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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:
23-09-2002 to 27-10-2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
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)
Remarks:
inspected: February 2000; signature: April 2000
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 0.0080, 0.025, 0.080, 0.25, 0.80 mg/L (nominal).
Time Weighted Average equivalent concentrations: 0 (control), of 0.00352, 0.00973, 0.0285, 0.106 and 0.387 mg/L (measured)
- 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. Where possible all samples were prepared for analysis on the day of sampling. Duplicate samples were taken and stored frozen (approximately -20°C) for further analysis if necessary.
- Sample storage conditions before analysis: See above.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct dissolution of test item in culture medium to prepare stock; serial dilution of stock to prepare test media During validation: Recoveries were in the region of 114 to 87 % at 0.008 and 0.8 mg/L concentrations. The results indicate the preparation procedure was consistent in preparing a saturated solution. The results also indicate that the test levels could be prepared at near the expected concentrations when compared to the original saturated solution concentration. During verification of the test item concentrations between 0.008 mg/L and 0.8 mg/L Accuracy was 52 – 134% (mean 106% ; n=7 and RSD 30%) and 74 – 103% (mean 87%; n=5 ; RSD 15%) respectively. This was within the acceptance targets of mean 80 – 120% and RSD < 20% respectively. The high RSD value of precision at 0.008 mg/L was considered to be a combination of analysis at low levels and some variation in the chromatographic conditions, however the GC conditions were amended and precision of analysis improved sufficiently to use the method for the whole range of test samples. Within the definitive test the test item was dissolved directly in reconstituted water. An amount of test item (1100 mg) was dispersed in 11 litres of reconstituted water and stirred with a propeller stirrer (approximately 2000 rpm) for a period of 48 hours at 25°C. After 48 hours stirring the mixture was cooled to approximately 21 °C prior to filtration through a 0.2 µm filter to give a saturated solution of approximately 2.2 mg/L (based on the results of a preceding acute toxicity study. Aliquots of the saturated solution (7.3, 22.7, 73,227 and 730 mL) were each separately dispersed in a final volume of 2 litres of reconstituted water to give nominal test concentrations of 0.0080, 0.025, 0.080, 0.25 and 0.80 mg/L. The concentration and stability of the test material in the test solutions were verified by chemical analysis on Days 0 (fresh media), 2, 5, 7, 9, 12, 14, 16, 19 (old and fresh media) and 21 (old media), with renewal 3 times a week. It was considered that the method was suitable for use in the definitive test. Analysis of the saturated solution used to prepare the test concentrations throughout the test showed the measured test concentrations to range from 1.55 mg/L to 2.27 mg/L. Analysis of the freshly prepared test solutions throughout the test showed the measured test concentrations to range from 0.00529 mg/L to 0.809 mg/L throughout the test.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No precipitate reported.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain: not reported
- Source: in-house laboratory cultures
- Age at study initiation (mean and range, SD): < 24 hours
- Method of breeding: Parthenogenesis
- Feeding during test: Yes. The daphnids were fed daily during the study ad libitum with algae suspension
- Food type: algae suspension [unicellular algal culture ( Chlorella sp.)]
- Amount: 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
- Acclimation period: None reported. Can be presumed to be < 2 hours in dilution water.
- Acclimation conditions (same as test or not): Yes.
- Type and amount of food: Not applicable.
- Feeding frequency: Not applicable.
- Health during acclimation (any mortality observed): No.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Remarks on exposure duration:
In accordance with the OECD TG 211 guideline.
Hardness:
Water hardness was observed to be in the range 205 to 260 mg/L as CaCO3 in the control and the highest surviving test group throughout the test
Test temperature:
Actual: 21ºC (throughout test period)
pH:
Control: 0 hours to 21 days: pH 7.9 – 8.0 ; test groups: 0 hours to 21 days: pH 7.8 – 8.1
It was considered that there was no treatment related differences to pH
Dissolved oxygen:
Control: 0 hours to 21 days: 8.3 - 8.4 mg O2/L (90 - 96% ASV) ; test groups: 0 hours to 21 days: 7.9 - 8.7 mg O2/L (90-97% ASV).
It was considered that there was no treatment related differences to oxygen concentration
Salinity:
Not applicable.
Conductivity:
Not applicable.
Nominal and measured concentrations:
Concentrations: 0 (control), 0.0080, 0.025, 0.080, 0.25, 0.80 mg/L (nominal).
Time Weighted Average equivalent concentrations: 0 (control), of 0.00352, 0.00973, 0.0285, 0.106 and 0.387 mg/L (measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL glass flasks
- Type (delete if not applicable): closed (covered to reduce evaporation)
- Material, size, headspace, fill volume: 100 mL fill volume, 50 mL headspace
- Aeration: No. Diluent water was aerated before use.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): 3 days renewal.
- No. of organisms per vessel: 1 per 100 mL vessel
- No. of vessels per concentration (replicates): 10 replicates.
- No. of vessels per control (replicates): 10 replicates.
- No. of vessels per vehicle control (replicates): Not applicable.
- Biomass loading rate: equivalent to 100 mL test media per animal.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water - analytical grade salts dissolved in purified water equivalent or similar to ISO Test water, according to OECD 202, Annex 3: composition (mg/L): KCl 5.8; NaHCO3: 64.8; CaCl2.2H2O: 294 ; MgSO4.7H2O: 123. pH 7.8 (adjusted if necessary).
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: <5 µS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: Daily, before and after renewal every other alternate day (3 day media renewal).

