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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:
2002-08-27 to 2002-09-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Analytical monitoring:
no
Details on sampling:
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.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION - Method:Due to the low aqueous solubility and complex nature of the test material for the purposes of the definitive test the test material was prepared as a Water Accommodated Fraction (WAF).Amounts of test material (10, 32, 100, 80 and 25 mg) were each separately added to the surface of 10, 10, 10, 2.5 and 2.5 litres of reconstituted water respectively to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates. After the addition of the test material, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. This was stirred for approximately 24 hours. The stirring was stopped after approximately 24 hours and the mixture allowed to stand for approximately 1 hour. A wide bore glass tube, covered at one end with Parafilm 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 Parafilm 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 WAF showed no microdispersions or undissolved test material, therefore a glass wool plug was not used to filter the WAF.:
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM- Source: 1st instar Daphnia magna derived from in-house laboratory cultures.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. 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 thetest, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
none
Hardness:
250 mg CaCO2/L
Test temperature:
20 - 21°C
pH:
7.7 - 8.1
Dissolved oxygen:
7.5 - 8.7
Salinity:
Not applicable
Nominal and measured concentrations:
nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L
Details on test conditions:
TEST SYSTEMFor 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 20 - 21°C with a photoperiod of 16 hours light (581 - 668 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. Each test group was randomly assigned to a position in the laboratory. The test vessels were not aerated. The diluent water only was aerated prior to use.Each daphnid received approximately 3.1 - 11 µl of a unicellular algal culture (Chlorella sp.) 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.The control group was maintained under identical conditions but not exposed to the test material.OTHER TEST CONDITIONSThe 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- Source/preparation of dilution water: deionised water with a conductivity of <5 µS cm-1. 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 valueEFFECT 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 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 ofoff-spring produced in the flasks.At the end of the test, the length of each surviving parent animal was determinedVEHICLE CONTROL PERFORMED: noRANGE-FINDING STUDY: No, test concentrations determined from acute toxicity test.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
11 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks on result:
other: 95% CI = 7.9 - 16 mg/L loading rate WAF
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% CI = 10 -25 mg/L loading rate WAF
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks on result:
other: loading rate WAF
Duration:
21 d
Dose descriptor:
LOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: loading rate WAF
Details on results:
See attached document for results tablesPhysico-chemical measurementsTemperature was maintained at 20 - 21°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 581 to 668 luxThe water hardness was observed to be in the range 240 to 257 mg/L as CaCO3 in the control and the highest surviving test group throughout the test.Observations on Test Material Solubility At the start of the stirring periods the loading rates were observed to have clear colourless water columns with oily globules of test material at the water surface and dispersed throughout the water column.After the stirring and standing periods the 1.0, 3.2 and 10 mg/L loading rates were observed to have clear colourless water columns with oily globules on the surface. The 32 and 100 mg/L loading rates were observed to have clear colourless water columns with very slight white emulsified globules on the surface.Microscopic inspection of the WAFs showed no micro-dispersions to be present. Therefore a glass wool plug was not used to siphon the WAF.Lethal Effects on the Parental Generation (P1) Mortality (immobilisation) occurred predominantly at the highest test concentration of 100 mg/L loading rate WAF resulting in 100% mortality by Day 9. Significant mortality (immobilisation) also occurred throughout the test in the 32 mg/L loading rate test group resulting in 60% and 100% mortality by Days 13 and 17 respectively indicating a prolonged toxic effect attributable to exposure of Daphnia magna to the test material.Mortality was also observed at the test concentration of 10 mg/L loading rate WAF on Days 12 (10%), 17 (20%) and 18 (40%).No mortalities occurred at the 1.0 and 3.2 mg/L loading rate WAFs throughout the test.The following EL50* (immobilisation) values based on nominal test concentrations were estimated or calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) at 8 and 21 days and the probit method (Finney 1971) at 14 days:EL50 (24h) > 100 mg/L loading rate WAFEL50 (48h) > 100 mg/L loading rate WAFEL50 (96h) > 100 mg/L loading rate WAFEL50 (8d) = 60 mg/L loading rate WAF (95% CI = 53 - 69 mg/L loading rate WAF)EL50 (14d) = 25 mg/L loading rate WAF (95% CI = 15 - 40 mg/L loading rate WAF)EL50 (21d) = 11 mg/L loading rate WAF (95% CI = 7.9 - 16 mg/L loading rate WAF)Although it was not possible to calculate the 24 and 48-Hour EL50* values (since no mortalities were observed) the 48-Hour EL50* value is consistent with the results of the acute toxicity test to 1st instar Daphnia magna which was found to be greater than 100 mg/L loading rate WAF.Sub-lethal Effects on the Parental Generation (P1) There was a significant effect on the colour of the surviving daphnid on Day 8 at the test concentration of 100 mg/L loading rate WAF as it was markedly paler in colour than the control animals. In addition the surviving daphnids in the 32 mg/L loading rate WAF were paler than the controls from Day 9 until Day 17 when 100% mortality was observed. Also one daphnid at the concentration of 10 mg/L loading rate WAF was observed to be paler than the controls on Day 17 prior to being observed as dead on Day 18.The daphnids at the remaining test concentrations were observed to be the same colour as the control animals.After 21 days there were no statistically significant differences between the control, 1.0 and 3.2 mg/L loading rate WAF test groups in terms of the number of live young produced per adult.The 10, 32 and 100 mg/L loading rate WAF test groups data were not included in the statistical analysis due to significant mortalities in the parental (P1) generation over the duration of the test.The EL50* (reproduction) value calculated by maximum-likelihood probit method (Finney 1971) on Day 21, based on nominal test concentrations was 16 mg/L loading rate WAF with 95% confidence limits of 10 - 25 mg/L loading rate WAF. The EL50 for reproduction is greater than the EL50 for immobilisation due to the observation that although some daphnids had died, daphnids remaining alive did produce some young.Analysis of the data obtained on Day 21 showed that the numbers of live young produced per adult by the control group were not significantly different (P≥0.05) from the 1.0 and 3.2 mg/L loading rate WAF test groups.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 and 3.2 mg/L loading rate WAF test groups in terms of length of the daphnids after 21 days exposure to the test material . The 10, 32 and 100 mg/L loading rate WAF test group data was not included in this analysis due to significant mortalities in the parental (P1) generation during the test.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 all the test groups were in the same general condition as the young produced by the controls over the duration of the test.Young were first produced in the control test group on Day 7 of the test.Numbers of unhatched eggs and dead young were low in all control and treatment groups surviving to maturation.Lowest Observed Effect Loading Rate The "Lowest Observed Effect Loading rate" (LOEL) was 10 mg/L loading rate WAF as this test group produced significantly fewer live young per adult than the control group and significant mortalities were observed in the adult (P1) generation.No Observed Effect Loading Rate The "No Observed Effect Loading rate" (NOEL) was 3.2 mg/L loading rate WAF as there were no significant mortalities (immobilisation) observed in the parental generation (P1), no significant differences (P≥0.05) in terms of the number of live young produced per adult and no significant differences in terms of length of surviving adult daphnids when compared to the control daphnids after 21 days.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
See text below.

