Glass scintillation vials (ca 25 mL) containing ca 20 mL of test media covered with a clear lid.
Environmental Conditions: Between 23 and 27ºC, not varying by more than 2°C, dissolved oxygen concentration ≤100% air saturation value, variation in pH of ≤ 1.5 and 16:8 hours light:dark photoperiod.
Preparation Details: An amount of reference substance (ca 1600 mg) was dissolved in 500 mL of Elendt M4 media to give the 3200 mg/L test concentration. Serial dilutions were prepared in Elendt M4 to give the remainder of the test concentrations. Control = Elendt M4 only.
Observations: Performed daily and the number of immobilised adult Ceriodaphnia and juveniles produced in each vessel recorded. Length of each surviving adult was not recorded at test termination in error.
Validity Criteria:
* Control parental mortality should not exceed 20% at the end of the test
* The proportion of adult males should be ≤10%
* Mean number of live offspring per surviving adult female should be ≥15
* 60% or more of adult females should produce three broods
Statistical Analysis System: CETIS v1.8.6.8, CETIS – Comprehensive Environmental Toxicity Information System. 2001-2012. Tidepool Scientific, LLC

RESULT: The 8-day EC50 value and NOEC for reproduction were determined to be 1789 and 1000 mg/L, respectively. The expected range required by Guideline (ISO 20665) for reproduction EC50 is 580-1560 mg/L. The EC50 in this test is slightly higher than this range, however as the coefficient of variation for data reproducibility in the guideline is high (30.3%), these results are still considered to be within the expected range. Therefore, these results indicated that the test organisms were responding as expected within the test system.

Reported statistics and error estimates:

EC10 (Adult survival) = 0.16 mg/L (95% confidence limit = 0.011 mg/L - NC)
EC20 (Adult survival) > 0.17 mg/L

EC10 (Juvenile production) = 0.12 mg/L (95% confidence limit = 0.013 mg/L - NC)
EC20 (Juvenile production) = 0.14 mg/L (95% confidence limit = 0.10 mg/L - NC)

EC10 (Growth) > 0.17 mg/L
EC20 (Growth) > 0.17 mg/L

The calculated EC10 and EC20 are considered not appropriate as reference values due to wide 95% confidence limits.
In addition, a 20% effect for a chronic study is not generally considered to be biologically significant given the complexity of the test system. It was therefore considered that the NOEC and LOEC values are the most relevant endpoints.

Summary of Biological Results

Nominal concentration (% saturated solution)	Time-weighted mean measured concentration (mg/L)	Survival of parental Ceriodaphnia dubia at Day 8 (%)	Mean cumulative live juvenile production at Day 8	Carapace measurements Mean length  (mm)
Control	Control	80	31	1.0
10	0.025	80	30	0.9
18	0.037	60	27	0.9
32	0.067	90	34	1.0
56	0.11	90	34	0.9
100	0.17	70	17	0.9

Analytical Results

The results of chemical analysis on the test media during the definitive test are presented in attached pdf Table.

Chemical analysis of the freshly prepared 100% saturated solution showed measured concentrations to range from 0.148 to 0.378 mg/L (mean: 0.246 mg/L, standard deviation: 0.0758 mg/L). These results were in-line with those observed during the range-finding test.

Variation was observed in the measured concentrations of the 100% saturated solution with a coefficient of variation (CV) of 31%. Given the difficult nature of the test substance (poor water solubility and high volatility) any slight variations in preparation of the saturated solution, handling of the test vessels or test conditions could impact on the reproducibility of the test concentrations. In addition, although the freshly prepared 100% saturated solution appeared to be a colourless solution, it cannot be fully excluded that no residual undissolved test substance was present in the saturated solution following centrifugation.

Comparing measured concentrations and data dispersion among the different series of samples at the same saturation degree, it must be noted that the overall uncertainty for sample preparation, sampling and chemical analysis would be expected to be quite large given the nature of the compound despite care being taken to apply best practices and verifying compliance with the quality control requirements.

