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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 7 September 2008 and 22 October 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of therelevant results.
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
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test material in cases where the test material is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (50 mg/l) of test material in dechlorinated tap water for a period of 4 hours prior to removing any undissolved test material present by filtration (0.2 µm Gelman AcroCap, first approximate 100 ml discarded in order to pre-condition the filter) to give a saturated solution of the test material.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Nominal definitive test concentrations: 0.0070, 0.022, 0.22 and 0.70 mg/l.

- 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, 2, 5, 7, 9, 12, 14, 16 and 19 and of the expired test preparations on Days 2, 5, 7, 9, 12, 14, 16, 19 and 21. Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.



Vehicle:
not specified
Details on test solutions:
Methods. Information supplied by the Sponsor indicated that the test material had a low solubility in water. Pre-study solubility work indicated that the test material was also insoluble in recognised auxiliary solvents. Based on this information the test material fell into the category of a 'difficult substance' as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.

An amount of test material (550 mg) was dispersed in 11 litres of dechlorinated tap water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C. After the stirring period samples were taken for chemical analysis after the following pre-treatments:

• Untreated
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 Jlm Gelman AcroCap filter (initial approximate 100 ml
discarded)
• Filtration through a 0.2 Jlm Gelman AcroCap filter (initial approximate 500 ml
discarded)

The above was prepared in duplicate with samples being taken after stirring for 24 hours and 48 hours.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: 1st instar Daphnia magna
- Source: in-house laboratory cultures.
- Age of parental stock (mean and range, SD):
- Food type: algae
- Frequency: daily

Each daphnid received approximately 5 to 10 IJI 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.

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 at a light intensity preferably not exceeding 600 lux. Each culture was fed daily with a suspension of algae (Chlorella sp.). 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.

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
No applicable.
Hardness:
Water hardness was determined to be between 108 and 160 mg/L
Test temperature:
Temperature was· maintained at approximately 20°C throughout the test
pH:
there were no treatment related differences for pH.
Dissolved oxygen:
there were no treatment related differences for oxygen concentration.
Salinity:
Not tested
Nominal and measured concentrations:
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.070 and 0.70 mg/l.
Based on the results of a preliminary range-finding test the following nominal test concentrations were assigned to the definitive test: 0.0070, 0.022, 0.070, 0.22 and 0.70 mg/l.
Details on test conditions:
TEST SYSTEM:
For each concentration a single daphnid was placed in 100 ml of the test preparation in
150 ml glass flasks which were then covered with a plastic lid to reduce evaporation. For
each test and control group ten replicate test vessels were prepared. The flasks were
maintained at approximately 20°C with a photoperiod of 16 hours light (504 to 524 lux)
and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days.
Each vessel was randomly assigned to a position in the laboratory. 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
material.

- Renewal rate of test solution:
The test preparations were renewed 3 times per week on Days 0, 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 using a stereo
microscope before being discarded.


TEST MEDIUM / WATER PARAMETERS:
The test water used for the range-finding and definitive tests was the same as that used to maintain the stock animals. Laboratory tap water was dechlorinated by passage through an activated carbon filter and partly softened giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchanges to achieve the required temperature.


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 daphnis 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: The control group was mainted under identical conditions but not exposed to the test material.

RANGE-FINDING STUDY:
The test concentrations to be used in the definitive test were determined by a preliminary
range-finding test. The results of the media preparation trial indicated that a test
concentration of 0.70 mg/I could be attained using a saturated solution method of
preparation. Therefore, in the range-finding test Daphnia magna were exposed to a
series of nominal test concentrations of 0.070 and 0.70 mg/1. The test material was
prepared as a saturated solution.

In the range-finding test 10 daphnids were placed in each test and control vessel and
maintained in a temperature controlled room at 21°C to 22°C with a photoperiod of
16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and
dusk transition periods. Some of the temperatures during the range-finding test were
observed to be slightly in excess of the range given in the protocol of 20 ± 1°C. This
deviation was considered not to have affected the outcome or the validity of the test as
no adverse effects of exposure were observed in the control daphnids throughout the
duration of the test and that the temperature range was within guideline specification.

The control group was maintained under identical conditions but not exposed to the test
material.

