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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:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From April 26, 1995 to May 17, 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the section 13 for details on the category justification.
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Single samples of 100 mg/L stock solution and duplicate samples of 0.032, 0.10 and 0.32 mg/L test solutions
- Sampling method: For the first of the two samples of the test solution and control groups, samples were always taken out of the actual test media (including the algal food) by pouring together the contents of the test beakers after the treatment periods. For the second sample, algal food was added in the same amount as in the actual test to the freshly prepared test media, and then these test media were incubated under the test conditions during the renewal periods.
- Sampling frequency: Day 0, 12 and 16
- Stability testing
- Concentration: Duplicate samples of 0.032, 0.10 and 0.32 mg/L test solutions and the control
- Sampling method: Both the samples could not be taken out of the actual test media, since at this test concentration only five of the Daphnia survived at the dates of the stability tests, and thus the available test media volumes were too low even for one analytical sample.
- Sampling frequency: Day 0, 12, 14, 16 and 19
- Sample storage conditions before analysis: Samples were treated with 8 % (v/v) of a Formalin solution (37 %), to preserve the samples until analysis

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance was homogenized by intense stirring. Then an adequate amount of the homogenized test substance was weighed into the test water for the preparation of a stock solution of 100 mg/L concentration. This stock solution was diluted with test water in a further step to result in a nominal concentration of 10 mg/L. Then adequate volumes of these intensively stirred stock solutions were mixed into test water to obtain the desired test concentrations before introduction of Daphnia.
- Controls: Yes, reconstituted water (test water)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No remarkable observations were made concerning the behaviour of the test substance in the test media.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Clone 5
- Source: BASF AG, D-67056 Ludwigshafen in January 1993
- Age of parental stock (mean and range, SD): Less than 24 h old
- Feeding during test
- Food type: Green algae (Scenedesmus subspicatus), cell freshly grown in the laboratories of RCC
- Amount: Days 0/1: 60,000 cells/mL (= 0.48*107 cells/80 mL): 0.05 mg TOC/80 mL
Days 2/12-15/19/20: 125,000 cells/mL (= 1.0 *107 cells/80 mL): 0.11 mg TOC/80 mL
Days 5-8: 100,000 cells/mL (= 0.8 *107 cells/80 mL): 0.09 mg TOC/80 mL
Day 9 (Friday): 200,000 cells/mL (= 1.6 *107 cells/80 mL): 0.18 mg TOC/80 mL
Day 16 (Friday): 250,000 cells/mL (= 2.0 *107 cells/80 mL): 0.23 mg TOC/80 mL
- Frequency: Daily (Monday to Friday)
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:
20.3-21.3°C

pH:
Ranged from pH 7.8 to 8.6
Dissolved oxygen:
>8.0 mg/L

Nominal and measured concentrations:
- Nominal: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass-beaker
- Type (delete if not applicable): Closed (i.e., covered with watch glass)
- Material, size, headspace, fill volume: 100 mL glass-beaker containing 80 mL test medium
- Aeration: Test water was aerated until oxygen saturation before the test, but not aerated during the test
- Renewal rate of test solution (frequency): Days 2, 5, 7, 9, 12, 14, 16 and 19 of the exposure period
- No. of organisms per vessel: Ten
- No. of vessels per concentration (replicates): Single
- No. of vessels per vehicle control (replicates): Single

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (Milli-Q-water) (For details on composition of the test water, please refer to the Table 1 in the appended word document under 'attached background material')
- Alkalinity: 0.9 mmol/L
- Conductivity: Lower than 0.1 µS cm-1

OTHER TEST CONDITIONS
- Photoperiod: A 16-h light to 8-h darkness photoperiod with a 15 minute transition period between light and darkness
- Light intensity: Approx. 405-700 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality, the number of young born and signs of intoxication
- Frequency: The mortality of adults and the number of young were recorded three times per week before renewal of test media

VEHICLE CONTROL PERFORMED: Yes

RANGE-FINDING STUDY
- Test concentrations: Not available
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
ca. 0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: survival rates and the reproduction rates
Remarks on result:
other: equivalent to a mean measured concentration of 0.07 mg/L
Key result
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
ca. 0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: equivalent to a mean measured concentration of 0.24 mg/L.
Details on results:
Survival of adults:
- In the control and at the test concentrations up to and including nominal 0.10 mg/L, the survival rate of the test animals (parent) after the exposure period of 21 days was at least 80% or higher.
- At the nominal concentration of 0.32 mg/L the mortality rate increased to 50%
- At the highest nominal test concentration of 1.0 mg/L all Daphnia were dead at the observation on Day 5. Thus, the survival rate of Daphnia magna after 21 days was clearly reduced (more than 20 %).
- A statistical test for the prove on significant differences of the mortality rates compared to the control is not available for the used test design with individual held ten test animals per concentration.

Reproduction rate:
- The first young Daphnia released from their parent animals were recorded in the control and at the test concentrations up to and including nominaltest concentration of 0.32 mg/L at the observation on Day 9. At the highest concentration of nominal 1.0 mg/L, no offspring were reproduced.
- The mean reproduction rate of those Daphnia in the control was 107.0 ± 14.7 (mean ± SD) alive offspring per adult. According to the results of a
WILLIAMS-Test (one-sided, α = 0.05) the reproduction rate of the surviving test animals was not significantly reduced up to and including the nominal test concentration of 0.32 mg/L.

Signs of intoxication: With exception of the reported mortality, no particular signs of intoxication were observed at the test animals during the test.
Reported statistics and error estimates:
Analysis of variance (ANOVA), followed by multivariate WILLIAMS-Test was applied to the reproduction parameters

Analytical determinations:

 - The analytically determined concentrations of the test substance test substance in the samples from the freshly prepared stock solutions ranged from 86% to 104% of the nominal concentration.

- The measured test substance concentrations in the samples from the freshly prepared test media of the very low test concentration of nominal 0.032 mg/L (in the range of the determination limit of the analytical method) varied from 85% to 171% of the nominal concentration. However, in this concentration range the measured values in the control samples must be taken into account. But for the biological results this low test concentration is of no importance.

- The analytically determined concentrations of test substance in the samples from the freshly prepared test media of the biologically relevant test concentrations of nominal 0.10 and 0.32 mg/L varied in the range from 85% to 109% of the nominal values. The mean measured values of all measurements of the freshly prepared test media of nominal 0.10 mg/L amounted to 100%, and at the test concentration of nominal 0.32 mg/L to 86% of the respective nominal value.

- During the test medium renewal periods of 48 and 72 h, the test substance concentrations decreased. At the test concentration of nominal 0.10 mg/L only 15% to 25% of the nominal value were found in the samples, taken out of the actual biological test after the 48 respectively 72 h exposure periods. However, 63% to 75% of nominal were found at that dates in test media, which were incubated with algae but without test animals under the test conditions in parallel to the actual test.

- The reason for these differences are unknown, but two possible reasons must be taken into consideration:

(a) firstly the algal cell densities were lower in the actual test media due to the filtration and consumption of algae by the test animals during the renewal periods, but the influence of the algae on the analytical results is unknown.

(b) Secondly, in the actual test media the test substance might have been stronger biodegraded due to the inocculation of bacteria with the drop of old test media at the transfer of the test animals from the old test media into the freshly prepared test media at each test medium renewal date. On the other side it is quite possible that the test substance adsorbed on the algal cells. In this case Daphnia can take up the test substance also via food. And toxic effects due to an uptake via food cannot be divided from the total toxic response determined in the biological test.

Since, the influence of all these factors cannot be quantified, the biological results (NOEC and LOEC) are based on the arithmetical mean value between the mean measured concentrations in the freshly prepared test media (as given in the tables with the analytical results), and the mean measured values in the 48 respectively 72 h old test media (i.e., at nominal 0.10 mg/L, this arithmetical mean measured concentration amounted to 0.073 mg/L, at nominal 0.32 mg/L to 0.244 mg/L).

