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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:
2007-08-28 to 2007-09-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Duplicate samples were taken from the freshly prepared test media of all test concentrations and the solvent control at the first treatment period (Day 0), at a treatment period in the second week (Day 7), and at a treatment period in the last week (Day 16). To determine the maintenance of the test item concentrations in the test media, stability control samples were taken at the end of two test medium renewal periods of 48 hours (Days 2 and 9) and at the end of one renewal period of 72 hours (Day 19).
- Sample storage conditions before analysis: The samples were extracted and stored at – 20 °C in a deep freezer until analysis was performed.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Due to the low solubility of the test item in test water, the organic solvent N,N Dimethylformamide (DMF) was used as a vehicle. The solvent was chosen based on its solubilizing properties and its relative non-toxicity to daphnids.
- Controls: a control and a solvent control
- Chemical name of vehicle: N,N Dimethylformamide (DMF)
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna Straus
- Strain/clone: clone 5
- Source: supplied by the University of Sheffield/UK in 1992 (Since this date, the clone is successfully bred in RCC’s laboratories in culture medium identical to the medium used for the test)
- Age of parental stock: less than 24 hours old
- Feeding during test: yes
- Food type: with green algae of the species Scenedesmus subspicatus and with a fish food suspension
- Frequency: three times a week

ACCLIMATION
- Acclimation period: The daphnids used for the test originated from parental daphnids that were at least 14 days old but not older than four weeks and were not first brood progeny.
- Acclimation conditions: same
- Type and amount of food: with green algae of the species Scenedesmus subspicatus and with a fish food suspension
- Feeding frequency: three times a week
- Health during acclimation (any mortality observed): No signs of stress were observed and the brood stock was healthy.

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
2.5 mmol/L (= 250 mg/L as CaCO3)
Test temperature:
20-21 °C
pH:
7.6 to 8.0
Dissolved oxygen:
at least 8.0 mg/L
Nominal and measured concentrations:
- nominal test concentration: 0.020, 0.063, 0.2, 0.63, 2.0 mg/L
- mean measured test item concentration: 0.019, 0.049, 0.13, 0.43, 1.4 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: in a 100-mL glass beaker containing 80 mL of test medium
- Type: closed
- Aeration: no
- Renewal rate of test solution: on Days 2, 5, 7, 9, 12, 14, 16, and 19 of the test period
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water; according to guideline
- Alkalinity: 0.9 mmol/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: At the beginning and end of each test medium renewal period, the pH and dissolved oxygen concentrations were measured in one replicate of each test concentration and the controls. At the same time, the water temperature was measured in one of the control replicates. Additionally, the room temperature was continuously monitored.
The appearance of the test media was visually recorded at the beginning and end of each test medium renewal period.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness, with a 30-minute transition period
- Light intensity: range of about 490 and 650 Lux