See attched document for tables of results.

Statistics

Analysis of Numbers of Live Young Produced per Adult

Results from the control, 1.0 and 3.2 mg/L loading rate WAF test groups data 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).

The 10, 32 and 100 mg/L loading rate WAF test groups data were not included in the statistical analysis due to the observation of significant mortalities in the parental (P1) generation.

Mean and standard deviation values derived from the number of live young produced per surviving adult in the control, 1.0 and 3.2 mg/L loading rate WAF test groups were used for this analysis and are given in the table below:

Nominal Concentration

(mg/L loading rate WAF)

Number of Live Young Produced per Adult by Day 21

Control

Mean

73.9

Standard deviation

11.9

1.0

Mean

70.1

Standard deviation

8.4

3.2

Mean

72.2

Standard deviation

13.4

No significant differences (P≥0.05) were found between the control, 1.0 and 3.2 mg/L loading rate WAF test groups in terms of the number of live young produced per adult by Day 21 using the above methods of statistical analysis.

Analysis of theDaphniaLength Data

Results from the control, 1.0 and 3.2 mg/L loading rate WAF test groupsDaphnialength data, determined for the surviving daphnids on termination of the test 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).

The 10, 32 and 100 mg/L loading rate WAF test group data was not included in the statistical analysis due to the observation of significant mortalities in the parental (P1) generation.

Mean and standard deviation values derived from the length of the surviving daphnids of the parental (P1) generation in the control, 1.0 and 3.2 mg/L loading rate WAF test groups were used for this analysis and are given in the table below:

Nominal Concentration

(mg/L loading rate WAF)

DaphniaLengths at Day 21 (mm)

Control

Mean

3.9

Standard deviation

0.1

1.0

Mean

3.9

Standard deviation

0.2

3.2

Mean

3.9

Standard deviation

0.1

No statistically significant differences (P≥0.05) were found between the control, 1.0 and 3.2 mg/L loading rate WAF test groups in terms of length of the surviving parental daphnids on Day 21 of the test, using the above methods of statistical analyses.