In general, a decrease in measured concentration was observed over each 24-hour exposure period with measured concentrations in the old media for the 100% saturated solution ranging from 0.0618 to 0.202 mg/L. These losses were considered to be due to the highly volatile nature of the test substance despite precautions being taken to prepare and seal the test vessels as soon as possible after preparation and sampling immediately upon re-opening the vessels.

In two instances, the measured test concentrations in the old media were equivalent or greater than the corresponding fresh media concentrations. This was observed for the 32, 56 and 100% saturated solution test concentrations on Days 1 and 2, where the Day 2 old media measured concentrations were higher than the Day 1 fresh media samples, and for the 18% saturated solution test concentration on Days 5 and 6 where the measured concentrations in the fresh and old samples were the same. A review of the data could not define a reason for these results. However, it should be noted that any slight variation in the preparation of the test vessels, for example, the time between adding the test substance to the vessel and sealing the vessel or the time taken to sample from the re-opened vessels, despite all precautions being taken to standardise the preparation method, could result in variations in measured concentration when the test substance is highly volatile.

Given the general decline in measured concentration over each exposure period, it was considered appropriate to base the results on time-weighted mean measured concentrations. These were calculated to be 0.025, 0.037, 0.062, 0.11 and 0.17 mg/L for the 10, 18, 32, 56 and 100% saturated solution test concentrations, respectively.

These values were again consistent with those calculated for the range-finding test indicating that the preparation method was relatively consistent given the difficulties associated with the handling of a highly volatile compound where any exposure to the air could result in losses of the test substance.

In some instances a small amount of test substance was measured in the control vessels. In general, the values recorded were at or close to the LOQ of the analytical method (0.010 mg/L). In addition, with the exception of Day 3 fresh media and the corresponding Day 4 old media, concentrations were not observed in fresh and old media from the same preparation period suggesting that the contamination was from an outside source and not present within the test vessels (for example, concentration was not observed in the control from the freshly prepared media on Day 5 but concentrations were observed in the corresponding old media on Day 6). Despite all precautions being taken, for example, sampling the control vessels prior to the test vessels and adding directly to the headspace vials, due to the highly volatile nature of the test substance contamination of the control can, on occasions be observed. In addition, the test substance has the potential to adsorb to organic matter which could result in difficulty in removing residues from equipment. It is therefore also possible that the contamination could be the result of carryover from equipment during sampling or analysis. The source of the contamination was finally not clarified. Even if the most probable explanation is that the observed control concentrations were due to possible contamination during sampling or analysis, contamination of the media during the preparation of the control replicates could not be excluded as the observed inconsistent concentrations between fresh and old media samples of the same renewal period may be a result of the variability in the preparation of individual replicates. No correction for the control values was made.

Validity criteria fulfilled:

yes

Conclusions:

Although the study meets the validity criteria set up in the OECD guideline No. 211 (2015) and ISO Guideline No. 20665 (2008) there are definitely some grounds of concern on the reproducibility of the results. In fact, various weak points were observed both in the measurement system and organisms system:

- Several technical and analytical difficulties (i.e. difficulties to generate reproducible test solutions, difficulties to maintain test concentrations at acceptable levels within the test period, test substance slight contamination in the control solutions) were observed due to the challenging execution because of practical reasons like high volatility and low water solubility of the test substance;

- Difficulties to maintain healthy and alive cultures of Ceriodaphnia dubia invertebrates were encountered by the laboratory. Indeed, the conditions in which the organisms appeared to be during all the experimental phase is questionable. This was apparent in the range finder study (high parental mortality rates during the range finding study wihout dose-response relationship, and failure of the culture after the test) as well as in the additional sensitivity tests (5 of 9 tests failed in fullfiling the acceptability criteria of the guidelines). The conditions are also questionable in the definitive study due to the high parental mortality rates in the negative control (at the boundary of the validity criteria) and at the different test concentrations without dose-response relationship.

In conclusion, uncertainty is present about the reproducibility of the results and therefore, the results are considered unsuitable for hazard assessment and classification.