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 16 h light / 8 hours dark, 20 minute dawn and dusk transition periods.
- Light intensity: 524 – 685 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Reproduction and Immobility (or adverse effects including mortality)
- On a daily basis the numbers of live and dead of the "Parental" (P1) generation, the numbers of live and dead "Filial" (F 1) Daphnids and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental Daphnids as compared with the controls.
- The number of Daphnids with eggs or young in the brood pouch were determined daily with the exception of days 3, 4, 17 and 18. 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 3.2 ; Based on preceding acute toxicity study (information in the full study report).
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study
- Test concentrations: Not applicable.
- Results used to determine the conditions for the definitive study: Not applicable.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.029 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: (95% CL. - mg/L)
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.11 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: (95% CL. - mg/L)
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.029 - < 0.11 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: (95% CL. - mg/L)
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.044 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks on result:
other: (95% CL. 0.033 - 0.060 mg/L)
Details on results:
- Behavioural abnormalities: None reported.
- Observations on body length and weight: In P1 generation: At 0.80 mg/L: there was a significant effect on size and colour of the daphnids in that the surviving daphnids were markedly smaller and paler in colour than the control animals prior to observing 100% mortalities by Day 7. In addition, it was observed that on days 2, 3 and 4 the daphnids were also moving slower than the controls. At 0.25 mg/L : there was also a significant effect on the size and colour of the daphnids prior to observing 100% mortalities by Day 19. The daphnids at the remaining test concentrations were observed to be the same size and colour as the control animals.
In F1 generation: there was no noted differences to controls.
- Other biological observations: None.
- Mortality of control: No mortalities in control.
- Other adverse effects control: None reported.
- Abnormal responses: None reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: It was speculated the decline in measured test concentrations was due to possible: losses to volatility, adsorption and accumulation of the test item in the test organisms or to any algae food/organic waste matter present in the test system.
- Effect concentrations exceeding solubility of substance in test medium: No.
Reported statistics and error estimates:
EC50 (immobilisation) values and associated confidence limits at 6, 7, 14 and 21 days were calculated by the trimmed Spearman-Karber method (Hamilton et al.,1977) using the ToxCalc software package (ToxCalc, 1999).
EC50 (reproduction) was estimated by inspection of the data.
NOEC was estimated 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)

Reproduction – live young per adult:
- Results from the control, 0.0080, 0.025 and 0.080 mg/L test groups 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).
- 0.25 and 0.80 mg/L test groups data were not included in the statistical analysis as the effects of exposure eliminated all the daphnids prior to termination of the test.
- No significant differences (P≥0.05) were found between the control, 0.0080, 0.025 and 0.080 mg/L test groups in terms of the number of live young produced per adult by Day 21

Length data:
- Results from the control, 0.0080, 0.025 and 0.080 mg/L test groups Daphnia length 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).
- 0.25 and 0.80 mg/L test groups data were not included in the statistical analysis as the effects of exposure eliminated all the daphnids prior to termination of the test.
- No significant differences (P≥0.05) were found between the control, 0.0080, 0.025 and 0.080 mg/L test groups in terms of the number of live young produced per adult by Day 21

All validity criteria were considered to be met:

1. Control mortality was ≤20% (actual 0%)

2. Dissolved oxygen was maintained ≥ 3 mg O2/L (actual ≥ 7.9 mg O2/L)

3. pH control group ≤ 1.5 deviation (actual 0.1)

5. Mean live young per surviving adult ≥ 60 after 21 days (actual 90)

6. CoV for control group ≤ 25% (actual 14%)

Validity criteria fulfilled:
yes
Conclusions:
The test item 21d-NOEC was 0.029 mg/L. The 21-d EC50 (reproduction) was between 0.029 and 0.11 mg/L. All effects were based on time weighted average concentrations.
Executive summary:

