Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 10 December 2015 and 15 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Waxy solid
Details on test material:
Identification: Acetalization product between glucose and C20/22 (even numbered)-alcohol
Physical state/Appearance: Beige waxy solid (pearls)
Batch: U44635
Purity: >99.9% (dry extract)
Expiry Date: 01 November 2017
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Verification of Test Concentrations
Water samples were taken from the control and 100 mg/L loading rate WAF test group (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 5, 9, 14 and 19, and of the expired test preparations on Days 2, 7, 12, 16 and 21. Duplicate samples were taken and stored frozen for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
Rane-finding test
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10, and 100 mg/L.
For the 1.0 mg/L test preparation, prior to addition of the test item a glass siphon tube was placed in the test media. Nominal amounts of test item (20, 20 and 200 mg) were each separately added to the surface of test water (20, 2 and 2 liters) to give the 1.0, 10, and 100 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. For the 1.0 mg/L test concentration, a length of Tygon tubing was attached to the top of the glass siphon tube. For the 10 and 100 mg/L test preparations 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. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs.

Definitives test
A nominal amount of test item (400 mg) was added to the surface of 4 liters of test water to give the 100 mg/L loading rate. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture 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. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 100 mg/L loading rate WAF.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Test System and Supporting Information
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None

Test conditions

Hardness:
The water hardness of the control and the 100 mg/L loading rate WAF was measured in the fresh (Days 0, 7 and 14) and old media (Days 2, 9, 16 and 21).
Test temperature:
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer. Measurements were made on one replicate for each test concentration. The temperature was also measured every hour in one replicate of the control using a Testo temperature logger up to day 20.
pH:
pH was recorded before and after each test media renewal using a Hach HQ30d Flexi handheld meterMeasurements were made on one replicate for each test concentration.
Dissolved oxygen:
Dissolved oxygen concentrations were recorded before and after each test media renewal using a Hach HQ30d Flexi handheld meterMeasurements were made on one replicate for each test concentration.
Nominal and measured concentrations:
Range-finding test: Nominal loading rates of 1.0, 10, and 100 mg/L.
Definitive test: Nominal loading rate of 100 mg/L
Details on test conditions:
Test Water
The reconstituted water (Elendt M7 medium) used for the range-finding and definitive tests was the same as that used to maintain the stock animals.

Experimental Design and Study Conduct
Validation of Mixing Period
Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.

Range-finding Test
In the range-finding test, for each concentration a single daphnid was placed in 100 mL of the test preparation in 150 mL glass vessels which were then covered with a plastic lid to reduce evaporation. For each test and control group five replicate test vessels were prepared. The water temperature was maintained at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for 10 days.
The control group was maintained under identical conditions, but not exposed to the test item.
The test preparations were renewed on Days 3, 5 and 7. The adult daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted before being discarded.
Each daphnid received approximately 5 to 10 µL of an algal suspension (Desmodesmus subspicatus) and approximately 20 µL of Tetramin® flake food suspension daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid. A sample of each test concentration was taken for chemical analysis on Days 0 and 5 from fresh media and on Day 7 from old media in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. A sample of each test concentration was taken on Day 3, however, the sample volume was incorrect therefore these samples were not analyzed. Only concentrations within the range to be used for the definitive test were analyzed. 

Definitive Test
Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a Water Accommodated Fraction (WAF) of the test item at single nominal loading rate of 100 mg/L for a period of 21 days. The test solution was renewed 3 times per week throughout the test.

Experimental Preparation
A nominal amount of test item (400 mg) was added to the surface of 4 liters of test water to give the 100 mg/L loading rate. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture 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. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 100 mg/L loading rate WAF.
The concentration and stability of the test item in the test preparations were verified by chemical analysis from the freshly prepared solutions on Days 0, 5, 9, 14 and 19 (from bulk test preparation), and from the old test media on Days 2, 7, 12, 16 and 21 (from pooled replicates) (see Annex 4).