Executive summary:

An 8-day Ceriodaphnia dubia reproduction study was conducted to determine the effects of exposure to test substance on Ceriodaphnia dubia re production, growth and survival. The study was conducted based on the OECD Chemicals Testing Guideline No. 211 Daphnia magna Reproduction Test (2 October 2012) and ISO 20665 Determination of Chronic Toxicity to Ceriodaphnia dubia (15 December 2008).

The test substance is known to be poorly soluble and highly volatile falling into the category of a “difficult substance” as defined by the OECD Guidance Document (OECD Series on Testing and Assessment, No. 23 (2000); Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures).

The rationale for the selection of this species was based on the small volumes of water required by Ceriodaphnia dubia compared to other species of daphnids. As the test substance is highly volatile from water, the use of small volumes of solutions during preparation, handling and transfer of media allows for minimisation of the possibility of losses of the test substance through volatilisation.

 

Prior to the main study extensive media preparation trials were performed to determine the most appropriate method of preparation for the test substance in order to achieve the maximum saturation concentration in the test medium avoiding the formation of emulsions.

It was considered that the most appropriate method of preparation was a saturated solution method whereby an initial loading rate of 676 mg/L was stirred for ca.4 hours followed by centrifugation at ca.1290 g for ca. 5 minutes. The supernatant was then used to prepare the test concentrations.

An analytical method for determination of the test substance in Elendt M4 medium was developed and validated according to SANCO/3029/99 rev. 4 for use in the study over a concentration range 0.01 to 0.5 mg/L.

The definitive test was conducted at nominal concentrations of 10, 18, 32, 56 and 100% saturated solution (corresponding to 0.025, 0.037, 0.062, 0.11 and 0.17 mg/L, respectively, based on time-weighted mean measured test concentrations). A control group was also included. The test was conducted using a semi-static design with daily renewal of the test media. As the test substance is known to be highly volatile, the test was conducted in completely closed system with minimised (vessels completely filled with media and sealed) head space volume in order to reduce losses of test substance due to volatilisation. Additional test vessels were prepared, for the control and each test concentration for analytical sampling only of the freshly prepared media. This was conducted to avoid any possible loss due to volatilisation from the test vessels due to sampling.

Chemical analysis of test solutions showed a decrease in measured concentration over each 24-hour exposure period (average decrease between 42% and 70% of the initial measured concentration). These losses were considered to be due to the highly volatile nature of the test substance despite precautions being taken to prepare and seal the test vessels as soon as possible after preparation and sampling immediately upon re-opening the vessels. In two instances, the measured test concentrations in the old media were equivalent or greater than the corresponding fresh media concentrations. A review of the data could not define a reason for these results. However, it should be noted that any slight variation in the preparation of the test vessels could result in variations in measured concentration when the test substance is highly volatile.

 

In some instances a small amount of test substance was measured in the control vessels. In general, the values recorded were at or close to the LOQ of the analytical method. The source of the contamination was finally not clarified. Even if the most probable explanation is that the observed control concentrations were due to possible contamination during sampling or analysis, contamination of the media during the preparation of the control replicates could not be excluded.No correction for the control values was made.

A high parental mortality rate (10 to 40%) was observed at all the tested concentrations without dose response relationship. In the control group, 20% parental mortality was observed, therefore the validity criterion for adult mortality not exceeding 20% over the duration of the test was satisfied. On comparison to the control group, there were no statistically significant effects on the mortality of the adult Ceriodaphnia at any of the concentrations employed. In addition, no significant differences were observed between treatments.

A significant reduction (p >0.05) in the number of juveniles produced was observed at the 100% saturated solution test concentration compared to the control. No reduction was observed at the other test concentrations.

 On comparison to the control group, there were no statistically significant effects on the growth of the adult Ceriodaphnia surviving to Day 8 at any of the concentrations employed.

