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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:
28 November 2019 - 06 June 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark.
- Stability under test conditions: Assumed stable.
- Solubility and stability of the test substance in the solvent/vehicle: Microscopic inspections of the WAFs showed no micro-dispersions or undissolved test item to be present. Stable over a period of 7 days in methanol.
- Reactivity of the test substance with the test medium: -

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid in THF co-solvent.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A

OTHER SPECIFICS: No
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1.2, 3.6, 10.8, 32.4, 97.2 mg/L loading rate WAF
- Sampling method: Collection
- Sample storage conditions before analysis: Frozen
Vehicle:
yes
Remarks:
methanol
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Solid phase extraction
- Eluate: 0.1 v/v% formic acid in methanol
- Differential loading: N/A
- Controls: N/A
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): methanol
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): N/A
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: derived from in-house laboratory cultures.
- Justification for species other than prescribed by test guideline: N/A
- Age at study initiation (mean and range, SD): less than 24hrs old
- Weight at study initiation (mean and range, SD): N/A
- Length at study initiation (length definition, mean, range and SD): N/A
- Stage and instar at study initiation: N/A
- Valve height at study initiation, for shell deposition study (mean and range, SD): N/A
- Peripheral shell growth removed prior to test initiation: N/A
- Method of breeding: in house
- Source: N/A
- Age of parental stock (mean and range, SD): N/A
- Feeding during test : Daily
- Food type: mixture of algal suspension (Raphidocelis subcapitata ) and GEMMA Micro 300 fish food suspension
- Amount: N/A
- Frequency: N/A

ACCLIMATION
- Acclimation period: N/A
- Acclimation conditions (same as test or not): N/A
- Type and amount of food: N/A
- Feeding frequency: N/A
- Health during acclimation (any mortality observed): N/A

QUARANTINE (wild caught)
- Duration: N/A
- Health/mortality: N/A

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
A Daphnia were maintained in 150 mL glass vessels containing Elendt M4 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.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
N/A
Hardness:
232 - 282 mg/L as CaCO3
Test temperature:
21 ºC
pH:
7.4 - 8.4
Dissolved oxygen:
7.2 - 9.3 mg O2 / L
Salinity:
N/A
Conductivity:
Not reported
Nominal and measured concentrations:
Not reported
Details on test conditions:
TEST SYSTEM
- Test vessel: 125ml glass vessel
- Type (delete if not applicable): oopen (covered with plastic lid)
- Material, size, headspace, fill volume: N/A
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): Daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): N/A
- Biomass loading rate: N/A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium
- Total organic carbon: N/A
- Particulate matter: N/A
- Metals: N/A
- Pesticides: N/A
- Chlorine: N/A
- Alkalinity: 0.9mmol/L
- Ca/mg ratio: N/A
- Conductivity: N/A
- Culture medium different from test medium: N/A
- Intervals of water quality measurement: N/A

OTHER TEST CONDITIONS
- Adjustment of pH: 7.9
- Photoperiod: 16hrs light-8hrs dark
- Light intensity: not exceeding 1500 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : N/A

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study: 1.0, 10. 100 mg/L Loading Rate
- Test concentrations: 1.2, 3.6, 10.8, 32.4, 97.2 mg/L Loading Rate
- Results used to determine the conditions for the definitive study: 1.2, 3.6, 10.8, 32.4, 97.2 mg/L Loading Rate
Reference substance (positive control):
not required
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
>= 97.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
>= 32.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 97.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 97.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Behavioural abnormalities: No effects observed in any test concentrations.
- Observations on body length and weight: No effects observed in any test concentrations.
- Other biological observations: No effects observed in any test concentrations.
- Mortality of control: 0 %
- Other adverse effects control: No
- Abnormal responses: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
N/A
Reported statistics and error estimates:
No

Table 1 Exposure based on the nominal test concentrations

 

Endpoint

Concentration

(mg/L LR WAF)

95% Confidence Limits (mg/L LR WAF)

 

 

 

Immobilization

EL10

19

Not applicable

EL20

Not determined

Not applicable

EL50

Not determined

Not applicable

No Observed Effect Loading Rate

97.2

-

Lowest Observed Effect Loading Rate

Not determined

-

 

 

 