Based on the results of a preliminary range-finding test the following nominal test
concentrations were assigned to the definitive test: 0.0070, 0.022, 0.070, 0.22 and
0.70 mg/l.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
1.6 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks:
Parental Daphnia (P1)
Remarks on result:
other: 95% confidence limits of 0.21 - 20 mg/l
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95 % confidence limits 0.51 - 9.3 mg/l
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
2.5 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
2.5 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.9 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.9 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
Range finding test:
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in Table 1 (see attached background material).

No immobilisation was observed at the nominal test concentrations of 0.070 and 0.70 mg/l.

Based on the results of a preliminary range-finding test the following nominal test concentrations were assigned to the definitive test: 0.0070, 0.022, 0.070, 0.22 and 0.70 mg/l.

Chemical analysis of the freshly prepared test preparations (see Appendix 3 - attached background material) showed measured concentrations in the saturated solution, equivalent to the highest test concentration, to be variable and in excess of the nominal concentration of 0.70 mg/I*. This was considered to be possibly due to slight variations in stirring speed and/or slight differences in water quality and the expected variation in the concentration of a saturated solution prepared on different occasions.

Based on these. results it was considered appropriate to express the exposure concentrations in terms of the time-weighted mean measured concentrations which were determined to be 0.025, 0.071, 0.24, 0.90 and 2.5 mg/l.

Definitive Test:
The observations for each test and control group and summarised in Tables 2 to 8 (see attached background material). The total cumulative production of live young is given in Table 9 (attached background material) and the number of live young produced per adult is shown in Table 10 (attached background material). The total number of offspring per parent daphnia (for each replicate) alive at the end of the test is shown in Figure 1 (see attached background material).


Reported statistics and error estimates:
Results from the control and each test group were compared using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955).

No significant differences (P>0.05) were found between the control and 0.025, 0.071, 0.24 and 0.90 mgtl test group in terms of the number of live young produced per adult by Day 21 using the above methods of statistical analysis.

However, significant differences (P <0.05) were found between the control and the 2.5 mg/l test group in terms of fewer numbers of live young produced per adult by Day 21.

corrected chi-squared statistic (Breslow and Day 1980) was performed to show whether the mortalities in the 0.071 and 0.90 mg/l test groups observed during the test, were statistically significant when compared to the control group.

The chi-squared table value for one degree of freedom is 3.84 at the 0.05 probability level.

Therefore, there was no significant difference (P>0.05) between the observed mortalities in the control and the 0.071 mg/l test group.

Therefore, there was no significant difference (P>0.05) between the observed mortalities in the control and the 0.90 mg/l test group.

Verification of Test Concentrations

Analysis (see Apendix 3 - attached background material) of the freshly prepared saturated solution, equivalent to the highest test concentration, showed measured concentrations to range from 1.75 to 4.22 mg/l. These concentrations were in excess of the 0.70 mg/l obtained from a saturated solution prepared in an identical manner during the pre-study media preparation trial. This was considered to be possibly due to slight variations in stirring speed and/or slight differences in water quality and the expected variation in the concentration of a saturated solution prepared on different occasions.

Analysis of the freshly prepared media showed measured concentrations of 0.0146 to 4.22 mg/l and analysis of the old media showed measured concentrations of 0.0153 to 4.40 mg/l.

Given the variability in the measured concentration of the saturated solution it was considered justifiable to base the results on the time-weighted mean measured test concentrations to give a "worst case" analysis of the data. The time-weighted mean

measured concentrations were calculated to be 0.025, 0.071, 0.24, 0.90 and 2.5 mg/l.

Observations on Test Material Solubility:

The freshly prepared test media were observed to be clear, colourless solutions whereas the old test media were observed to be green tinged due to the presence of algal cells used as feed for the daphnids.

Physico-chemical Measurements:

Temperature was· maintained at approximately 20°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Throughout the test the light intensity was observed to be in the range 504 to 524 lux. The water hardness was observed to be in the range 108 to 160 mg/l as CaC03 in the control and the highest surviving test group throughout the test.

Validation criteria: The validation criteria was me throughout the test.

Lethal Effects on the Parental Generation (P1)

Mortality (immobilisation) occurred predominantly at the highest test concentration of 2.5 mg/l resulting in 70% mortality by Day 21.

