Fatty acids, C7-9, tetraesters with pentaerythritol

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 - 19 Nov 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study with acceptable restrictions (no purity, no reference substance)

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
- Sampling method: Samples were collected on Day 2 of dry mixing, Day 6 of hydration equilibration, Day 0, prior to the addition of worms for the control and treatment and at test termination (Day 14). The samples were collected using a stainless steel spatula and weighed into 40 mL VOA vials. Three replicates were collected from the treatment soil and duplicate from the control. The samples collected at termination were from a pooled sample where all four replicates for each treatment group were composited.
- Sample storage conditions before analysis:

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): The treated artificial soil was prepared by spiking 300 grams of peat with the appropriate amount of test material using volumetric pipettes and/or stainless steel and glass syringe and mixed prior to the addition of clay and sand. The treated soil and control soil was mixed on a roller mill for three days. After hydration with distilled water the soils were placed in an environment chamber at test temperature to equilibrate for 8 days.
- Controls: A control soil was prepared without the test substance.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: Lot W-43, Carolina Biological Supply Co. 2700 York Road, Burlington, NC 27215-3398
- Age at test initiation (mean and range, SD): Worms with clitellum.
- Weight at test initiation: 300 - 600 mg

ACCLIMATION
- Acclimation period: 12 October - 05 November 2012
- Acclimation conditions (same as test or not): Worms were held and evaluated in worm bedding moistened with distilled water from 03 October 2012 until 12 October 2012. All study worms were transferred from worm bedding and conditioned in an artificial soil bed from 12 October 2012 until study initiation on 05 November 2012.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

14 d

Test temperature:

19.87°C -20.16°C

pH:

Control: 6.43 (Day 0); 6.41 (Day 14)
1000 mg/kg: 6.31 (Day 0); 6.43 (Day 14)

Moisture:

Control: 27.4 - 30.6% (Day 0)
1000 mg/kg: 24.2 - 29.9% (Day 0)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 1 L glass jars
- Amount of soil or substrate: 750 g of soil
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- Other: Each chamber was covered with perforated plastic film during the study to maintain hydration and allow some air circulation.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 70% sand, 20% kaolin clay, 10% peat (according to OECD 207)
- Other: Distilled water- UV sterilized, deionized well water is distributed throughout the testing facility via PVC stainless steel pipes.

OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity: 719 - 728 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Observations for mortality were performed on Days 7 and 14 of the test. Each test chamber was weighed after the addition of hydrated soil and worms on Day 0 to obtain a starting weight for each test chamber. On Day 7 and 14, the chambers were re-weighed to monitor any water weight loss. All surviving worms were pooled per replicate and weighed at the end of the test.

Nominal and measured concentrations:

nominal: 1000 mg/kg dw
measured:
991 mg/kg (average initial concentration prior to soil hydration)
205 mg/kg (final analysis of the soil concentration)
see table 1 under "Any other information on materials and methods" for detailed information

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Mortality at end of exposure period: No mortality was observed at test termination in both the control and treatment group.
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: The weight indicate that there were no sublethal effects observed.

Table 2: Worm Percent Survival and Worm Weights

Treatment Group (mg/kg)	Percent Worm Survival		Mean Wet Worm Weight(g)
	Day 7*	Day 14*	Day 0**	Day 14**
Control
Replicate1	10/10 (100%)	10/10 (100%)	4.2498	3.1220
Replicate2	10/10 (100%)	10/10 (100%)	4.7216	3.6122
Replicate3	10/10 (100%)	10/10 (100%)	4.3836	3.3593
Replicate4	10/10 (100%)	10/10 (100%)	4.9141	4.1418
1000 mg/kg
Replicate1	10/10 (100%)	10/10 (100%)	4.7145	3.3925
Replicate2	10/10 (100%)	10/10 (100%)	4.4095	3.5429
Replicate3	10/10 (100%)	10/10 (100%)	4.6580	3.6534
Replicate4	10/10 (100%)	10/10 (100%)	4.5076	3.5073

*Worms were observed, counted and placed back in original soil and chamber
**Worms were carefully rinsed with de-ionized water, bottled dry and weighed by replicate. The mean wet weight was determined an average of the replicate weights for each group.

