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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:
07 Nov 2012 to 12 Jan 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom, UK
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Water samples were taken from the solvent and each surviving test group (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 5, 13 and 19 and of the expired test preparations on Days 3, 7, 14 and 21. Samples at the No Observed Effect Concentration and above only were analyzed.
- Sample storage conditions before analysis: Samples were stored at approximately -20 °C prior to analysis. Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. A test concentration of 1.0 mg/L was obtained using a preliminary solution in dimethylformamide.
Based on this information the test item was categorized as being 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 item under test conditions. The test material preparation of the final test is based on the outcome of the media preparation trial.
Preparation of test solution (final trial):
An amount of test item (100 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give a 100 mg/10 mL solvent stock solution. An aliquot (500 µL) of the solvent stock solution was dispersed in a final volume of 5 liters of Elendt M7 media with the aid of a magnetic stirrer for approximately 10 minutes to give a 1.0 mg/L stock solution.
On Days 3, 7, 10, 14 and 17, the 1.0 mg/L stock solution was centrifuged at 40000g for 30 minutes to give the 0.040 mg/L test concentration. 0.04 mg/L was thus the highest attainable concentration under the test conditions. Aliquots (100, 180, 325 and 550 mL) of the 0.040 mg/L test concentration were added to a final volume of 1 liter of Elendt M7 media to give the further test concentrations of 0.0040, 0.0072, 0.013 and 0.022 mg/L respectively.
On Days 0, 5, 13 and 19 when fresh media samples were required for analysis, 1 liter of the 1.0 mg/L stock solution was centrifuged at 40000g for 30 minutes to give the 0.040 mg/L test concentration. Aliquots (200 and 360 mL) of the 0.040 mg/L test concentration were then added to final volumes of 2 liters of Elendt M7 media to give the 0.0040 and 0.0070 mg/L test concentrations respectively. A further 3 liters of the 1.0 mg/L stock solution was centrifuged at 10000g for 160 minutes, as per the manufacturer's guidelines, to give a further volume of the 0.040 mg/L test concentration. Aliquots (650 and 1100 mL) of this 0.040 mg/L test concentration were then added to a final volume of 2 liters of Elendt M7 media to give the further test concentrations of 0.013 and 0.022 mg/L respectively.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 5, 13 and 19 (fresh media) and on Days 3, 7, 14 and 21 (old media).
- Controls: A solvent control was included in the test.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 100 µL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: In-house laboratory culture. Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at 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. Culture conditions ensured that reproduction was by parthenogenesis
- Feeding during test
- Food type: Mixture of algal suspension and Tetramin® flake food suspension
- Frequency: The Daphnia were fed daily
- Amount: Each daphnid received approximately 2 to 7 µL of an algal suspension (Desmodesmus subspicatus) and approximately 10 to 32 µL of Tetramin® flake food suspension 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.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
254-310 mg/L CaCO3 in the solvent control and the highest surviving test concentration
Test temperature:
19-21°C
pH:
7.7-8.2
Dissolved oxygen:
> 3 mg/L
Nominal and measured concentrations:
0, 0.0040, 0.0072, 0.013, 0.022 and 0.040 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 150 mL glass flasks filled with 100 mL of test preparation and covered with a plastic lid
- Aeration: none (the dilution water only was aerated prior to use)
- Renewal rate of test solution (frequency/flow rate): Test solutions were renewed 3 times per week on Days 0, 3, 5, 7, 10, 13, 14, 17 and 19.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (Elendt M7 medium)

OTHER TEST CONDITIONS
- Photoperiod: photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition
- Light intensity: 748 to 807 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily: 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 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 immobilization 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.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: No toxic effects of the test item at the limit of solubility.
Details on results:
See any other information in results.
Reported statistics and error estimates:
See any other information in results.

Lethal Effects on the Parental Generation (P1)

Single mortalities were observed in the solvent control on Day 18 and at the test concentration of 0.022 mg/L on Day 9. However, no further mortality occurred throughout the test and, hence, no prolonged effects attributable to exposure of Daphnia magna to the test item were observed. It was not possible to calculate the EC50 value since less than 50% mortalities were observed.