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the nominal 21 d NOEC and LOEC values were 0.10 and 0.32 mg/L, equivalent to mean measured values of 0.07 and 0.24 mg/L, respectively.
Executive summary:

A study was carried out to determine the chronic toxicity of the read across substance, C12-18 and C18-unsatd. DEA, to Daphnia magna according to OECD Guideline 211, in compliance with GLP. Groups of ten daphnids were exposed to the substance (dispersed in test medium by intensive stirring) in a concentration range of 0.010 to 1.0 mg/L for 21 d under semi-static conditions. Survival and reproduction were determined three times per week. Single samples of the 100 mg/L stock solution and duplicate samples of 0.032, 0.10 and 0.32 mg/L test solutions were collected on Days 0, 12 and 16 for analytical dose verification by HPLC-UV. In the controls and at the test concentrations up to and including 0.10 mg/L, the survival rate of parental animals after 21 d was ≥ 80%. At 0.32 mg/L, the mortality rate increased to 50%. At the highest test concentration of 1.0 mg/L, all Daphnia were dead at the observation on Day 5. With exception of the reported mortality, no particular signs of intoxication were observed during the study. The first young Daphnia released from their parents were recorded in the control and at the test concentrations up to and including 0.32 mg/L at the observation on Day 9. At the highest concentration of 1.0 mg/L, no offspring were recorded. The mean reproduction rate of control Daphnia was 107.0 ± 14.7 alive offspring per adult. According to the results of a Willians Test (one-sided, α = 0.05), the reproduction rate of surviving test animals was not significantly reduced up to and including 0.32 mg/L. The corresponding analytically determined concentrations at 0.10 and 0.32 mg/L were 0.07 and 0.24 mg/L, respectively. Under the study conditions, the nominal 21 d NOEC and LOEC values were 0.10 and 0.32 mg/L, equivalent to mean measured values of 0.07 and 0.24 mg/L, respectively (Memmert, 1995). Based on the results of the read across study, the test substance is also expected to have similar toxicity to invertebrates

Description of key information

Key value for chemical safety assessment

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

Additional information

A study was carried out to determine the chronic toxicity of the read across substance, C12-18 and C18-unsatd. DEA, to Daphnia magna according to OECD Guideline 211, in compliance with GLP. Groups of ten daphnids were exposed to the substance (dispersed in test medium by intensive stirring) in a concentration range of 0.010 to 1.0 mg/L for 21 d under semi-static conditions. Survival and reproduction were determined three times per week. Single samples of the 100 mg/L stock solution and duplicate samples of 0.032, 0.10 and 0.32 mg/L test solutions were collected on Days 0, 12 and 16 for analytical dose verification by HPLC-UV. In the controls and at the test concentrations up to and including 0.10 mg/L, the survival rate of parental animals after 21 d was ≥ 80%. At 0.32 mg/L, the mortality rate increased to 50%. At the highest test concentration of 1.0 mg/L, all Daphnia were dead at the observation on Day 5. With exception of the reported mortality, no particular signs of intoxication were observed during the study. The first young Daphnia released from their parents were recorded in the control and at the test concentrations up to and including 0.32 mg/L at the observation on Day 9. At the highest concentration of 1.0 mg/L, no offspring were recorded. The mean reproduction rate of control Daphnia was 107.0 ± 14.7 alive offspring per adult. According to the results of a Willians Test (one-sided, α = 0.05), the reproduction rate of surviving test animals was not significantly reduced up to and including 0.32 mg/L. The corresponding analytically determined concentrations at 0.10 and 0.32 mg/L were 0.07 and 0.24 mg/L, respectively. Under the study conditions, the nominal 21 d NOEC and LOEC values were 0.10 and 0.32 mg/L, equivalent to mean measured values of 0.07 and 0.24 mg/L, respectively (Memmert, 1995).