 

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Oct 2012 - 20 Feb 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Qualifier:

equivalent or similar to guideline

Guideline:

ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss Federal Office of Public Health, Bern, Switzerland

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): A stock solution was prepared by dissolving the test substance in acetone (250 mg test substance/mL acetone). The required test substance concentrations were prepared by a serial dilution of the stock solution. The test solutions were subsequently mixed with sand. The mixture was left for evaporation of the solvent for 2 hours. Subsequently the sand/test substance mixture was mixed again. Before test start the mixture was added to artificial soil. For each test vessel, 496.4 g fresh weight of the prepared artificial soil was thoroughly mixed with 10 g sand/test item mixture, 2.8 g calcium carbonate, 5 g food, and 143.6 mL purified water in a laboratory mixer.
- Chemical name of vehicle: acetone
- Evaporation of vehicle before use: yes

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Source: Blades Biological Ltd, Cowden, United Kingdom
- Age at test initiation: adult, with clitellum
- Weight at test initiation (range): 310 - 594 mg

ACCLIMATION
- Acclimation period: 3 days
- Acclimation conditions: same as in test

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Remarks:

After 28 days the adult earthworms were removed and the cocoons and juveniles remained in the test substrate for another 28 days

Test temperature:

19 - 22 °C

pH:

5.8 - 6.1

Moisture:

29 - 38% of water holding capacity (WHC: 50.3 g /100 g dry soil).

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Plastic boxes (16.5 cm x 12 cm x 6.5 cm, area about 200 cm²)
- Amount of soil or substrate: 650 g
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8 (incl. vehicle control)

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 10% Sphagnum peat, 20% Kaolin clay, 69% Sand
- Maximum water holding capacity (in % dry weigth): 50.3%

OTHER TEST CONDITIONS
- Photoperiod: 16:8 h day-night regime
- Light intensity: 656-749 lux

EFFECT PARAMETERS MEASURED:
Mortality and body weight of adult earthworms after 28 d, reproduction rate after 56 d

Nominal and measured concentrations:

Nominal: control, 62.5, 125, 250, 500 and 1000 mg/kg dry soil.

Reference substance (positive control):

yes

Remarks:

Carbendazim, Batch No.: 12001/03

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality at end of exposure period: control: no mortality observed; test substance: 5%

Results with reference substance (positive control):

10% mortality at a reference substance concentration of 8.33 mg/kg dry soil

Reported statistics and error estimates:

Changes in body weight and differences regarding mortalilty between treatment and control were evaluated by multiple Dunnett t-test (Dunnett, 1955; Dunnett, 1964) after a one-way analysis of variance (ANOVA).
The body weight change of the surviving worms in the positive control was compared to the control and was statistically evaluated by means of a Student-t test (Sachs, 1984).
Reproduction rates were analysed using a multiple Dunnett t-test [Dunnett, 1955; Dunnett, 1964] after a one-way analysis of variance (ANOVA).

Table 1: Mortality, number of hatched juveniles and changes in body weight

Treatment (mg/kg dry weight)	Mortality (%) after a 4-week exposure	Juveniles per vessel (mean)	Juvenils per vessel (% of control)	Mean body weight per worm (% difference from test start)
Control	0	117	100	21
62.5	0	149	127	16
125	5	130	111	18
250	0	106	90	22
500	0	111	94	21
1000	0	113	96	17
Reference substance	10	0	0	-17

Reference 3

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

05 - 19 Nov 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study with acceptable restrictions (no purity, no reference substance)

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to IUCLID Section 13 for the detailed justification of the Analogue Approach.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
- Sampling method: Samples were collected on Day 2 of dry mixing, Day 6 of hydration equilibration, Day 0, prior to the addition of worms for the control and treatment and at test termination (Day 14). The samples were collected using a stainless steel spatula and weighed into 40 mL VOA vials. Three replicates were collected from the treatment soil and duplicate from the control. The samples collected at termination were from a pooled sample where all four replicates for each treatment group were composited.
- Sample storage conditions before analysis:

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): The treated artificial soil was prepared by spiking 300 grams of peat with the appropriate amount of test material using volumetric pipettes and/or stainless steel and glass syringe and mixed prior to the addition of clay and sand. The treated soil and control soil was mixed on a roller mill for three days. After hydration with distilled water the soils were placed in an environment chamber at test temperature to equilibrate for 8 days.
- Controls: A control soil was prepared without the test substance.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: Lot W-43, Carolina Biological Supply Co. 2700 York Road, Burlington, NC 27215-3398
- Age at test initiation (mean and range, SD): Worms with clitellum.
- Weight at test initiation: 300 - 600 mg

ACCLIMATION
- Acclimation period: 12 October - 05 November 2012
- Acclimation conditions (same as test or not): Worms were held and evaluated in worm bedding moistened with distilled water from 03 October 2012 until 12 October 2012. All study worms were transferred from worm bedding and conditioned in an artificial soil bed from 12 October 2012 until study initiation on 05 November 2012.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