EFFECT PARAMETERS MEASURED: The test replicates were observed for mortality of adults at the start of the test, on the first and second day after the start of the study and thereafter three times per week before renewal of test media. On the same observation dates, the number of offspring was recorded. The test beakers were also checked for the presence of aborted eggs or dead offspring.
The reproduction rate was calculated as the total number of living offspring produced per parent female surviving until the end of the test.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- The test concentrations were based on the results of a range finding test (without GLP) and on the results of a pre-experiment to determine the solubility and the stability of the test item in test water.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.049 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
In the control, the solvent control and at the lower test concentrations up to and including 0.13 mg/L (mean measured) the survival of the test animals at the end of the test was at least 90% or higher. At the concentration of 0.43 mg/L, the survival was 50%. At the highest test concentration of 1.4 mg/L, all test animals were dead at observation on Day 9.
Thus, the survival of Daphnia magna after 21 days was significantly reduced (>20%) first at the test concentration of 0.43 mg/L.
The first young offspring released from their parent animals were recorded in the control, solvent control and at all test concentrations with surviving animals (0.019 – 0.43 mg/L) at observation on Day 9. Thus, the time of the first brood was not affected up to and including the test concentration of 0.43 mg/L. However, at the test concentration 0.43 mg/L, the highest test concentration with surviving animals, only 3 juvenile daphnids were produced at the observation at Day 9.
The mean reproduction rate of the daphnids in the solvent control was 62.1 ± 14.1 living offspring per adult (mean ± standard deviation). The corresponding value in the control was 71.2 ± 7.5. The control and the solvent control were pooled, since their mean reproduction rates were not statistically significantly different to each other.
According to the results of a Williams’ test (one-sided smaller, α = 0.05) no significant inhibitory effect of the test item on the mean reproduction rate was determined up to and including the test concentration of 0.049 mg/L. At the next higher test concentration of 0.13 mg/L, the mean reproduction rate statistically significantly reduced to 79.7% compared to the pooled controls.
The 21-day EC50 for the inhibition of the reproduction rate of Daphnia magna was calculated to be 0.32 mg/L (95% confidence limits: 0.30 – 0.35 mg/L).
With the exception of the reported mortality and the reduced reproduction rates, no visible abnormalities were observed at the test animals during the test.
Reported statistics and error estimates:
The NOEC and the LOEC of the reproduction rate were statistically evaluated by testing the mean reproduction rate at the test concentrations for statistically significant differences to the pooled control and solvent control value by the multiple Williams’ test.
Additionally, the EC50 for the inhibition of the reproduction rate after 21 days was calculated by Probit Analysis
Validity criteria fulfilled:
yes
Conclusions:
The 21-day NOEC based on reproduction was calculated to be 0.049 mg/L (measured concentration). The LOEC was calculated to be 0.32 mg/L (measured concentration).
Executive summary:

The long-term toxicity to daphnia was tested in accordance to OECD guideline 211. Daphnids were exposed for 21 day under semi-static conditions to nominal concentrations of 0.020, 0.063, 0.2, 0.63, 2.0 mg/L resulting in measured concentrations of 0.019, 0.049, 0.13, 0.43, 1.4 mg/L. Taking into account the survival and the reproduction of the test animals, the highest concentration of tert-Butyl peroxyneodecanoate tested without toxic effects after the exposure period of 21 days (21-day NOEC) was determined to be 0.049 mg/L. The lowest concentration tested with toxic effects (21-day LOEC) was determined to be 0.13 mg/L due to the statistically significantly reduced mean reproduction rate of Daphnia magna observed at this test concentration. The 21-day EC50 for the inhibition of the reproduction rate of Daphnia magna was calculated to be 0.32 mg/L (95% confidence limits: 0.30 – 0.35 mg/L).

Description of key information

The long-term toxicity to invertebrates was assessed according to OECD guideline 211. In this study the 21-day NOEC based on reproduction was calculated to be 0.049 mg/L (measured concentration). The LOEC was calculated to be 0.32 mg/L (measured concentration).

Key value for chemical safety assessment

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

Additional information

The long-term toxicity to daphnia was tested in accordance to OECD guideline 211. Daphnids were exposed for 21 day under semi-static conditions to nominal concentrations of 0.020, 0.063, 0.2, 0.63, 2.0 mg/L resulting in measured concentrations of 0.019, 0.049, 0.13, 0.43, 1.4 mg/L. Taking into account the survival and the reproduction of the test animals, the highest concentration of tert-Butyl peroxyneodecanoate tested without toxic effects after the exposure period of 21 days (21-day NOEC) was determined to be 0.049 mg/L. The lowest concentration tested with toxic effects (21-day LOEC) was determined to be 0.13 mg/L due to the statistically significantly reduced mean reproduction rate of Daphnia magna observed at this test concentration. The 21-day EC50 for the inhibition of the reproduction rate of Daphnia magna was calculated to be 0.32 mg/L (95% confidence limits: 0.30 – 0.35 mg/L).