Mortality was also observed at the test concentrations of 0.071 mg/l on Day 4 and 0.90 mg/l on days 6, 15 and 19. However, statistical analysis of the mortality data using the corrected chi-squared statistic (Breslow and Day 1980) showed that the observed mortalities in the 0.071 and 0.90 mg/l test groups were not significantly different (P0.05) when compared to the control group. No mortalities occurred at the 0.025 and 0.24 mgtl test concentrations throughout the test.

Sub-lethal Effects on the Parental Generation (P1)

There was a significant effect on size and colour of the daphnids in that 100% of the surviving daphnids on Day 21 at the test concentration of 2.5 mg/l were markedly smaller and paler in colour than the control animals. The daphnids at the remaining test concentrations were observed to be the same size and colour as the control animals.

After 21 days there were no statistically significant differences between the control and the 0.025, 0.071, 0.24 and 0.90 mg/l test groups in terms of the number of live young produced per adult. The 2.5 mg/l test group showed a statistically significant difference from the control and the remaining test groups after 21 days in terms of producing fewer numbers of live young per adult.

The EC50 (reproduction) value calculated by the maximum-likelihood probit method (Finney 1971) on Day 21, based on time-weighted mean measured test concentrations was 1.2 mg/l with 95% confidence limits of 0.51 - 9.3 mg/l.

After 21 days the length of each suriving adult was determined. The results showed that there were no statistically significant differences between the control and the 0.025, 0.071, 0.24 and 0.90 mg/l test groups in terms of length of the daphnids after 21 days exposure to the test material. The 2.5 mg/l test group data showed the surviving daphnids to be significantly smaller compared to the control.

Effects on the Filial Generation (F1):

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 the 0.025, 0.071, 0.24 and 0.90 mg/l test groups were in the same general condition as the young produced by the controls over the duration of the test. The young produced by the 2.5 mg/l test group, however, were observed to be small and pale when compared to the control young throughout the test. Young were first produced in the control test group on Day 7 of the test.

The numbers of unhatched eggs and dead young were zero in all control and treatment groups surviving to maturation.

Lowest Observed Effect Concentration

The "Lowest Observed Effect Concentration" (LOEC) was 2.5 mg/l as this test group produced significantly fewer live young per adult (P<0.05) than the control group.

No Observed Effect Concentration:

The "No Observed Effect Concentration" (NOEC) was 0.90 mg/l 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.

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test material resulted in significant mortalities at the time weighted mean measured test concentration of 2.5 mg/l resulting in 70% mortalities by Day 21.

The 21-Day EC50 (immobilisation) value, based on time-weighted mean measured test concentrations, for the parental Daphnia generation (P1) was calculated to be 1.6 mg/l with 95% confidence limits of 0.21 - 20 mg/l.

The 21-Day EC50 (reproduction) value, based on time-weighted mean measured test concentrations, was 1.2 mg/l with 95% confidence limits of 0.51 - 9.3 mg/l.
Executive summary:

Introduction: 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) "Daphnia magna, Reproduction Test", referenced as Method C.20 of Commission Directive 2001/59/EC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods: Information supplied by the Sponsor indicated that the test material had a low solubility in water. Pre-study solubility work indicated that the test material was also insoluble in recognised auxiliary solvents. Based on this information the test material fell into the category of a 'difficult substance' as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.

The pre-study media preparation trial indicated that the use of a saturated solution method of preparation followed by filtration to remove the undissolved test material was the most appropriate method of preparation for the test material.

Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to an aqueous solution of the test material over a range of time-weighted mean measured test concentrations of 0.025, 0.071, 0.24, 0.90 and 2.6 mg/l for a period of 21 days. The test material solution was prepared by stirring an excess (50 mg/l) of test material in dechlorinated tap water using a propeller stirrer at approximately 1500 rpm at a temperature of approximately 21°C for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm Gelman AcroCap filter, first approximate 100 ml discarded in order to pre-condition the filter) to produce a saturated solution of the test material. The test solutions 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: Analysis of the freshly prepared saturated solution, equivalent to the highest test concentration, showed measured concentrations to range from 1.75 to 4.22 mg/l.

These concentrations were in excess of the 0.70 mg/l obtained from a saturated solution prepared in an identical manner during the pre-study media preparation trial. This was considered to be possibly due to slight variations in stirring speed andtor slight differences in water quality and the expected variation in the concentration of a saturated solution prepared on different occasions.