14 d

Test temperature:

19.87°C -20.16°C

pH:

Control: 6.43 (Day 0); 6.41 (Day 14)
1000 mg/kg: 6.31 (Day 0); 6.43 (Day 14)

Moisture:

Control: 27.4 - 30.6% (Day 0)
1000 mg/kg: 24.2 - 29.9% (Day 0)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 1 L glass jars
- Amount of soil or substrate: 750 g of soil
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- Other: Each chamber was covered with perforated plastic film during the study to maintain hydration and allow some air circulation.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 70% sand, 20% kaolin clay, 10% peat (according to OECD 207)
- Other: Distilled water- UV sterilized, deionized well water is distributed throughout the testing facility via PVC stainless steel pipes.

OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity: 719 - 728 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Observations for mortality were performed on Days 7 and 14 of the test. Each test chamber was weighed after the addition of hydrated soil and worms on Day 0 to obtain a starting weight for each test chamber. On Day 7 and 14, the chambers were re-weighed to monitor any water weight loss. All surviving worms were pooled per replicate and weighed at the end of the test.

Nominal and measured concentrations:

nominal: 1000 mg/kg dw
measured:
991 mg/kg (average initial concentration prior to soil hydration)
205 mg/kg (final analysis of the soil concentration)
see table 1 under "Any other information on materials and methods" for detailed information

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Mortality at end of exposure period: No mortality was observed at test termination in both the control and treatment group.
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: The weight indicate that there were no sublethal effects observed.

Table 2: Worm Percent Survival and Worm Weights

Treatment Group (mg/kg)	Percent Worm Survival		Mean Wet Worm Weight(g)
	Day 7*	Day 14*	Day 0**	Day 14**
Control
Replicate1	10/10 (100%)	10/10 (100%)	4.2498	3.1220
Replicate2	10/10 (100%)	10/10 (100%)	4.7216	3.6122
Replicate3	10/10 (100%)	10/10 (100%)	4.3836	3.3593
Replicate4	10/10 (100%)	10/10 (100%)	4.9141	4.1418
1000 mg/kg
Replicate1	10/10 (100%)	10/10 (100%)	4.7145	3.3925
Replicate2	10/10 (100%)	10/10 (100%)	4.4095	3.5429
Replicate3	10/10 (100%)	10/10 (100%)	4.6580	3.6534
Replicate4	10/10 (100%)	10/10 (100%)	4.5076	3.5073

*Worms were observed, counted and placed back in original soil and chamber
**Worms were carefully rinsed with de-ionized water, bottled dry and weighed by replicate. The mean wet weight was determined an average of the replicate weights for each group.

 

Reference 4

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

25 Oct 2012 - 20 Feb 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (ECHA, 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to IUCLID Section 13 for the detailed justification of the Analogue Approach.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Qualifier:

equivalent or similar to guideline

Guideline:

ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss Federal Office of Public Health, Bern, Switzerland

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): A stock solution was prepared by dissolving the test substance in acetone (250 mg test substance/mL acetone). The required test substance concentrations were prepared by a serial dilution of the stock solution. The test solutions were subsequently mixed with sand. The mixture was left for evaporation of the solvent for 2 hours. Subsequently the sand/test substance mixture was mixed again. Before test start the mixture was added to artificial soil. For each test vessel, 496.4 g fresh weight of the prepared artificial soil was thoroughly mixed with 10 g sand/test item mixture, 2.8 g calcium carbonate, 5 g food, and 143.6 mL purified water in a laboratory mixer.
- Chemical name of vehicle: acetone
- Evaporation of vehicle before use: yes

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Source: Blades Biological Ltd, Cowden, United Kingdom
- Age at test initiation: adult, with clitellum
- Weight at test initiation (range): 310 - 594 mg

ACCLIMATION
- Acclimation period: 3 days
- Acclimation conditions: same as in test

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Remarks:

After 28 days the adult earthworms were removed and the cocoons and juveniles remained in the test substrate for another 28 days

Test temperature:

19 - 22 °C

pH:

5.8 - 6.1

Moisture:

29 - 38% of water holding capacity (WHC: 50.3 g /100 g dry soil).

