Reaction product of 3,5,5-trimethyl-hexanoic acid and 2-methylpropanoic acid and pentaerythritol

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

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)

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.