The 21-d reproduction toxicity to Daphnia magna was carried out according to OECD TG 211 and EU Method C.20 guidelines under GLP. Based on the results of an acute toxicity test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to an aqueous solution of the test item over a range of nominal test concentrations of 0.0080, 0.025, 0.080, 0.25 and 0.80 mg/L for a period of 21 days. Test concentrations were prepared by dilution of a saturated solution of the test item. The saturated solution was prepared by stirring an excess of test item in test medium for a period of 48 hours prior to removal of the excess test item by filtration (0.2 µm). The test solutions were renewed 3 times per week. Daphnids were fed daily with algal suspension. The numbers of live and dead adult (P1) daphnids and (F1) young daphnids was determined daily. It was considered validity criteria of the test guideline were fulfilled. Analysis of the saturated solution used to prepare the test concentrations throughout the test showed the measured test concentrations to range from 1.55 mg/L to 2.27 mg/L. These results were consistent with the results from the acute toxicity test (2.2 mg/L) and the pre-test preparation trials (1.75 mg/L). Analysis of the freshly prepared test solutions throughout the test showed the measured test concentrations to range from 0.00529 mg/L to 0.809 mg/L throughout the test. Analysis of the old or expired test solutions showed a marked decline in measured test concentrations. Measured test concentrations were shown to range from 0.000421 mg/L to 0.264 mg/L of nominal. On this basis it was considered appropriate to base effect level estimates on time weighted mean measured concentrations.The test item 14-d EC50 (immobilisation) was 0.11 (C.I. 0.067 – 0.17) mg/L and 21-d EC50 (immobilisation) was 0.044 (C.I. 0.33 – 0.060) mg/L based on time weighted average concentrations. The test item 21-d EC50 (reproduction) was between 0.029 and 0.11 mg/L based on TWA concentrations. There was no significant difference (P ≥ 0.05) in terms of the number of young produced per adult when compared to the control and that the adult daphnids at 0.11 mg/L were eliminated from the test by Day 19 due to a prolonged toxic effect of the test item. The test item 21d-NOEC was 0.029 mg/L based on TWA concentrations.

Description of key information

21d-NOEC (invertebrates, reproduction) = 0.029 mg/L based time weighted average concentrations, 21-days freshwater, OECD TG 211, 2003

Key value for chemical safety assessment

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

Additional information

Key study : OECD TG 211, 2003: The 21-d reproduction toxicity to Daphnia magna was carried out according to OECD TG 211 and EU Method C.20 guidelines under GLP. Based on the results of an acute toxicity test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to an aqueous solution of the test item over a range of nominal test concentrations of 0.0080, 0.025, 0.080, 0.25 and 0.80 mg/L for a period of 21 days. Test concentrations were prepared by dilution of a saturated solution of the test item. The saturated solution was prepared by stirring an excess of test item in test medium for a period of 48 hours prior to removal of the excess test item by filtration (0.2 µm). The test solutions were renewed 3 times per week. Daphnids were fed daily with algal suspension. The numbers of live and dead adult (P1) daphnids and (F1) young daphnids was determined daily. It was considered validity criteria of the test guideline were fulfilled. Analysis of the saturated solution used to prepare the test concentrations throughout the test showed the measured test concentrations to range from 1.55 mg/L to 2.27 mg/L. These results were consistent with the results from the acute toxicity test (2.2 mg/L) and the pre-test preparation trials (1.75 mg/L). Analysis of the freshly prepared test solutions throughout the test showed the measured test concentrations to range from 0.00529 mg/L to 0.809 mg/L throughout the test. Analysis of the old or expired test solutions showed a marked decline in measured test concentrations. Measured test concentrations were shown to range from 0.000421 mg/L to 0.264 mg/L of nominal. On this basis it was considered appropriate to base effect level estimates on time weighted mean measured concentrations.The test item 14-d EC50 (immobilisation) was 0.11 (C.I. 0.067 – 0.17) mg/L and 21-d EC50 (immobilisation) was 0.044 (C.I. 0.33 – 0.060) mg/L based on time weighted average concentrations. The test item 21-d EC50 (reproduction) was between 0.029 and 0.11 mg/L based on TWA concentrations. There was no significant difference (P ≥ 0.05) in terms of the number of young produced per adult when compared to the control and that the adult daphnids at 0.11 mg/L were eliminated from the test by Day 19 due to a prolonged toxic effect of the test item. The test item 21d-NOEC was 0.029 mg/L based on TWA concentrations.