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Plastic boxes (16.5 cm x 12 cm x 6.5 cm, area about 200 cm²)
- Amount of soil or substrate: 650 g
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8 (incl. vehicle control)

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 10% Sphagnum peat, 20% Kaolin clay, 69% Sand
- Maximum water holding capacity (in % dry weigth): 50.3%

OTHER TEST CONDITIONS
- Photoperiod: 16:8 h day-night regime
- Light intensity: 656-749 lux

EFFECT PARAMETERS MEASURED:
Mortality and body weight of adult earthworms after 28 d, reproduction rate after 56 d

Nominal and measured concentrations:

Nominal: control, 62.5, 125, 250, 500 and 1000 mg/kg dry soil.

Reference substance (positive control):

yes

Remarks:

Carbendazim, Batch No.: 12001/03

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality at end of exposure period: control: no mortality observed; test substance: 5%

Results with reference substance (positive control):

10% mortality at a reference substance concentration of 8.33 mg/kg dry soil

Reported statistics and error estimates:

Changes in body weight and differences regarding mortalilty between treatment and control were evaluated by multiple Dunnett t-test (Dunnett, 1955; Dunnett, 1964) after a one-way analysis of variance (ANOVA).
The body weight change of the surviving worms in the positive control was compared to the control and was statistically evaluated by means of a Student-t test (Sachs, 1984).
Reproduction rates were analysed using a multiple Dunnett t-test [Dunnett, 1955; Dunnett, 1964] after a one-way analysis of variance (ANOVA).

Table 1: Mortality, number of hatched juveniles and changes in body weight

Treatment (mg/kg dry weight)	Mortality (%) after a 4-week exposure	Juveniles per vessel (mean)	Juvenils per vessel (% of control)	Mean body weight per worm (% difference from test start)
Control	0	117	100	21
62.5	0	149	127	16
125	5	130	111	18
250	0	106	90	22
500	0	111	94	21
1000	0	113	96	17
Reference substance	10	0	0	-17

Description of key information

NOEC (56 d) ≥ 1000 mg/kg soil dw based on reproduction (OECD 222); read-across
NOEC (14 d) ≥ 1000 mg/kg soil dw based on mortality (OECD 207); read-across

Additional information

Since no studies investigating the long-term toxicity of Tetraesters of pentaerythritol with heptanoic acid and 3,5,5-trimethylhexanoic acid (List number 805-289-4) to soil macroorganisms are available, in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5 a read-across to the structurally related source substances Decanoic acid, mixed esters with heptanoic acid, octanoic acid, pentaerythritol and valeric acid (CAS 71010-76-9) and 2,2-bis[[(1-oxoisononyl)oxy]methyl]-1,3-propanediyl diisononanoate (CAS 93803-89-5) was conducted. Decanoic acid, mixed esters with heptanoic acid, octanoic acid, pentaerythritol and valeric acid (CAS 71010-76-9) is characterized by pentaerythritol tetra-esterified with fatty acids of C5-C10 (even and uneven numbered). The second source substance 2,2-bis[[(1-oxoisononyl)oxy]methyl]-1,3-propanediyl diisononanoate (CAS 93803-89-5) is a mono-constituent substance characterized as a tetraester of pentaerythritol and 3,5,5-trimethylhexanoic acid. This read-across is justified in detail in the overall summary (IUCLID Section 6.1) and within the analogue justification in IUCLID Section 13. In this case of read-across, the best suited (highest degree of structural similarity, nearest physico-chemical properties) read-across substance was used for the assessment.

The first study with the read-across substance 2,2-bis[[(1-oxoisononyl)oxy]methyl]-1,3-propanediyl diisononanoate (CAS 93803-89-5) was performed according to OECD 222 (GLP) with the earthworm Eisenia fetida (Eisner, 2013). Five nominal concentrations were mixed into artificial soil and mortality/growth (after 4 weeks) and reproduction (after 8 weeks) were measured. No significant effects on mortality/growth and reproduction were found resulting in a NOEC ≥ 1000 mg/kg soil dw.

The second study was performed with the source substance decanoic acid, mixed esters with heptanoic acid, octanoic acid, pentaerythritol and valeric acid (CAS 71010-76-9). It was conducted according to OECD 207 under GLP conditions (Kelley, 2013). Eisenia fetida was exposed to a test concentration of 1000 mg/kg dw in artificial soil for 14 d. No mortality was recorded at the end of the study. 100% survival was observed in both the control group and treatment group resulting in a NOEC (14 d) for mortality of ≥ 1000 mg/kg soil dw.

Based on the results from structurally related read-across substances (in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5) which is characterized by an equal ecotoxicological profile and a similar structure, it can be concluded that Tetraesters of pentaerythritol with heptanoic acid and 3,5,5-trimethylhexanoic acid (List number 805-289-4) will not exhibit toxic effects to soil macroorganisms.