(1R)-1-[(4R,4aR,8aS)-2,6-bis(3,4-dimethylphenyl)tetrahydro[1,3]dioxino[5,4-d][1,3]dioxin-4-yl]ethane-1,2-diol

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:

10th June 2002 to 1st July 2002 (definitive test)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

Sponsor's indentification: Experimental 11334-18
Lot No.: Y7977

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The concentration and stability of the test material in the test preparations were verified by chemical analysis on Days 0 (fresh media), 2, 4, 7, 9, 11, 14, 16, 18 and 21 (old media).

- Concentrations: Water samples were taken from the solvent control and each surviving test group (replicates pooled) for quantitative analysis.
- Sampling method: Duplicate samples were taken on the days specified above.
- Sample storage conditions before analysis: Samples were stored frozen at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:

yes

Details on test solutions:

Identity and concentration of auxiliary solvent for dispersal: dimethylformamide

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Amounts of test material (37.5 and 120 mg) were each separately dissolved in dimethylformamide, with the aid of ultrasonication for approximately 5 minutes, prior to adjusting the volume to 250 mL to give 37.5 and 120 mg/250 mL solvent stock solutions respectively. These solvent stock solutions were diluted to give further solvent stock solutions of 0.48, 1.5 and 4.8 mg/100 mL. An aliquot (500 μL) of each of the solvent stock solutions was separately dispersed in reconstituted water and the volume adjusted to 5 litres to give the 0.00048, 0.0015, 0.0048, 0.015 and 0.048 mg/L test concentration.
Each solvent stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
- Controls: The control and the solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 µl/L of dimethylformamide.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not specified
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None specified

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Strain/clone: Daphnia magna
- Justification for species other than prescribed by test guideline: Not applicable
- Source: in-house laboratory culture
- Age of parental stock (mean and range, SD): Adult Daphnia
- Feeding during test:
- Food type: Unicellular algal culture (Chlorella vulgaris)
- Amount: Each daphnid received approximately 2.3 – 4.7 μL of food daily. Feeding was at a level of approximately 0.1 – 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.
- Frequency: Daily

ACCLIMATION: Not specified - culture conditions are described below

QUARANTINE (wild caught)
Not applicable

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water at a temperature of 21°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 at a light intensity of 560 - 612 lux. Each culture was fed daily with a suspension of algae (Chlorella vulgaris.). 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:

Not performed

Test conditions

Hardness:

Theoretical total hardness of the water of 250 mg/L as CaCO3.
Measured water hardness values were observed to be in the range 226 to 357 mg/L as CaCO3 in the solvent control and the highest surviving test group throughout the test.

Test temperature:

21°C

pH:

Measured values throughout the test: pH 7.9-8.0

Dissolved oxygen:

Measured values throughout the test were from 8.1-8.5 mg O2/L.
Dissolved oxygen concentration as a % of the air saturation volume = 91-96%

Salinity:

Not specified

Nominal and measured concentrations:

Based on the results of an acute toxicity test to 1st instar Daphnia magna test and the limit of water solubility value (0.048 mg/L, based on data supplied by the Sponsor) the following test concentrations were assigned to the definitive test: 0.00048, 0.0015, 0.0048, 0.015 and 0.048 mg/L.
Chemical analysis of the freshly prepared test media on Day 0 showed measured test concentrations to range from 84% to 116% of nominal. Analysis of the expired test media at Days 2 to 21 showed measured test concentrations to range from 64% to 118% of nominal. The mean values for each test concentration (over Days 0-21) were calculated to range from 82% to 107% of nominal and so it was considered justifiable to base the results on nominal test concentrations only. The low and variable results obtained throughout the test were considered to be due to possible adsorption to algal feed the test organisms and/or their waste products.
Chemical analysis of the solvent control showed a measured test concentration of 0.000362 mg/L on Day 21. This was less than the lowest nominal test concentration used during the study and given that no test material was shown in the solvent control on all other sampling occasions it was
considered that this may have been due to post sampling contamination of the sample. Therefore, this was considered not to affect the validity or integrity of the study given that no mortalities or adverse effects of exposure were observed in the parental generation (P1) and no significant difference in number of young produced when compared to the control group.

Details on test conditions:

TEST SYSTEM
- Test vessel: For each concentration a single daphnid was placed in 120 mL of the test preparation in 150 mL glass flasks.
- Type: Closed - the flasks were covered with a plastic lid to reduce evaporation.
- Material, size, headspace, fill volume: Not specified
- Aeration: The test vessels were not aerated. The diluent water only was aerated prior to use.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable - semi-static test
- Renewal rate of test solution (frequency/flow rate): The test preparations were renewed 3 times per week on Days 2, 4, 7, 9, 11, 14, 16 and 18.
- No. of organisms per vessel: 1 daphnid/ vessel
- No. of vessels per concentration (replicates): 10 vessels/ concentration
- No. of vessels per control (replicates): 10 vessels/ control
- No. of vessels per vehicle control (replicates): 10 vessels/ control
- Biomass loading rate: Not specified

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: An aliquot (25 mL) of each of the solutions (CaCl2.2H2O, MgSO4.7H2O, NaHCO3 and KCl) was added to each litre (final volume) of deionised water. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
- Total organic carbon: Not specified
- Particulate matter: Not specified
- Metals: Not specified
- Pesticides: Not specified
- Chlorine: Not specified
- Alkalinity: Not specified
- Ca/mg ratio: Not specified
- Conductivity: <5 μS cm-1
- Salinity: Not specified
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature of the test preparations and light intensity were recorded daily throughout the test. Dissolved oxygen concentrations, pH and temperature were recorded before and after each test media renewal. Measurements were made on one replicate for each test concentration. The water hardness of the solvent control and the highest surviving test concentration in the fresh and old media was measured once per week.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days
- Light intensity: 560 - 612 lux

EFFECT PARAMETERS MEASURED:
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.
The number of Daphnia with eggs or young in the brood pouch was determined at each media renewal. Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Daphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilisation 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.