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test material resulted in significant mortalities at the test concentrations of 10, 32 and 100 mg/L loading rate resulting in 40%, 100% and 100% mortalities by Day 21 respectively. The 14 and 21-Day EL50* (immobilisation) values, based on nominal test concentrations, for the parental Daphnia generation (P1) were calculated to be 25 and 11 mg/L loading rate WAF with 95% confidence limits of 15 - 40 mg/L loading rate WAF and 7.9 - 16 mg/L loading rate WAF respectively. The 21-Day EL50* (reproduction) based on nominal test concentrations was 16 mg/L with 95% confidence limits of 10 - 25 mg/L loading rate WAF. The "Lowest Observed Effect Loading rate" (LOEL) and the "No Observed Effect Loading rate" (NOEL) based on nominal test concentrations were 10 and 3.2 mg/L loading rate WAFs respectively.
Executive summary:

Test Guidance

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.

Based on the results of an acute toxicity test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to Water Accommodated Fractions (WAF) of the test material over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L for a period of 21 days. The 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 14 and 21-Day EL50 (immobilisation) values, based on nominal test concentrations, for the parental Daphnia generation (P1) were calculated to be 25 and 11 mg/L loading rate WAF with 95% confidence limits of 15 - 40 mg/L and 7.9 - 16 mg/L loading rate WAF respectively. The 21-Day EL50 (reproduction) value based on nominal test concentrations was calculated to be 16 mg/L loading rate WAF with 95% confidence limits of 10 - 25 mg/L loading rate WAF. The EL50 for reproduction is greater than the EL50 for immobilisation due to the observation that although some daphnids had died, daphnids remaining alive did produce some young. The "Lowest Observed Effect Loading rate" was considered to be 10 mg/L loading rate WAF on the basis that at this test concentration significantly fewer live young per adult were produced when compared to the control and significant mortalities were observed in the adult (P1) generation. The "No Observed Effect Loading rate" was considered to be 3.2 mg/L loading rate WAF on the basis that at this test concentration there were no significant mortalities (immobilisation) observed in the parental generation (P1), no significant differences (P≥0.05) between the control and the 3.2 mg/L loading rate WAF test group in terms of numbers of live young produced per adult and there were no significant differences in terms of length of the surviving adult daphnids when compared to the controls after 21 days.

Description of key information

Exposure of Daphnia magna to the test material resulted in significant mortalities at the test concentrations of 10, 32 and 100 mg/L loading rate resulting in 40%, 100% and 100% mortalities by Day 21 respectively.
The 14 and 21-Day EL50* (immobilisation) values, based on nominal test concentrations, for the parental Daphnia generation (P1) were calculated to be 25 and 11 mg/L loading rate WAF with 95% confidence limits of 15 - 40 mg/L loading rate WAF and 7.9 - 16 mg/L loading rate WAF respectively.
The 21-Day EL50* (reproduction) based on nominal test concentrations was 16 mg/L with 95% confidence limits of 10 - 25 mg/L loading rate WAF.
The "Lowest Observed Effect Loading rate" (LOEL) and the "No Observed Effect Loading rate" (NOEL) based on nominal test concentrations were 10 and 3.2 mg/L loading rate WAFs respectively.

Key value for chemical safety assessment

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

Additional information

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).

Based on the results of an acute toxicity test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to Water Accommodated Fractions (WAF) of the test material over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L for a period of 21 days. The 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.

The 14 and 21-Day EL50* (immobilisation) values, based on nominal test concentrations, for the parental Daphnia generation (P1) were calculated to be 25 and 11 mg/L loading rate WAF with 95% confidence limits of 15 - 40 mg/L and 7.9 - 16 mg/L loading rate WAF respectively. The 21-Day EL50* (reproduction) value based on nominal test concentrations was calculated to be 16 mg/L loading rate WAF with 95% confidence limits of 10 - 25 mg/L loading rate WAF. The EL50 for reproduction is greater than the EL50 for immobilisation due to the observation that although some daphnids had died, daphnids remaining alive did produce some young. The "Lowest Observed Effect Loading rate" was considered to be 10 mg/L loading rate WAF on the basis that at this test concentration significantly fewer live young per adult were produced when compared to the control and significant mortalities were observed in the adult (P1) generation. The "No Observed Effect Loading rate" was considered to be 3.2 mg/L loading rate WAF on the basis that at this test concentration there were no significant mortalities (immobilisation) observed in the parental generation (P1), no significant differences (P≥0.05) between the control and the 3.2 mg/L loading rate WAF test group in terms of numbers of live young produced per adult and there were no significant differences in terms of length of the surviving adult daphnids when compared to the controls after 21 days.