Exposure Conditions
For each concentration a single daphnid was placed in 100 mL of the test preparation in 150 mL glass vessels which were then covered with a plastic lid to reduce evaporation. For each test and control group ten replicate test vessels were prepared. The test vessels were maintained in a temperature controlled room at 18 to 22 C with a maximum deviation of
±1 °C with a photoperiod of 16 hours light (not exceeding 1500 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. The test vessels were not aerated. The diluent water only was aerated prior to use.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were renewed 3 times per week on Days 2, 5, 7, 9, 12, 14, 16 and 19. The adult daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted before being discarded.
From Days 0 to 6, each daphnid received approximately 5 µL of an algal suspension (Desmodesmus subspicatus) and approximately 20 µL of Tetramin® flake food suspension daily. From Day 7 onward, each daphnid received approximately 20 µL of an algal suspension (Desmodesmus subspicatus) daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

Assessments
Test Organism Observations
On a daily basis the numbers of live and dead of the "Parental" (P1) generation, the numbers of live and dead "Filial" (F1) daphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental daphnia as compared with the controls.
Filial daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Parental daphnia which were unable to swim for approximately 15 seconds after gentle agitation (i.e. immobile) were considered to be dead. An immobilization criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.
At the end of the test, the length of each surviving parent animal was determined.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
Range-finding Test
No immobilization or sub-lethal effects of exposure were observed at the test concentrations of 1.0, 10 and 100 mg/L loading rate WAF.
Chemical analysis of the water accommodated fraction of the test item in the 10 mg/L loading rate WAF test preparations on Days 0, 5 and 7 showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.0021 mg/L. Measured test concentrations in the
100 mg/L loading rate WAF test preparations from fresh media on Days 0 and 5 ranged from 0.00484 to 0.0254 mg/L. The measured test concentration in the old media on Day 7 was observed to have declined to less than the LOQ indicating that the water accommodated fraction of the test item was unstable under test conditions.
Based on this information a single nominal loading rate of 100 mg/L was selected for the definitive test.

Definitive Test
Lethal Effects on the Parental Generation (P1)
No mortalities occurred in the 100 mg/L loading rate WAF group throughout the test.
It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Sub-lethal Effects on the Parental Generation (P1)
The daphnids in the 100 mg/L loading rate WAF test concentration were observed to be the same size and color as the control daphnids.
After 21 days the length of each surviving adult was determined. The results showed that there were no statistically significant differences (P≥ 0.05) between the control and the 100 mg/L loading rate WAF test group in terms of length of the daphnids after 21 days exposure to the test item.

Effects on Reproduction
After 21 days there were no statistically significant differences between the control and the 100 mg/L loading rate WAF group in terms of the number of live young produced per adult.
The following ELx (reproduction) values based on nominal loading rates were estimated from inspection of the immobilization data at 21 days:
EL10 >100* (mg/L Loading Rate WAF)
EL50 >100* (mg/L Loading Rate WAF)
It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Effects on the Filial Generation (F1)
Information on the effects of the test item on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by the
100 mg/L loading rate WAF group were in the same general condition as the young produced by the control over the duration of the test.
Young were first produced in the control test group on Day 7 of the test.
There were no unhatched eggs or dead young recorded in the control and treatment groups.

Lowest Observed Effect Loading Rate
The "Lowest Observed Effect Loading Rate" (LOEL) was considered to be greater than 100 mg/L on the basis that this loading rate had no significant mortalities (immobilisation) observed in the parental generation (P1) and that there were no significant differences (P≥0.05) between the control and the 100 mg/L loading rate WAF group in terms of numbers of live young produced per adult by Day 21.

No Observed Effect Loading Rate
The "No Observed Effect Loading Rate" (NOEL) was 100 mg/L as there were no significant mortalities (immobilization) observed in the parental generation (P1) and there were no significant differences (P≥0.05) in terms of the number of live young produced per adult when compared to the control after 21 days.
Reported statistics and error estimates:
An estimate of the 21-Day EL50 value (immobilization) was given by inspection of the data.
The EL50 (reproduction) value after 21 days was estimated by inspection of the data.
For the estimation of the "Lowest Observed Effect Loading Rate" (LOEL) and the "No Observed Effect Loading Rate" (NOEL) the numbers of live young produced per adult over the duration of the test for the control and 100 mg/L loading rate WAF test group were compared using a Student-t test for Homogenous Variances incorporating Levene’s Test on Variance Homogeneity and Shapiro-Wilk’s Test on Normal Distribution. Results from the control and 100 mg/L loading rate WAF test group daphnia length data, determined for the surviving daphnids on termination of the test, were compared using a Student-t test for Homogenous Variances incorporating Levene’s Test on Variance Homogeneity and Shapiro-Wilk’s Test on Normal Distribution. All statistical analyses were performed using the ToxRat professional computer software package (TOXRAT).