Sub-lethal Effects on the Parental Generation (P1)

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 solvent control and each test group. 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 solvent control and the each test groups in terms of length of the daphnids after 21 days exposure to the test item.

Effects on the Filial Generation (F1)

Information on the effects of the test item on the Fi 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 solvent controls over the duration of the test.

Young were first produced in the solvent control test group on Day 8 of the test. There were no unhatched eggs and dead young in all control and treatment groups surviving to maturation.

Lowest Observed Effect Concentration

The "Lowest Observed Effect Concentration" (LOEC) based on time-weighted mean measured test concentrations was considered to be greater than 0.10 mg/L on the basis that at this concentration no significant mortalities (immobilisation) were observed in the parental generation (P1) and that there were no significant differences (P > 0.05) between the solvent control and the 0.10 mg/L time weighted mean measured test concentration in terms of numbers of live young produced per adult by Day 21.

No Observed Effect Concentration

The "No Observed Effect Concentration" (NOEC) based on time-weighted mean measured test concentrations was 0.10 mg/L as there were no significant mortalities (immobilization) 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 solvent control after 21 days.

Verification of Test Concentrations

Samples from the No Observed Effect Concentration (NOEC) and above only were analyzed.

Analysis of the freshly prepared test media for the 0.040 mg/L nominal test concentration on Days 0, 5, 13 and 19 showed measured test concentrations of 0.09, 0.13, 0.14 and 0.18 mg/L respectively. This declined in the old or expired media on Days 3, 7, 14 and 21 to 0.046, 0.074, 0.10 and 0.098 mg/L respectively. This was considered to be due to the unstable nature of the test item.

It was therefore considered justifiable to base the results on the time-weighted mean measured test concentration of the test media to give a "worst case" analysis of the data, which was calculated to be 0.10 mg/L.

Table: Summary of Findings Following the Exposure of Daphnia magna for 21 Days

Nominal Concentration (mg/1)

%

Survival of P1

Number of Live Young*

Number of Dead Young

Number of Unhatched Eggs

Total

Per Female (cumulative)

Total

Per Female (cumulative)

Total

Per Female (cumulative)

Control

100

1165

117

0

0

0

0

Solvent Control

90

1194

133

0

0

0

0

0.0040

100

1199

120

0

0

0

0

0.0072

100

1369

137

0

0

0

0

0.013

100

1426

143

0

0

0

0

0.022

90

1406

156

0

0

0

0

0.040

100

1290

129

0

0

0

0

Description of key information

No adverse effects up to the limit of water solubility (< 0.05 mg/L in aqua dest; <= 0.04 mg/L under test conditions) for Daphnia magna (OECD 211)

Key value for chemical safety assessment

Additional information

A GLP guideline study conducted according to OECD 211 on the chronic toxicity of Propane-1,2,3-triyl 2-ethylhexanoate (CAS No. 7360-38-5) is available. Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. A pre-study media preparation trial indicated that a dissolved test item concentration of approximately 0.040 mg/L was obtained from a solvent spike method of preparation indicating this to be the limit of water solubility of the test item under test conditions. Therefore, for the final study Daphnia magna (10 replicates of a single daphnid per group) were exposed to the following test concentrations: 0.0040, 0.0072, 0.013, 0.022 and 0.040 mg/L. The test was conducted under semi-static conditions. Analysis of the freshly prepared test media for the 0.040 mg/L nominal test concentration on Days 0, 5, 13 and 19 showed measured test concentrations of 0.09, 0.13, 0.14 and 0.18 mg/L respectively. This declined in the old or expired media on Days 3, 7, 14 and 21 to 0.046, 0.074, 0.10 and 0.098 mg/L respectively. This was considered to be due to the unstable nature of the test item. It was therefore considered justifiable to base the results on the time-weighted mean measured test concentration of the test media to give a "worst case" analysis of the data, which was calculated to be 0.10 mg/L. No significant effects were observed in the treatment groups, thus the EC50 and the NOEC values for immobilization and reproduction were determined to be > 0.1 mg/L and >= 0.1 mg/L , respectively.

In conclusion, chronic ecotoxicological effects on aquatic invertebrates are not expected up to the limit of water solubility of Propane-1,2,3-triyl 2-ethylhexanoate (CAS No. 7360-38-5).