Analysis of the freshly prepared media showed measured concentrations of 0.0146 to 4.22 mg/l and analysis of the old media showed measured concentrations of 0.0153 to 4.40 mg/l.

Given the variability in the measured concentration of the saturated solution it was considered justifiable to base the results on the time-weighted mean measured test concentrations to give a "worst case" analysis of the data. The time-weighted mean measured concentrations were calculated to be 0.025, 0.071, 0.24, 0.90 and 2.5 mg/l.

The 21-Day EC50 (immobilisation) value, based on time-weighted mean test concentrations, for the parental Daphnia generation (P1) was calculated to be 1.6 mg/l with 95% confidence limits of 0.21 - 20 mg/l.

The 21-Day EC50 (reproduction) value, based on time-weighted mean measured test concentrations, was calculated to be 1.2 mg/l with 95% confidence limits of 0.51 - 9.3 mg/l.

The "Lowest Observed Effect Concentration" was considered to be 2.5 mg/l on the basis that at this test concentration significantly fewer live young per adult (P <0.05) were produced when compared to the control.

The "No Observed Effect Concentration" was considered to be 0.90 mg/l on the basis that at this test concentration 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 0.90 mg/l test group in terms of numbers of live young produced per adult by Day 21.

Description of key information

Mortality (immobilisation) occured predominantly at the highest test concentration of 2.5 mg/l, resulting in 70% mortality by Day 21.
The "Lowest Observed Effect Concentration" was considered to be 2.5 mg/l.
The "No Observed Effect Concentration" was considered to be 0.90 mg/l.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.9 mg/L

Additional information

Introduction:

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) "Daphnia magna, Reproduction Test", referenced as Method C.20 of Commission Directive 2001/59/EC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods:

Information supplied indicated that the test material had a low solubility in water. Pre-study solubility work indicated that the test material was also insoluble in recognised auxiliary solvents. Based on this information the test material fell into the category of a 'difficult substance' as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.

The pre-study media preparation trial indicated that the use of a saturated solution method of preparation followed by filtration to remove the undissolved test material was the most appropriate method of preparation for the test material.

Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to an aqueous solution of the test material over a range of time-weighted mean measured test concentrations of 0.025, 0.071, 0.24, 0.90 and 2.6 mg/l for a period of 21 days. The test material solution was prepared by stirring an excess (50 mg/l) of test material in dechlorinated tap water using a propeller stirrer at approximately 1500 rpm at a temperature of approximately 21°C for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm Gelman AcroCap filter, first approximate 100 ml discarded in order to pre-condition the filter) to produce a saturated solution of the test material. The test solutions 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:

Analysis of the freshly prepared saturated solution, equivalent to the highest test concentration, showed measured concentrations to range from 1.75 to 4.22 mg/l.

These concentrations were in excess of the 0.70 mg/l obtained from a saturated solution prepared in an identical manner during the pre-study media preparation trial. This was considered to be possibly due to slight variations in stirring speed andtor slight differences in water quality and the expected variation in the concentration of a saturated solution prepared on different occasions.

Analysis of the freshly prepared media showed measured concentrations of 0.0146 to 4.22 mg/l and analysis of the old media showed measured concentrations of 0.0153 to 4.40 mg/l.

Given the variability in the measured concentration of the saturated solution it was considered justifiable to base the results on the time-weighted mean measured test concentrations to give a "worst case" analysis of the data. The time-weighted mean measured concentrations were calculated to be 0.025, 0.071, 0.24, 0.90 and 2.5 mg/l.

The 21-Day EC50(immobilisation) value, based on time-weighted mean test concentrations, for the parental Daphnia generation (P1) was calculated to be 1.6 mg/l with 95% confidence limits of 0.21 - 20 mg/l.

The 21-Day EC50 (reproduction) value, based on time-weighted mean measured test concentrations, was calculated to be 1.2 mg/l with 95% confidence limits of 0.51 - 9.3 mg/l.

The "Lowest Observed Effect Concentration" was considered to be 2.5 mg/l on the basis that at this test concentration significantly fewer live young per adult (P <0.05) were produced when compared to the control.

The "No Observed Effect Concentration" was considered to be 0.90 mg/l on the basis that at this test concentration 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 0.90 mgtl test group in terms of numbers of live young produced per adult by Day 21.