 

The Day 8 EC50 values in terms of reproduction, adult survival and adult growth based on time-weighted mean measured test concentrations, were >0.17 mg/L. The corresponding NOEC and LOEC values for adult survival and growth, based on time-weighted mean measured test concentrations, were considered to be 0.17 and >0.17 mg/L, respectively. The NOEC and LOEC values for reproduction, based on time-weighted mean measured test concentrations, were considered to be 0.11 and 0.17 mg/L, respectively.

EC10 and EC20 values are considered not reliable due to the wide 95% confidence limits.

 

The validity criteria set up in the OECD guideline No. 211 (2015) and ISO Guideline No. 20665 (2008) were satisfied. However, although the study meets  the validity criteria there are some grounds of concern on the reproducibility of the results.

The healthy conditions in which the clone appeared to be during all the experimental phase is questionable. Indeed, difficulties were encountered to maintain alive cultures of invertebrates. This was apparent in the range finder study where a high parental mortality rates without dose response relationship was observed as well as in the additional sensitivity tests performed before to launch the main study. In fact, after the range-finding study, the culture of C.dubia crushed and a new culture was initiated using a new batch. Acute and chronic sensitivity tests were launched on the new batch of C. dubia. In 5 on 9 instances the chronic sensitivity studies with the reference substance Sodium Chloride conducted according the TG ISO 20665 failed in fulfilling the validity criteria defined in the test guideline As a consequence, the sensitivity tests were repeated several times, and the main study was launched after obtaining two consecutive valid sensitivity tests.

A high parental mortality rate was observed at all the tested concentrations during the definitive test too. The most probable cause of death was supposed to be mechanical stress due to handling. The high mortality rate observed at all the tested concentration and in the control, just at the boundary of the validity criteria, add additional uncertainty on the reproductibility of the results.

In conclusion,although the study meets  the validity criteria set up in the OECD guideline No. 211 (2015) and ISO Guideline No. 20665 (2008) there were various weak points both in the measurement system and organisms system:

- Difficulties to generate reproducible test solutions;

- Difficulties to maintain test concentrations at acceptable levels within the test period;

- Test substance slight contamination in the control;

- Difficulties encountered by the laboratory to maintain healthy cultures of invertebrates;

- High maternal mortality in controls and treatment vessels but without dose response relationship;

- Only significant effects on reproduction in the highest tested dose;

Uncertainty is therefore present about the reproducibility of the resultsand therefore, the results are considered unsuitable for hazard assessment and classification.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Data waiving:

exposure considerations

Justification for data waiving:

other:

Description of key information

No reliable long term studies on aquatic invertebrates are available. However, according to the environmental fate assessment, GALDEN LMW is not expected to distribute significantly into water compartment and therefore exposure of aquatic organism is considered negligible.

Additional information

Although GALDEN LMW, according to the environmental fate assessment, is not expected to distribute significantly into water compartment and therefore exposure of aquatic organism is considered negligible, a long-term toxicity study on invertebrates was conducted in order to assess the potential toxic effects on aquatic organisms following long term exposure against the EU GHS criteria.

However, several technical difficulties and weak points were encountered during the performance of that study which finally prevented to obtain reliable results suitable for hazard assessment:

- Difficulties to generate reproducible test solutions (Coefficient of variation of 31% was observed in the measured concentrations of the 100% saturated solution , CV% between 29% and 56% in the other dilutions.);

- Difficulties to maintain test concentrations at acceptable levels within the test period (Decrease in measured concentration was observed over each 24-hour: 100%SS ca 45%; 56%SS ca 42%;32%SS ca 45%;18%SS ca 58%; 10%SS ca 70%]);

- Test substance contamination in the control media;

- Difficulties encountered by the testing laboratory to maintain healthy cultures of invertebrates.

- High maternal toxicity in controls and treatment vessels (at different concentrations) but without dose response relationship;

- Only significant effects on reproduction in the highest tested dose.

 

Based on all of these observations and difficulties combined, the reproducibility of the test is questionable and the results are not considered reliable and suitable for hazard asses.

The technical difficulties encountered during the generation of test solutions and analytical system were fundamentally due to the difficult nature of the substance (high volatility and low water solubility), confirming the tendency of the test substance to not remain in the water compartment.