VEHICLE CONTROL PERFORMED: Yes - the solvent control group was exposed to 100 μl/L of dimethylformamide.

RANGE-FINDING STUDY
A range-finding study was not performed. Test concentrations were chosen based on the results from an acute toxicity test to Dapnia magna.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.048 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: Parental (P1) generation

Effect concentrations 2

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.048 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Parental (P1) generation

Effect concentrations 3

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 0.048 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: Parental (P1) generation

Effect concentrations 4

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.048 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: Parental (P1) generation

Details on results:

- Mortality of parent animals: No mortalities occurred at the 0.00048, 0.0015 and 0.015 mg/L test concentrations throughout the test. However, a single mortality was observed at each of the test concentrations of 0.048 and 0.0048 mg/L on Days 11 and 18 respectively.
Statistical analysis of the mortality data using the corrected chi-squared statistic (Breslow and Day 1980) showed that the observed mortality in each of the 0.0048 and 0.048 mg/L test groups were not significant (P≥0.05) when compared to the solvent control group.
However, no further mortality occurred throughout the test and, hence, no prolonged effects attributable to exposure of Daphnia magna to the test material were observed.
- No. of offspring produced per day per female: See Table 1
- Body length and weight of parent animals: After 21 days the length of each surviving adult was determined, the results of which are given in
Table 2. The results showed that there were no statistically significant differences (P≥ 0.05) between the control, solvent control and all the test groups in terms of length of the daphnids after 21 days exposure to the test material.
- Type and number of morphological abnormalities: None reported
- Type and number of behavioural abnormalities: None reported
- Number of males and females (parental): Not specified
- Time to first brood release or time to hatch: Young were first produced in the solvent control test group on Day 7 of the test.
- Egg development time: Numbers of unhatched eggs and dead young were low in all control and treatment groups surviving to maturation.
- Brood size: After 21 days there were no statistically significant differences between the solvent control, control and all the test groups in terms of the number of live young produced per adult.
- Time to sexual maturity: Not specified
- Type and magnitude of biochemical changes: Biochemical changes were not observed.
- Other biological observations: Information on the effects of the test material 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 all the test groups were in the same general condition as the young produced by the solvent controls over the
duration of the test.
- Effect concentrations exceeding solubility of substance in test medium: Not applicable - concentrations were chosen to be below the limit of solubility.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

No data available for error estimates.
Statistics are discussed in the results section.

Any other information on results incl. tables

The analytical detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test material in test medium. A method of analysis was validated and proven to be suitable for use. The test material was stable in the test medium for the duration of the test period.

Table 1: Summary of findings following exposure of Daphnia magna for 21 days

Nominal concentration (mg/L)	% survival of P1	Number of live young		Number of dead young		Number of unhatched eggs
		Total	Per female (cumulative)	Total	Per female (cumulative)	Total	Per female (cumulative)
Control	100	891	89	0	0	0	0
Solvent control	100	922	92	0	0	1	<1
0.00048	100	1002	100	0	0	5	1
0.0015	100	953	95	0	0	1	<1
0.0048	90	934	97	0	0	1	<1
0.015	100	955	96	0	0	1	<1
0.048	90	926	101	0	0	1	<1

Table 2: Mean length of the surviving daphnids of the P1 generation

Nominal concentration (mg/L)		Day 21 (mm)
Control	Mean	4.0
	Standard deviation	0.3
Solvent control	Mean	3.9
	Standard deviation	0.2
0.00048	Mean	3.9
	Standard deviation	0.2
0.0015	Mean	3.9
	Standard deviation	0.2
0.0048	Mean	3.9
	Standard deviation	0.3
0.015	Mean	3.9
	Standard deviation	0.3
0.048	Mean	4.0
	Standard deviation	0.3

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test material resulted in no significant mortalities at all the test concentrations employed during the test.
The 14 and 21-Day EC50 (immobilisation) values, based on nominal test concentrations, for the parental Daphnia generation (P1) were estimated to be greater than 0.048 mg/L.
No significant impairment of reproduction was observed at the test concentrations employed during the test.
The 21-Day EC50 (reproduction) based on nominal test concentrations was greater than 0.048 mg/L.
The LOEC and the NOEC based on nominal test concentrations were considered to be greater than 0.048 mg/L and 0.048 mg/L respectively.

Executive summary:

A study was performed to assess the effect of the test material on the reproduction of Daphnia magna over a 21-day period. The method followed that described in the OECD Guidelines for Testing of Chemicals No 211 (1998), referenced as Method C.20 of Commission Directive 2001/59/EC.

The 14 and 21-Day EC50 (immobilisation) values, based on nominal test concentrations, for the parental Daphnia generation (P1) were estimated to be greater than 0.048 mg/L.

The 21-Day EC50 (reproduction) value based on nominal test concentrations was estimated to be greater than 0.048 mg/L.

The LOEC based on nominal test concentrations was considered to be greater than 0.048 mg/L on the basis that at this test concentration no significant mortalities (immobilisation) were observed in the parental generation (P1) and that there were no significant differences (P≥0.05) between the solvent control and the 0.048 mg/L test group in terms of numbers of live young produced per adult by Day 21.

The NOEC based on nominal test concentrations was considered to be 0.048 mg/L on the basis that at this test concentration no significant mortalities (immobilisation) were observed in the parental generation (P1) and that there were no significant differences (P≥0.05) between the solvent control and the 0.048 mg/L test group in terms of numbers of live young produced per adult by Day 21.