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:
26th October 2010 to 22nd November 2010
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 relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 20th July 2010 Date of signature: 29th October 2010

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification :DVS005u (aka Weston 705)
Description : clear colourless viscous liquid
Chemical name :Tris (amylated phenyl) phosphate mixture
Purity : not supplied
Batch number : MW9F23A901
Date received : 31 December 2009
Expiry date : 15 January 2011
Storage conditions :approximately 4°C in the dark under nitrogen

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: (nominal loading rates - mg/l): 1.0, 3.2, 10, 32 and 100

- Sampling method: Water samples were taken from the control and each surviving test group (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 5, 9 and 19 and of the expired test preparations on Days 2, 7, 12 and 21.

- Sample storage conditions before analysis: Samples were stored at approximately -20°C prior to analysis. Duplicate samples and samples on Days 2 (fresh media), 5 (old media), 7 (fresh media), 9 (old media), 14, 16 (old and fresh media) and 19 (old media) were taken and stored at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Due to the low aqueous solubility and complex nature of the test item, for the purpose of the definitive test the test item was prepared as a Water Accommodated Fraction (WAF).
Amounts of test item (10, 32, 100, 320 and 1000 mg) were each separately added to the surface of 10 litres of dechlorinated tap water to give the 1.0, 3.2, 10, 32 and 100 mg/l loading rates. After the addition of the test item, the dechlorinated tap water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into each vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phases or WAFs were removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
The concentration of the test item in the test preparations were verified by chemical analysis on Days 0, 5, 9 and 19 (fresh media) and 2, 7, 12 and 21 (old media).

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain/clone: not stated
- Justification for species other than prescribed by test guideline: not applicable
- Source: Derived from in-house laboratory culture
- Age of parental stock (mean and range, SD): age described only as adult
- Feeding during test: Each daphnid received approximately 5 to 10 µl of a unicellular algal culture (Chlorella sp.), 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.


ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not): Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of dechlorinated tap water at a temperature of 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.
- Type and amount of food and Feeding frequency: Each culture was fed daily with a suspension of algae (Chlorella sp.).
- Health during acclimation (any mortality observed): no mortality observed

QUARANTINE (wild caught)
- Not applicable

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: 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 using a stereo microscope before being discarded.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
15 seconds

Test conditions

Hardness:
The water hardness was observed to be in the range 118 to 158 mg/l as CaCO3 in the control and the highest surviving test group throughout the test.
Test temperature:
Temperature of the test preparations were recorded daily throughout the test using a Hanna Instruments HI 93510 digital thermometer

Temperature was maintained at approximately 19¿C to 22°C throughout the test.
Some of the temperatures during the definitive test were measured to be slightly in excess of the 20 ± 1°C given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed throughout the duration of the test and that the temperatures were within the test guideline specification.
pH:
pH was recorded before and after each test media renewal. The pH was measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

No treatment-related differences were observed for pH.
Dissolved oxygen:
The dissolved oxygen concentration was measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100 %. This was considered to be due to the presence of microscopic air bubbles in the media supersaturating the diluent and was considered not to have had an impact on the outcome or validity of the test as no adverse effects were observed in the control daphnids.
Salinity:
Not applicable, as freshwater study
Nominal and measured concentrations:
Nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beaker
- Type (delete if not applicable): closed - covered with plastic lid to reduce evaporation
- Material, size, headspace, fill volume: 100 ml of the test preparation in a 150 ml glass beaker
- 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
- Renewal rate of test solution (frequency/flow rate): The test preparations were renewed 3 times per week on Days 0, 2, 5, 7, 9, 12, 14, 16 and 19.
- No. of organisms per vessel: single Daphnid
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: not determined

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the test was the same as that used to maintain the stock animals.
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardeness of approximately by 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.

- Total organic carbon:
Average: 0.985 mg/l
Range: 0.640 - 1.390 mg/l

- Metals: no total value for metals reported

- Pesticides: (Total)
Average: 0.014 µg/l
Range: 0.006 - 0.031 µg/l

- Chlorine:
Free chlorine:
Average: 0.250 mg/l
Range: 0.010 - 0.460 mg/l

Total chlorine
Average: 0.312 mg/l
Range: 0.050 - 0.540 mg/l

- Alkalinity:

- Ca/mg ratio:

- Conductivity:
Average: 358.885 µS/cm at 20°C
Range: 269.000 - 487.000 µS/cm at 20°C

- Salinity: Not applicable, as freshwater study

- Culture medium different from test medium: No

- Intervals of water quality measurement: 8 samples taken over a period of 1 year

OTHER TEST CONDITIONS
- Adjustment of pH: not required
- Photoperiod: photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 422 to 503 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 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 daily. 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 beakers.
At the end of the test, the length of each surviving parent animal was determined.


VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: Acute Toxicity study to Daphnia magna - please reference Section 6.1.3 of IUCLID
- Results used to determine the conditions for the definitive study: Yes
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.
Remarks:
loading rate WAF
Basis for effect:
immobilisation
Remarks on result:
other: Parental generation (P1)
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
other: LOELR
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
immobilisation
Remarks on result:
other: P>=0.05
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
immobilisation
Remarks on result:
other: P>=0.05
Details on results:
- Mortality of parent animals: No mortalities occurred at the 1.0 and 100 mg/l loading rate WAF test groups throughout the test.
Mortality was observed at the 3.2, 10 and 32 mg/l loading rate WAFs. However, statistical analysis of the mortality data using the corrected chi-squared statistic (Breslow and Day 1980) showed that the observed mortalities were not significantly different (P¿0.05) when compared to the control group.
No further mortality occurred throughout the test and, hence, no prolonged effects attributable to exposure of Daphnia magna to the test item were observed.

- No. of offspring produced per day per female: After 21 days there were no statistically significant differences between the control and all the test groups in terms of the number of live young produced per adult

- Body length and weight of parent animals: 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 1.0, 3.2, 10, 32 and 100 mg/l loading rate WAF test groups in terms of length of the daphnids after 21 days exposure to the test item.

- Type and number of morphological abnormalities:

- Type and number of behavioural abnormalities:

- Number of males and females (parental): Not determined

- Time to first brood release or time to hatch: Please refer to attached Appendix

- Egg development time: Please refer to attached Appendix

- Brood size: Please refer to attached Appendix

- Time to sexual maturity: Not determined

- Type and magnitude of biochemical changes: Not determined

- Other biological observations:
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 all the test groups were in the same general condition as the young produced by the controls over the duration of the test.

- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Coefficient of variation for control group: <=25% required, 18% achieved

Any other information on results incl. tables

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 1.0, 3.2, 10, 32 and 100 mg/l loading rates were observed to be clear, colourless water columns with oily test item floating at the surface. After 23 hours stirring and a 1-Hour standing period the 1.0, 3.2, 10, 32 and 100 mg/l loading rates were observed to remain clear, colourless water columns with oily test item floating at the surface. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the 1.0, 3.2, 10, 32 and 100 mg/l loading rates were observed to be clear, colourless solutions.

Lowest Observed Effect Loading Rate

The "Lowest Observed Effect Loading rate" (LOEL) was considered to be greater than 100 mg/l loading rate WAF on the basis that at this loading rate no significant mortalities (immobilisation) were 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 test group in terms of numbers of live young produced per adult by Day 21. 5.2No Observed Effect Loading Rate The "No Observed Effect Loading rate" (NOEL) was 100 mg/l loading rate WAF as there were no significant mortalities (immobilisation) 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.

Verification of Test Concentrations

Analysis of the freshly prepared test concentrations on Days 0, 5, 9 and 19 showed measured concentrations to range from less than the limit of quantitation (assessed as 0.00044 mg/l) to 0.0258 mg/l. Analysis of the old or expired media on Days 2, 7, 12 and 21 showed measured concentrations to range from less than the limit of quantitation to 0.0254 mg/l. The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item resulted in no significant mortalities at all the loading rates employed during the test.
Executive summary:

Introduction.

A study was performed to assess the effect of the test item 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) "Daphnia magna, Reproduction Test", referenced as Method C.20 of Commission Regulation (EC) No. 440/2008.

Methods.

Based on the results of an acute toxicity to Daphnia magna test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a Water Accommodated Fraction (WAF) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/l for a period of 21 days. WAFs were renewed 3 times per week. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with an algal suspension.

Results.

The 21-Day EL*50 (immobilisation) 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 EL*50 (reproduction) value based on nominal loading rates was estimated to be greater than 100 mg/l loading rate WAF.

The "Lowest Observed Effect Loading rate" was considered to be greater than 100 mg/l loading rate WAF on the basis that at this loading rate no significant mortalities (immobilisation) were 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 test group in terms of numbers of live young produced per adult by Day 21.

The "No Observed Effect Loading rate" was considered to be 100 mg/l loading rate WAF on the basis that at this loading rate there were 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 test group in terms of numbers of live young produced per adult by Day 21.

Analysis of the freshly prepared test concentrations on Days 0, 5, 9 and 19 showed measured concentrations to range from less than the limit of quantitation (assessed as 0.00044 mg/l) to 0.0258 mg/l. Analysis of the old or expired media on Days 2, 7, 12 and 21 showed measured concentrations to range from less than the limit of quantitation to 0.0254 mg/l.

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