Any other information on results incl. tables

Validation of Mixing Period

The measured concentration of the water accommodated fraction of the test item in the test media was shown not to increase with an extended stirring period. In fact, increasing the preparation time from 24 to 96 hours resulted in a reduced measured concentration, therefore the WAF’s for the range-finding and definitive tests were prepared using a 23-Hour stirring period followed by a 1-Hour settlement period prior to removal of the aqueous phase for testing.

Verification of Test Concentrations

Chemical analysis of the fresh test preparations showed measured concentrations of the water accommodated fraction of the test item to range from below the limit of quantification (LOQ) to 0.0144 mg/L. Chemical analysis of the aged test preparations showed measured concentrations of less than the LOQ of the analytical method employed were obtained indicating that the water accommodated fraction of the test item was unstable under test conditions. The LOQ was determined to be 0.0021 mg/L. 

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Validation Criteria

The following validation criteria were achieved during the test

 

Required

Actual

a)     Control mortality

20%

0%

b)     Mean number of live young per surviving adult (control group)

60 after 21 days

144

c)     Coefficient of variation for control group

25%

11.1%

d)    No ephippia produced

0

0

e)     Dissolved oxygen

> 3 mg O2/L

≥8.3 mg O2/L

f)      pH (control group)

6 to 9
Variation < 1.5

7.9 to 8.2
< 0.3

Water Quality Criteria

Temperature was maintained at 21 to 23°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

The water hardness was observed to be in the range 250 to 278 mg/L as CaCO3in the control and the 100 mg/L loading rate WAF test group throughout the test.

Throughout the test the light intensity was observed to be in the range 482 to 598 Lux.

The water temperature was also recorded in the control vessel every hour using a Testo temperature logger.

Vortex Depth Measurements

The vortex depth was recorded at the start and end of the mixing period and was observed to be approximately 1% of the media column height.

Observations on Test Item Solubility

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

At the start of the mixing period the 100 mg/L loading rate was observed to be a clear colorless water column with test item floating at the surface. After 23 hours stirring and a
1-Hour standing period the 100 mg/L loading rate was observed to remain as at the start of stirring. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the 100 mg/L loading rate was observed to be a clear, colorless solution.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The test item was a poorly water soluble multi-component substance; therefore test exposure solutions were prepared as Water Accommodated Fractions. Chemical analysis of the exposure solutions showed measured concentrations of the water accommodated fraction of the test item to range from less than the Limit of Quantification of the analytical method to 0.0144 mg/L. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.
Exposure of Daphnia magna to the test item resulted in no significant mortalities at the loading rate employed during the test.
The 21-Day EL50 (immobilization) value, based on nominal loading rates, for the parental daphnia generation (P1) was estimated to be greater than 100 mg/L.
No significant impairment of reproduction was observed at the loading rate employed during the test. The 21-Day EL50 (reproduction) based on nominal loading rates was greater than 100 mg/L.
The "Lowest Observed Effect Loading Rate" (LOEL) and the "No Observed Effect Loading Rate" (NOEL) based on nominal loading rates were 100 mg/L.
Executive summary:

Introduction

A study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211, "Daphnia magna Reproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008.

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 (WAF).

Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a Water Accommodated Fraction (WAF) of the test item at single nominal loading rate of 100 mg/L for a period of 21 days. The test solutions were renewed 3 times per week throughout the test. 

The numbers of live and dead adult daphnia and young daphnids (live and dead) were determined daily. The daphnia were fed daily with a mixture of algal suspension and Tetramin®flake food suspension until day 6 and with an algal suspension from day 7 to the end of the test.

 Results

Chemical analysis of the fresh test preparations showed measured concentrations of the water accommodated fraction of the test item to range from below the limit of quantification (LOQ) to 0.0144 mg/L. Chemical analysis of the aged test preparations showed measured concentrations of less than the LOQ of the analytical method employed were obtained indicating that the water accommodated fraction of the test item was unstable under test conditions. The LOQ was determined to be 0.0021 mg/L

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

The 21-Day EL50 (immobilization) value, based on nominal loading rates, for the parental Daphnia generation (P1) was estimated to be greater than 100 mg/L loading rate WAF.

The 21-Day EL50(reproduction) value, based on nominal loading rates, was estimated to be greater than 100 mg/L loading rate WAF.

The "No Observed Effect Loading Rate" was considered to be 100 mg/L on the basis that at this test concentration there were no significant mortalities (immobilization) observed in the parental generation (P1) and that there were no significant differences (P³0.05) between the control and the 100 mg/L loading rate WAF test group in terms of numbers of live young produced per adult by Day 21.

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