Reproduction

EL10

>97.2

Not applicable

EL20

>97.2

Not applicable

EL50

>97.2

Not applicable

No Observed Effect Loading Rate

32.4

-

Lowest Observed Effect Loading Rate

97.2

-

 

 

 

Body Length

EL10

>97.2

Not applicable

EL20

>97.2

Not applicable

EL50

>97.2

Not applicable

No Observed Effect Loading Rate

97.2

-

Lowest Observed Effect Loading Rate

Not determined

-

Table 2 Summary of Observations for 21 Days

Nominal Loading Rate (mg/L)

Control

1.2

3.6

10.8

32.4

97.2

Adults Surviving

Live Young

Adults Surviving

Live Young

Adults Surviving

Live Young

Adults Surviving

Live Young

Adults Surviving

Live Young

Adults Surviving

Live Young

10

1612

10

1665

10

1612

8

1601

10

1553

9

1365

Table 3 Body Length of Surviving Adults at Day 21

Nominal Loading Rate

(mg/L)

Individual Daphnia Lengths (mm)

1

2

3

4

5

6

7

8

9

10

Control

4.6

4.6

4.4

4.7

4.7

4.6

4.5

4.5

4.5

4.6

1.2

4.4

4.5

4.6

4.6

4.6

4.7

4.7

4.4

4.5

4.5

3.6

4.5

4.2

4.4

4.5

4.3

4.5

4.5

4.6

4.6

4.4

10.8

4.4

4.5

4.6

-

-

4.3

4.4

4.4

4.5

4.4

32.4

4.7

4.5

4.5

4.4

4.6

4.4

4.5

4.5

4.5

4.5

97.2

4.4

4.6

4.8

4.8

-

4.9

4.4

4.2

4.2

4.4

Table 4 Number of Live Young Produced per Surviving Adult by Day 21

Nominal Loading Rate (mg/L)

Number of Live Young Produced per Surviving Adult by Day 21

 

Control

Mean

208.1

Standard Deviation

13.8

 

12

Mean

143.4

Standard Deviation

16.4

 

20

Mean

187.0

Standard Deviation

30.3

 

32

Mean

164.3

Standard Deviation

26.3

 

50

Mean

144.3

Standard Deviation

8.8

 

80

Mean

161.2

Standard Deviation

31.7

Validity criteria fulfilled:
yes
Remarks:
The mortality of the parent animals did not exceed 20 % at the end of the test; - the mean number of living offspring produced per parent animal surviving at the end of the test is > 60.
Conclusions:
No mortalities occurred at the 1.2, 3.6 and 32.4 mg/L loading rate WAF groups throughout the test. However, for effects on Reproduction, the 97.2 mg/L loading rate WAF group showed a statistically significant difference from the control after 21 days in terms of producing fewer numbers of live young per adult. The resulting endpoints are;

Parental mortality and sub-lethal effects: 21days
EL50 >97.2mg/L Loading Rate
NOEL 97.2 mg/L Loading Rate
Executive summary:

OECD 211 (2012) - The 21-day-chronic toxicity of Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid to Daphnia magna was studied under semi-static conditions. Test species were exposed to control, solvent control, and test chemical at mean measured concentrations of 1.2, 3.6, 10.8, 32.4, 97.2 mg/L Loading Rate.


 


The 21-day EL50 based on mortality/sublethal effect was >97.2mg/L Loading Rate as no effects were observed at any of the test concentrations.  The 21-day NOEL based on sublethal effects was therefore 97.2 mg/L Loading Rate.


 


An effect on the reproduction were observed only at 97.2mg/L loading rate. The 21-day NOEC for reproduction was therefore 32.4 mg/L Loading Rate.


 


This study is classified as acceptable and satisfies the guideline requirements for a chronic toxicity study with freshwater invertebrates.

Description of key information

Mortality and sublethal effects: 21-day EL50 = >97.2 mg/L and 21 -day NOELR = 97.2 mg/L; OECD 211; K, Hull. (2020)

Reproduction: 21-day NOEC = 97.2 mg/L; OECD 211; K, Hull. (2020)

Key value for chemical safety assessment

Additional information

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as Water Accommodated Fractions (WAFs).Daphnia magna were exposed (10 replicates of a single daphnid per group) to a WAF of the test item over a range of nominal loading rates of 1.2, 3.6, 10.8, 32.4 and 97.2 mg/L for a period of 21 days.  The test solutions were renewed daily throughout the test.  The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily.